IAEA-195

RADIONUCLIDE-LABELLED ANTIGENS INSEROLOGICAL EPIDEMIOLOGY

REPORT OF A FIVE-YEAR INVESTIGATION BY O. FELSENFELD AND M.W. PARROTT TULANE UNIVERSITY DELTA REGIONAL PRIMATE RESEARCH CENTER COVINGTON, LOUISIANA UNITED STATES OF AMERICA

| a@A TECHNICAL DOCUMENT ISSUED BY THE (]) INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1977 RADIONUCLIDE-LABELLED ANTIGENS IN SEROLOGICAL EPIDEMIOLOGY, IAEA, VIENNA, 1977 Printed by the IAEA in Austria February 1977 PLEASE BE AWARE THAT ALL OF THE MISSING PAGES IN THIS DOCUMENT WERE ORIGINALLY BLANK The IAEA does not maintain stocks of reports in this series. However, microfiche copies of these reports can be obtained from INIS Microfiche Clearinghouse International Atomic Energy Agency Kdmtner Ring 11 P.O. Box 590 A-1011 Vienna, Austria on prepayment of US $0.65 or against one IAEAmicrofiche service coupon. FOREWORD

Radioactive tracer techniques m microbial immunology have yet to be fully exploited. Of particular interest in relation to the health problems of developing countries is the possible use of radionuclide-labelled antigens as tracers in serological tests for specific antibodies. From 1970 to 1975, the International Atomic Energy Agency (IAEA) organized a co-ordinated research programme on "The Use of Labelled Antigens in Serological Epidemiology", aimed at the development of such labelled antigen tests and the investigation of their suitability for routine epidemiological studies. A leading role in this programme was played by the group at the Tulane University Delta Regional Primate Research Center, Covington, Louisiana, USA, led by Oscar Felsenfeld, who collaborated in it under IAEA Research Contract No. 828/RB: "Uses of Antigens Labelled with Radioisotopes in Serological Epidemiology". The present report describes in detail the results obtained by Oscar Felsenfeld and his colleagues under the above-mentioned research contract. The IAEA wishes to express its gratitude to Professor Felsenfeld and his colleagues at Tulane University and at other collabo- rating institutions. Marshall W. Parrott assisted in writing the report; his present address is Radiation Control Section, Health Division, Department of Human Resources, Portland, Oregon, USA. The book should be of interest to all who are working in, or who propose to work In, the field concerned.

CONTENTS

Page GENERAL PRECAUTIONS IN WORK WITH RADIONUCLIDE-LABELLED ANTIGENS

I. INTRODUCTION : METHODOLOGICAL ASPECTS A. LIMITATIONS OF LABELLED ANTIGEN TESTS ...... 1 B. CHOICE OF RADIONUCLIDE AND RADIOACTIVITY MEASUREMENT METHOD ...... ,...... 2 C. TEST PROCEDURES ... ****...... 3 D. EXPRESSION AND EVALUATION OF RESULTS ...... 3 E. COLLECTION, PRESERVATION, AND SHIPMENT OF SPECIMENS ...... *...... 5 1. Serum ...... 6 2. Faeces ...... * 7 3. Cerebrospinal fluid ...... 7 4. Sputum ...e.....ee ...... ee.....e...... e.....e 7 5. Other materials ...... 7 F. APPLICATIONS ...... 8 II. EXPERIMENTS WITH BACTERIA AND BACTERIAL TOXINS A. ENTEROBACTERIACEAE ...... 8 1. Salmonella ...... 8 2 Shigella ...... 19 3. Escherichia coli ...... 24 B. VIBRIOS ...... 27 1. Vibrio cholerae ...... 27 2. Vibrio parahaemolyticus ...... 31 C. STAPHYLOCOCCI ...... 34 D. MENINGOCOCCI ...... 37 E. "SMALL GRAM NEGATIVE BACTERIA" 41 P. TETANUS AND DIPHTHERIA TOXINS ...... 44 III. EXPERIMENTS WITH PROTOZOA AND METAZOA A. ENTAMOEBA HISTOLYTICA ...... 47 B. SCHISTOSOMA MANSONI ...... 50 C. TRYPANOSOMA CRUZI ...... 51 D. PLASMODIA ...... 55 E. MISCELLANEOUS PARASITES ...... 58 CONTENTS (continued) Page

IV. EXPERIMENTS WITH VIRUSES A. VACCINiA VIRUS ...... 58 B. ADENOVIRUSES ...... 61 C. POLIOVIRUSES ...... 63 D. INFILUENZA VIRUSES ...... 65 ...... E. IISCELLANEOUS VIRUSES ...... 66

V. FBXPEJRIMEITS WITH FUNGI ...... 68

AC OfOWEIDG] ITS CN' ...... C...... 71

REFERENCES ...... 71. GENlERA L PRECAUTIONS I1T .ORKT WITH

RADI O~TU CLI DE-LABTELLEID ANTIGE'S

The paragraphs that follow constitute no more than a short reminder of precautionary measures whici should be known to and followed by all persons engaged in work with radio- nuclide-labelled antigens.

1. In all such work, the relevant rules and regulations concerning the handling, storage, and disposal of both patho- genic micro-organisms and radioactive materials must be strictly obeyed.

2. Bacteriology, and microbiology technicians and others accustomed to work with pathogenic micro-organisms must be trained in the safe handling of radioactive materials arc' correspondingly, cherlistry ard physics technicians and others accustomed to work with radioactive materials must be trained in the safe handling of pathogenic micro-organisms.

3. Particularly dangerous are certain viruses and ray.cotic agerts which may be airborne, as elements of Coccidioidos imrritis, respiratory viruses, plague bacilli, etc. Constant monitoring of the air for such agents is a conditio sine aiia non in laboratories where they are handled. I. INTRODUCTIOlN : ivETIiUDOiOGICA.L ASLiCTS

The purpose of the project to which this report relates was to explore the feasibility of tests employing radionuclide-labelled antigens in serological surveys. In the choice and development of appropriate methodology, particular attention was paid to the likely availability of facilities and personnel in the tropics and arctics, where the function of instruments for radioactivity measurement may be disturbed by climatic influences. Because a particular aim of the project was to devise methods suited to the examination of large numbers of sera, as required in epidemiological surveys, the methodology was kept as simple as possible and rapidity was an important consideration.

A. LIMITATIONS Ok' LABELLED ANTIGEN TESTS The characteristics of sub-cultures of a given micro-organism may vary. The methods described in this report relate to specific procedures with specific reagents including antigens prepared from specific strains. The required conditions of incubation, centrifuga- tion, dialysis, extraction etc. may vary from strain to strain even in the same laboratory. Therefore the methods described may require modification, especially when reagents of different origin are used or when antigens are prepared from different strains. The reagents used in the methods described were purchased from the firms of Eastman, Fisher or Mallinckrodt unless it is indicated otherwise. Other brands may yield different results. All the micro-organisms studied are available from the American Type Culture Collection. It must be borne in mind that antigens retain their character- istics only for a limited time and that this time may be shortened by radionuclide labelling. It cannot be too strongly stressed that the potency of a labelled antigen preparation must be checked against known positive and negative sera on each occasion of use. The project by no means covered all available methods for the preparation and use of labelled antigens. For example, enzyme- mediated labelling methods were not utilized, nor were coated-tube or solid-phase immunoradiometric assay methods. The aim throughout, however, was to develop simple test methodology. In view of the administrative difficulties inherent in the shipment of radioactive - 1 - materials, the hse of "home-made" labelled antigen preparations was preferred. The radioactive materials required for labell.ed-antigen tests are admittedly still expensive. The more extensive use of these materials may, however, result in a decrease in their price, The equipment required for the radioactivity measurements involved, though complex, is already available in most medical :in-sti tutions.

B. CHOICE OF' RADIOI.iUCLIDE AliD f-,DXIOOACTIVITi ;MEASURihEINT ,ETHIOD

A labelled antigen preparation for use in labelled anti;en tests must satisfy two often mutually opposing requirements, hith antigenicity and high specific activity. In the early Lsta-es of the project, preference wvas ,iven to pure beta-ray emitters, partic- ularly 14C, as radionuclides for antigen labelling, whilst automatic low-background gas-flow Getier-?.ller counter . steims were consid- ered the most suitable for radioactivity mebesurerpertns by virtue of their sturdiness and reliability. Attempt.s to usse portable radio- activity monitors incorporating Ceiger-l:uller counters for radio- activity measurements did not meet with success, primarily because of the low sensitivities of such ir;strum-nts an.r the low counti:ng rates that they afford. The introduction of automatic liquid scintillation counter sy atems for such meo]suremcnts did, ihowever, extend the scope of the tests, enhancing their sensitivity and allowing labellin-g vith 3Ii, though the require.nert for liquid scintillator then increased costs.

The pure beta-ray enitters H and 14C retmained the favourite: for antigen labelling, because of their long half-lives and the ease with which they could be handled. Gamma-ray emitting radio- 51 125 nuclides, notably 51Cr and 15I, were used to some extent, however, in. situations in which the use of 3H or 1 4 C proved infeasible. The disadvantages of these radionuclides were found to lie in their shorter half-lives, necessitating frequent preparation of batches of labelled antigen, and the more stringent measures reouired for their handling. On the other hand, the radioactivity measurements are simplified by the use of manuval or automatic thallium-act:ivated sodium iodide crystal scintillation counter s:.stems.

- 2 - C. TEST PROCEDURES The two main test procedures that have been used in the project have been the following: (i) Micro-organisms or their exotoxins are labelled with H or 1C by incubation with a labelled metabolite during their growth phase. In some instances, labelled exotoxins are released to the culture supernate which may then yield a labelled antigen prepara- tion. In others, the micro-organisms may be disintegrated in a bacterial mill, by ultrasonication or in a microbial press, and a labelled antigen extract prepared. Such extracts must be absolutely free from particulate matter. An aliquot of the labelled antigen preparation may be added to the test serum and the mixture incubated and then filtered through a membrane. Alternatively, the antigen- antibody reaction may be allowed to take place directly on the membrane. The membrane is thoroughly washed and radioactivity retained on it, representing added labelled antigen bound by antibody in the test serum, is measured in a low-background gas-flow Geiger- Muller counter system or in a liquid scintillation counter system. The unbound labelled antigen should not be retained on the membrane to a significant extent when the latter is washed, otherwise erratic results may ensue.

(ii) Micro-organisms or their exotoxins are labelled externally with 5 1 Cr or 125I and an aliquot of the resulting labelled antigen preparation incubated with the test serum. After incubation, the precipitated antigen-antibody complex is separated by centrifugation. The precipitation may be enhanced by the addition of ammonium sulphate (Farr, 1958) or antiserum against the test serum. Either bound radio- activity in the precipitate or unbound radioactivity in the supernate may be measured in a thallium-activated sodium iodide crystal scintilla- tion counter system. This elegant method is applicable to practically all microbial determinants, but may require considerable manipulation of the antigen, as in parasitological and virological studies.

D. EXPRESSION AND EVALUATiOS OF RESULTS

Radioactivity measurements are usually measurements of counting rate (counts per minute, c/min), these being either "total" or "net" - the latter after subtraction of the "background" counting - 3 - rate of the measurement system. A simple method of expressing the results of any labelled antigen test is to calculate the percentage of the added radioactivity retained in the antigen-antibody complex, that is, to express the "net" counting rate representing the bound moiety as a percentage of the initial "net" counting rate. Experience has shown that this percentage may be up to 10 % in the absence of any true binding of antigen by antibody. However, when results giving higher percentages are considered, an ogival calibra- tion curve relating the percentage of added labelled antigen bound to the antibody titre may usually be drawm. This curve usually levels off at high antibody titres, with less than 100 % of added labelled antigen bound. In the evaluation of the results of serological tests, a clear dividing line between negative (-) and positive (+) is often difficult to establish. For instance, early work with diphtheria antitoxin led to the belief that if 60 % of the population are immune there will be no diphtheria outbreak. The application of this principle showed it to be fallacious. Nevertheless, it has crept into numerous textbooks and scientific papers. Further un- certainties arise since it rarely occurs in such tests that a titre signifyingthe lower limit of susceptibility can be established. Immunity depends on numerous factors which may be non-specific (e.g. genetic constitution, presence of particular digestive enzymes and other factors in the gastrointestinal tract), para-specific (e.g. cross-immunity against a related infection) or specific. The latter are usually characterized either by the estimation of antibody or by the cellular defence mechanisms. At present, precipitating and bactericidal antibodies, but few neutralizing antibodies, can be estimated by techniques making use of labelled antigens. A disadvantage of tests making use of labelled antigens lies in the fact already mentioned that their ogival calibration curves usually level off at a certain percentage of added labelled antigen bound. Because of this, the results of labelled antigen tests may be identical whether, for example.,the end-point of the corresponding haemagglutination test is 1:800, 1:6 400 or 1:25 600. This is only a limited disadvartage if the minimal protective level can be established and if it lies below the plateau of the curve, since one may then still establish whether the serum under test contains or - 4 - Per Cent Radioactivity Retained S typhi 70 ----

B. hermsii

60- o0

e50 V. parohaemolyticus

50 ------A ^

30- A yA

20 _ \ \ \ Zone

drum diktion 0 1/10 120 1/40 1/80 1/160 1/320 1/640 1/1280 12560

Fig. I. Variation of percentage radioactivity retained in antigen-antibody conplex in labelled antigen tests for . typhi, B. herisii and V. parahaemolyticus antibodies with serum dilution expressed as titre in haemagglutination test.

does not contain sufficient antibody to protect the individual. In numerous instances, there will still be a "gray zone", usually in the region of "lOto 20 % bound" in which the outcome of the test is doubtful (±). However, as Fig.l shows, provided that the "gray zone" and the region of the first positive (+) rcsults fall on the descending part of the curve, gradation of the results is still possible.

In many serological tests, antigenic surplus may cause negative results. This is rarely the case, in the experience of the authors, in routine labelled antigen tests,

E. COLLECTION, PRESERVATION, AND SHIPIMENT OF SPECIM1ENS

The rules and regulations governing the collection, preserva- tion, and shipment of specimens for labelled antigen tests are

- 5- identical with those for potentially infectious materials collected for other examinations, particularly microbiological studies. Thus, sera which may contain infective agents, as those from patients with infective hepatitis A, yellow fever, plague, and other easily transmissible and highly dangerous invaders of the bloodstream must be collected under precautions satisfying the requirements of proper protection of the collectors, including immediate steriliza- tion of all instruments and materials used and the use of appropriate collecting vessels to preclude leakage and outside contamination. Specimens must be shipped according to the rules of local authorities, which usually require the use of double containers, with absorbent material between collecting vessels and the two containers, Appropriate labels calling attention to the possible infectiosity of the shipment must be affixed.

If shipment is to be made by messenger and large numbers of specimens have been collected, commercially available double con- tainers with several spaces for tubes and vials may be used.

1. Serum

Methods for collection of serum include the following:

(a) Filter paper method Blood is collected on a filter paper, the paper enclosed in a sterile plastic envelope and the envelope then sealed by a hand machine and shipped. This method is not yet so effective as to be suited to large-scale operations. It may, however, be applied to small numbers of specimens provided that these are not to be exposed to excessive heat. The most feasible procedure is then to collect a drop of blood on a Gelman GA-6 or similar membrane, dry the specimen and then ship it. The specimen is subsequently eluted with phosphate-buffered saline (PBS), the eluate concentrated in a micro- diffusion apparatus, the globulins precipitated with ammonium sulphate and the precipitate dissolved in PBS, dialysed and concen- trated. The concentrate may then be used for labelled antigen tests after determination of the globulin content. (b) Capillary tube method

A finger or earlobe is pricked and blood is collected in a capillary tube by suction. The capillary is sealed. Serum is - 6 - permitted to separate and the tube is centrifuged, then broken near the interface of serumr and clot. The part of the tube containing the serum is closed with silicone or a piece of clay and shipped. (c) Syringe method The syringes, needles and collecting tubes used to draw the blood should be dry to prevent haemolysis. NIo anticoagulant should be used. The blood is allowed to stand for 4 hr at room temperature, then overnight at +2 to +4°C, after which the serum is separated. Lyophilization may affect large molecular weight anti- bodies; therefore, shipment in ice is recomirended. Cooling also diminishes the multiplication of eventual microbial contaminants. This is the preferred method for collection of serum. If haemolysis has occurred, the serum may subsequently be decolourized by the addition of a few drops of hydrogen peroxide solutton or the haemoglobin may be excluded by column filtration.

2. Faeces Proteolytic enzymes present in the intestines may destroy antibodies. Faeces should therefore be collected while fresh and antitrypsin, approximately 100 000 U per g of stool, should be thoroughly mixed with the specimen in a blender. The specimen should 'be immediately refrigerated and kept at 0 to +4°C until processed. Processing should be done within the possible shortest time.

3. Cerebrospinal fluid After aseptic collection, cerebrospinal fluid should be treated as serum.

4. Sputum Whole sputum is heavily contaminated and may contain extraneous particles. Therefore, the most feasible method is to ship washings frozen at -70°C in dry ice. The specimen should be carefully thawed and the antibodies concentrated.

5. Other materials The preferred method for the preservation of tissue biopsy specimens is to dust them with talcum and at once freeze them in liquid nitrogen. They should be shipped in liquid nitrogen or, if the time between collection and work-up is short, in dry ice.

Lymph, exudates, cyst fluids, ano similar materials shoulc( be collected aseptically and preserved in dry ice.

P. APPLICATIONS

The chapters that follow present the results of experiments with various labelled antigen tests carried out by the authors and of surveys based on such tests also carried out by them. MI¥any of the applications described are not yet suitable for large-scale use, Other attempted applications, for example in helminthic infections, proved infeasible because of particular features of the antigens involved and the antibodies that they elicit. Consideration of the results shows conclusively that the sensitivities of labelled antigen tests are 3 to 10 times higher than those of conventional serological tests. This is a considerable advantage. On the other hand, with increased sensitivity, non-specificity is an increasing problem.

II. EXPERIMENTS WITH BACTERIA ADI BACTERIAL TOXINS

A. E ITEROIBACTEERIACEAE 1. Salmonella

(a) Culture and label.l.ing procedures

,Enterobacteriaceaecausing disease in man, including salmone.llae, shigellae and enteropathogenic Escherichia coli, were grorwn in a medium containing a high quality peptone (Tryptose, Trypticase, \Z-Case, Whitte, or similar) 10 g, yeast extract 2 g, sodium chloride 5 g, distilled water to 1 litre, with the p). adjusted to 7.0 to 7.2. After bottling, the broth was sterilized by autoclaving for 30 min at 15 lb pressure.

Internal labelling with 14C was the preferred method for labelling Enterobacteriaceae antigens, though other labels were occasionally used. For labelling with 14C, the labelling agent could be 14C-acetate or a single 1 4 C-labelled amino acid. However, for the sake of simplicity and to assure uptake of the label in multi-purpose use, preference was given to 1 4 C-labelled yeast extract or 14C- labelled amino acid mixture. The labelling agent was added to the - 8 - extent of 2 mCi per litre of medium just before inoculation of the latter, by means of a syringe equipped with a small filter containing a membrane (Gelman, Millipore or similar) to withold bacterial con- taminants. The syringe was then filled 2 or 3 times in succession with sterile broth and the contents pushed each time through the filter into the medium to wash out retained agent. The medium containing the labelling agent was inoculated with the culture of the micro-organism, preferably 0.1 ml of a.16 -to 18 hr growth per litre of medium, and the whole incubated at +37°C for 20 to 22 hr.

The bacterial growth and the supernate (the latter containing the exotoxins, if any) were separated by centrifuging at 1 000 to 5 000 x g and +2 to +4°C for 1 to 2 hr. The time and the centrifugal force required were found to vary according to the mean size and shape of the bacteria and with the presence or absence of a bacterial capsule. Typhoid bacilli usually sedimented well at 1 500 x ~ in 1 hr. The supernate was decanted and, if it was to be used for the isolation of exotoxins, was either immediately processed, frozen at -70°C, or lyophilized. The bacterial sediment was taken up in an equal volume of PBS made up by dissolving anhydrous monopotassium phosphate 0.36 g, disodium phosphate dodecahydrate 1.43 g, and sodium chloride 5 g in 1 litre of distilled water, the pH being adjusted if necessary to 7.0 to 7.2. The bacteria were then disintegrated. Ultrasonic vibration in a Sonifer apparatus was first used, later a Bronwill bacterial mill at 8 000 cycles and under carbon dioxide gas cooling for 5 min. The disintegration was repeated until microscopic examination of the emulsion did not show particles 0.05 im in size. The emulsion was centrifuged at 25 000 x g and +2°C for 30 min. The sediment contained the glass beads used in the disintegration, and sometimes small bacterial particles.

The supernate was collected and concentrated at +2 to +4°C in cellophane (Viscose) bags on which dry polyvinylpyrrolidone or powdered refined sucrose had been heavily dusted. Lyphogel (Gelman) did not reduce the counting rate of the extract in radioactivity measurements, but reduced its volume to approximately one tenth. - 9 TABLE I

Effect of Purification of Labelled Antigen Preparation on Performance in Labelled Antigen (Membrane) Tests for S. typhi Antibodies

Counting rate of labelled antigen retained on membrane a

Reagent Undiluted Antiserum diluted Antiserum diluted antiserum to 50% of to 25% of (c/mn?) initial titre initial titre (c/min) (c/min)

Untreated extract 330 ± 12 358 ± 25 211 ± 11

+ Extractagainst dialyesdPBS 345 + 18 348 ± 19 233 13 Extract dialysed against 0.1 M 335 + 20 321 + 16 203 +'14 phosphate buffer at pH 7.0

Extract washed 260 + 14 246 10 207 l 10 with PBS ±

a Measurements in low-background gas-flow Geiger-Mller counter (Beckman Lowbeta) system. Geometric mean± standard deviation of results in 10 tests.

As is seen from Table I, dial.yojs of the extract to excJude any unbound 4C did not yield improved resulta in tests ithl sera of norwn agglutinin titres, whilst repeated washi.n ani centrifuging :,ave inferior results. The -test procediure used is described in II.A.2(b). (b) Test procedures

The mnembrane test procedure used was developed after co-.icidera- tion of the studies and reviews of Sch.reiber and P;eigelson (1967), Schreiber et al. (1968) an'd Borella (1968), wiio investigated the inClucnce of different factors such as pore size, incubati.oni time, incubetion temperature ard washing fluid on the retention of antigen- an.t.ibody complexes on cellulose membranes. Whatman 33F!W,Gelman GA-6

- 10 - and Millipore HA (average pore size 0.45 rlm) and Gelman GA-8 and Millipore GS (average pore size 0.22 nim),and later also Millipore Celotate EG membranes were used. The labelled antigen preparation was first diluted to give a counting rate of 1 000 to 3 000 c/min per ml. Next, 0.1 ml aliquots of each test serum were permitted to soak into membranes of 22 to 25 cm diameter. Then 0.2 ml aliquots of labelled antigen preparation were added. The membranes were incubated for 2 to 3 hr at +50 C in a wet chamber. After incubation, the membranes were placed in a Gelman or Millipore filter apparatus and washed with 3 to 5 ml PBS under mild suction. Several filter apparatuses could be connected to one pump. The Mlillipore 3025 Sampling Manifold permits washing 30 membranes of 24 to 25 mm diameter simultaneously.

An alternative procedure was mixing 0.2 ml aliquots of serum with 0.2 to 0.3 ml aliquots of labelled antigen preparation in small tubes, incubating for 2 to 3 hr at +50°C, pouring the contents on to membranes positioned in the filter apparatus, washing the tubes with 3 to 4 aliquots of PBS and pouring the washings on to the membranes. The unreacted antigen and antibody were washed out with 2 to 3 ml aliquots of the same buffer under mild suction.

The membranes were dried at +37°C on the tips of vertical spikes, or at ambient temperature after immersion into a mixture of equal volumes of absolute ethanol and diethyl ether. "Background" controls were set up with 0.1 ml aliquots of serum without labelled antigen. Other controls were set up with respectively 0.1, 0.2, 0.3 ml aliquots of labelled antigen preparation without serum. In these cases, the membranes were not washed, so that all the added radioactivity was retained.

If the low-background gas-flow Geiger-Muller counter (Beckman Lowbeta) system was used for radioactivity measurements, the membranes were fastened to the inside of the planchets with a small amount of starch paste, care being taken that their surfaces remained even.

If the liquid scintillation counter (Beckman LS-150C) system was used, the membranes were generally simply dried and immersed in liquid scintillator in counting vials. A liquid scintillator modified

- 11 - from that of Tye et al. (1965) was most often used. This consisted of naphthalene 100 g, PPO 16 g and POPOP 1 g, dissolved in dioxane 1 litre and toluene 50 ml. NCS, Spectrafluor (Amersham-Searle), Aqua- fluor, Liquifluor, Protosol, and Aquasol (New England Nuclear) and Scintisol (Isolab) were also ocassionally employed. The xylene-based Aquasol and the dioxane-based Aquafluor were particularly useful because of their tolerance of small amounts of water and their low chemi- luminescence. Membranes consisting of mixed cellulose esters, if properly dried, became transparent and dissolved directly in toluene-based liquid scintillators. 'oi sterlin; with a dioxane-based liquid scintillator was sufficient in the case of Celotate membranes. Other membranes could be solubilized or made transparen byyt treatieelt with 1 r; .Illyamine (Roeihm anid llaas) irn letlhanol. It has to be emphasiz.ed that the cournting efficienicy of the low- background gas-fl ow Geiger-Tul] er cour-toer (B3ecknr'm Lowbeta) system. for 1 4 C was 19 %, while that of the liquid. sc.itillation counter (Beckman LS-150C) system reactied 85 %. The results were expressed as "net" counting, rate (c/rwin) or as percent radioactivity retained or thle m::'rbrane.

Tables II and III illustrate the di ffereonces in results achiie e with different brands of membranes, solvents ard liqui.d scintillalo;"..., ag;ain in tests V;rith S3 typhi. In these tests, 'he serur was from a convalescent typhoid fever patJent. The serum agglut;in-in "0" titre was 1:640, the "H"1 titre 1:2 560. The labelled antigen preparation used waa from a!tig--en isolated from the ae pati ent. Uptake of tLh 4C during labelling was ai:proximately 3 %. (c) Results

The aut ors have publi shed the first practical applicati onc of these firndings in preliminary commurn cations (Pel serfeld e'-d Parrott, 1969, 1970), showing that if the labelled antigen preparatioln gave 3 050 ± 170 c/min per ml in the low-back'ro'und gas-flow ;eiger-l]iuller counter system and 0.2 to 0.3 ml of it were sed in tests with un- diluted sera, the radioactivity retained on T.ilip ore or Gelm.r membranes gave 310 ± 18 c/min for sera from patients wjith typhoid fever; 18 to 280 c/min for sera from convalescets from typhloid (3 to 14 months after infection), and 50 to 76 c/min for sera from non- infected controls. These results ran parallel to those of tube and - 12 - TABLE II Results of Labelled Antigen (Membrane) Tests for S. typhi Antibodies with Different Proportions of Labelled Antigen Preparation and Different Membranes

Labelled Counting rate of Meb eSerum Labnelled labelled antigen Membrane (Ml)(ml) prearionpreparation retained on (ml ) (c/min)a

0 0 8 0.1 0 9 0.2 0 6 0 0.05 70 Whatman 0 0.1 128 3MM 0 0.2 204 0 0.3 286 0.1 0.1 66 0.1 0.2 54 0.1 0.3 62 0 0 6 0.1 0 9 Millipore 0 0.2 110 HA 0 0.3 199 0.1 0.2 71 0.1 0.3 79 0 0 9 0.1 0 5 Millipore 0 0.2 133 GS 0 0.3 203 0.1 0.2 84 0.1 0.3 88 0 0 7 0.1 0 5 Gelman 0 0.2 130 GA-6 0 0.3 217 0.1 0.2 75 0.1 0.3 .78 0 0 7 0.1 0 5 Celotate 0 0.2 185 EG 0 0.3 249 0.1 0.2 91 0.1 0.3 98

(a) Measurement in low-background gas-flow Geiger-Muller counter (Beckman Lowbeta) system. Mean of results in three tests. Membranes on which labelled antigen preparation alone was deposited were not washed, so that all added radioactivity was retained.

- 13 - TABLE III Results of Labelled Antigen (Membrane) Tests for S. typhi Antibodies with Different Proportions of Labelled Antigen Preparation, Different Membranes and Different Liquid Scintillators

Labelled Counting rate of Labeled labelled antigen Membrane Liquid Serum scintillator (ml) preparationantigen retainedmembrane on (m) (c/min)a

0 0 12 0.1 0 11 0 0.1 369 Tye 0 0.2 622 0 0.3 825 0.1 0.2 410 0.1 0.3 406 0 0 10 0.1 0 17 0 0.1 305 Millipore uaso 0.2 589 GS quasol 0.3 781 0,1 0.1 210 0.1 0.2 337 0.1 0.3 379 0 0 15 0.1 0 9 0 0.1 370 Aquafluor 0 0.2 789 0 0.3 858 0.1 0.2 333 0.1 0.3 397 0 0 15 0.1 0 11 0 0.2 617 Tye 0 0.3 844 0.1 0.2 470 _0.1 0.3 497 0 0 9 0.1 0 18 Gelman Aq l 0 0.2 601 GA-6 Aq0 0.3uasol 867 0.1 0.2 408 0.1 0.3 437 0 0 10 0.1 0 5 0 0.2 602 Aquafluor °0 0.3 871 0.1 0.2 488 0.1 0.3 499

Contd. - 14 Table III - continued

Labelled Counting rate of labelled antigen Membrane iLiquid scintillator Serum(ml) preparationantigen retainedmembrane on (ml) (c/min)a

0 0 14 0.1 0 12 Tye 0 0.2 649 ye ° 00 0.3 861 0.1 0.2 440 0.1 0.3 481 0 0 12 0.1 0 13 Celotate 0 0.2 663 EG Aquasol 0 0.3 892 0.1 0.2 550 0.1 0.3 560 0 0 17 0.1 0 10 Aquafluor 0 0.2 680 0 0.3 802 0.1 0.2 449 0.1 0.3 489

(a) Measurements in liquid scintillation counter (Beckman LS-150C) system. Mean of results of three tests. Membranes on waich labelled antigen preparation alone was deposited were not washed, so that all added radioactivity was retained.

slide agglutination tests, but were positive (>180 c/min) in 5 out of 8 instances of typhoid fever before the routine serological tests showed elevated titres, and in 3 out of 4 instances in which S. typhi was excreted by symptomless carriers. Further comparison with the bentonite and capillary tube agglutination tests (Rossen et al., 1967; Severson and Thompson, 1966) yielded simil.]ar results.

Since 1969, the authors have extended their studies by collecting and examining 100 sera from patients with typhoid fever, 100 from convalescents, 200 from persons immunized against typhoid and 500 from individuals without a history of typhoid or immunization against it. The labelled antigen preparation used was from S. typhi strain No. 2, which is available from any national bacterial type

- 15- TABLE IV

Results of Labelled Antigen and Other Tests for S. typhi Antibodies

Serum or other test No. TA HA oatril GtestedGM LS ma~terial~ (c/min) (c/min)

Serum alone 900 7 + 2 11 5 0 0 Antigen alonea 270 + 18 682 ± 32 - 440 21 4 62 ± 9 0 0 Control subjectsb 31 22 + 6 64 ± 8 29 31 11 76 19 117 + 29 0 1 18 137 + 15 271 + 31 8 11 93 140 0 460 33 90 91 PatientsC 4 131 + 11 420 + 22 0 0 3 27 + 8 53 i 11 3 3

83 110 18 440 + 29 78 80 Convalescentsd 10 105 10 399 + 26 0 2 7 35 + 5 107 i 8 0 1 3 25 3 90 ±6 1 1 163 138 + 1 450 + 23 163 163 ImmunizedsubjectP 11 104 ± 10 398 ± 42 0 3 18 41 ± 7 ;181 + 12 18 18 8 20 3 70 8 0 0 _~~~~~~~ . _ ...... ,,,,.

LA : Labelled antigen (membrane) test with 1 4 C-labelled bacterial antigen. Retained radioactivity. Geometric mean and standard deviation of results in three tests. GM : Radioactivity measurements in low-background gas-flow Geiger- Muller counter (Beckman Lowbeta) system. LS : Radioactivity measurements in liquid scintillation counter (Beckman LS-150C) system. TA : Tube agglutination test. No. tested with reciprocal titre > 20. HA : Haemagglutination test. No. tested with reciprocal titre > 40. (a) Not washed; all radioactivity retained, (b) No history of typhoid or immunization against it. (c) From 2 to 4 weeks after infection. (d) From 2 to 6 months after infection. (e) From 2 to 4 weeks after immunization.

- 16 - culture collection. With 1 4 C-labelled yeast extract as the labelling agent, uptake during labelling was 2 to 3 %. The results of tests with this preparation are shown in Table IV. Millipore GS filters were used in pairs, a 0.1 ml aliquot of serum and a 0.2 ml aliquot of labelled antigen extract being deposited on each membrane. After washing, one of each pair was measured in the low-background gas-flow Geiger-NMller counter system and the other in the liquid scintillation counter system. The data shown are geometric means and standard deviations of the results of three such tests. Comparison was made with the results of the routine tube agglutination test using live S. typhi strain No. 2 at +37°C for 20 hr, and with those of the micro-plate haemagglutination test using formolized sheep red blood cells to which unlabelled S. typhi No. 2 extracts were adsorbed. The results showed that false positive and negative results occurred both with the labelled antigen and the routine serological tests, and that in the former the "gray zone" was between 25 and 35 c/min (8 to 12 %) with the low-background gas-flow Geiger-Muller counter system and between 50 and 80 c/min (7 to 12 %) with the liquid scintillation counter system. These results were better than was expected. However, the control sera could not be evaluated with confidence because an individual may not know whether or not he has had typhoid'or is a symptomless carrier.

The results of studies with more recently developed membranes and liquid scintillators are too few at present to be reported. Cross-reactions have been observed. Table V shows examples of salmonellosis in man in which the results of both labelled antigen and routine serological tests were positive with heterologous antigens.

Cross-reactions between Salmonella antigens and sera from patients with salmonellosis are well known and discussed in practically every textbook of bacteriology. They may be due to several factors, including common antigens not revealed in the rabbits used for the preparation of Salmonella typing sera.

Attempts to use purified "0" and "H" antigens in these tests have not yet been successful. They cannot therefore be longer regarded as specific for the determination of the causative Salmonella in human salmonellosis.

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- 18 - It was not necessary in these tests to use external labelling of the culture supernates because the results with the more con- venient internal labelling appeared to be satisfactory. Further development of the methodology is desirable to avoid false negative and positive reactions.

2. Shigella

The main interests here lay in labelled antigen tests with the four groups of shigellae and tests with the exotoxin produced by Sh. dysenteriae 1.

(a) Culture and labelling procedures For the preparation of labelled bacterial antigers, internal labelling with 4 C was used. At first, labelling was carried out with specific labelling agents in synthetic media desirned to assure selective utilization of the agent, but subsequently labelling with 4C-labelled yeast extract or a 4 C-labelled amino acid mixture in a chemically not fully characterized medium, as described for salmonellae, was found to give satisfactory results. F'or the preparation of labelled exotoxins, either internal or external labelling could be used.

For the preparation of internally la.belled Sh. dysenteriae, after culture with the labelling agent, the bacterial growth and the supernate were separated by centrifuging, usually at 1 500 x g and -2°C. All subsequent operations were carried out at +2 to +4°0C The supernate was purified by elution through a Sephndex G-50 column, the eluate being monitored for protein content by UV spectrophoto- metry at 280 nm and also for rad:ioactivity to identify the labelled exotoxin peak. The largest amount of exotoxin could be collected when 0.1 M phosphate buffer (pH 7.8) containing 0.14 : sodium chloride was used as eluant. The exotoxin fraction could be purified further by chloroform extraction, but gave satisfactory titres in the rabbit caecal test in the raw form.

The exotoxin could be kept at -20°C for 2 to 3 weeks. Treated with 1 % formol, it lost 90 % of its toxicity, but not its anti- genicity, in 2 weeks.

- 19 - Sh. dysenteriae exotoxin could also be externally labelled with 4C as 1 4 C-formaldehyde, by the method of Rice and Means (1971). This procedure produced minimal changes in the protein and was, therefore, also employed in the labelling of other toxins. The bacteria were grown in the usual medium, but without labelling agent. Sh. dysenteriae 1 (as Corynebacterium diphtheriae) formed exotoxin at an optimal rate when about 0.15 mg ferrous ion and 7 mg magnesium ion per litre were added. The precise amounts varied with the strains and had to be determined in advance. After 24 to 48 hr incubation, the bacteria were centrifuged at 2 000 x E for 1 to 2 hr and -2°C. The supernate was eluted through a Sephadex G-50 or G-75 column, the eluate monitored for protein content by UV spectro- photometry, and the fractions tested for toxicity by the Ouchterlony method against a known (and commercially available) antitoxin serum, by cross-electrophoresis, or by the rabbit caecal test. Positive fractions were pooled, evaporated with a fan in the cold room, concentrated in Viscose bags by diffusion against polyvinyl- pyrrolidone, Carbowax, or powdered refined sucrose, and, if necessary, further concentrated by adding a pearl of Lyphogel (Gelman). The concentrate was adjusted to contain 5 mg protein per ml. It was diluted with 5 vol 0.85 % saline and sufficient 0.1 M sodium hydroxide to adjust the pH to 8.8 to 9.0. The reaction vessel was put into an ice bath and 100 >il 0.04 M 1 4 C-formaldehyde (specific activity 10 to 20 mCi/miV) added per ml of contents. Thirty seconds later, four doses of 20 il sodium boronydride (5 mg/ml) were sequentially added, followed by a further 100 1 1 min later. Dialysis of the labelled exotoxin was not necessary, but was desirable when the method was used for labellirg viruses.

An alternative procedure for external labelling of the exotoxin was with 125I by the method of Gruber and Wright (1967). This was tested particularly because Habermann (1970, 1972) emphasized the loss of toxicity of tetanus toxin, which according to Van Heyninger (1960) is related in several. of its characteristics to Sh. dysenteriae exotoxin, on labelling with 125I.

125 For labelling with 125I, a slightly modified form of the method of Gruber and Wright (1967) offered several advantages. A 2.5 cm high and 1 cm wide glass tube was sealed in a vertical position to

- 20 - the bottom of a 50 ml Erlenmeyer flask to form a separate (central) compartment. Silicone glue could be used to seal the bottom end of the tube to the flask. Two-tenths of a ml of 0.002 M (0.332 %) potassium iodide was pipetted into the central compartment and 50 to 100 liCi 125I in the form of sodium iodide added. One ml of the exotoxin preparation, containing 5 to 10 ig protein per ml, was pipetted into the outer compartment. Then the Erlenmeyer flask was sealed with a skirt-type vaccine stopper. One tenth to 0.2 ml of a solution of freshly prepared (from 10-times concentrated stock) 0.03 N (0.09 %) sodium dichromate dihydrate in 1.8 M (3.6 %) sulphuric acid was added to the central compartment with a glass syringe fitted with a 3 inch (7.5 cm) long needle. The flask was kept at +23 to +25°C for 1 hr, during which it was gently rotated from time to time. The contents of the outer compartment, containing the labelled exotoxin, were then withdrawn by means of a syringe again fitted with a long needle. Free 125I was removed by elution with PBS through a Sephadex G-25 column, or by dialysis. In most instances, 75 to 80 % of the 125I was converted into free iodine, and of this approximately 12 % became bound to the protein, usually at a level of 1 ± 0.25 atoms iodine per molecule of exotoxin, giving counting rates of about 106 c/min per fg of antigen in the thallium- activated sodium iodide crystal scintillation counter (Nuclear Chicago Autogamma 1185) system. Labelling at a level of 5 atoms per molecule caused loss of antigenicity by 70 to 80 %. (b) Test procedures

Tests with internally labelled Shigella antigen preparations could be carried out by the membrane procedure, the radioactivity measurements being carried out in either the low-background gas-flow Geiger-Miller counter system or the liquid scintillation counter system. Tests with Sh. dysenteriae exotoxin externally labelled with 14C as 4C-formaldehyde could also be carried out by the membrane procedure. It was found in the latter case, however, that an incubation time of 4 hr was preferable to 2 hr, and that the low counting rates obtained necessitated the use of the liquid scintilla- tion counter system for radioactivity measurements.

The exotoxin externally labelled with 125I could be used in tube radioimmunoprecipitation (co-precipitation) procedures as

- 21 - 0C F O c o000 mi CM CM r N 0d- .r4 12 OD Hr- 0 cOC O OC-00 C6N CV CM } -4-

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COt 0) c (C'pC) Cd PCd 0.*r * * C co _ co J

- 22 - described by Farr (1958). One tenth ml aliquots of serum were diluted with PBS to 0.5 ml and mixed with equal volumes of the 1 2 5 I-labelled exotoxin preparation. The tubes were incubated for 30 to 60 min, according to the concentration of the antigen, and 1 ml 3.2 M (42.32 %) ammonium sulphate then added to each. After 18 to 20 hr at +2 to +4°C, the tubes were centrifuged at 2 000 x g. The radio- activity of a 0.1 ml aliquot of each supernate, representing added labelled exotoxin not bound by antibody in the test serum, was measured in the thallium-activated sodium iodide crystal scintillation counter system and the corresponding percentage of added radioactivity retained in the precipitate calculated.

This method was later used with smaller amounts of the reagents (0.1 ml serum, 0.1 ml labelled toxin preparation and 0.2 ml 3.2 M ammonium sulphate), with results differing by ± 5 to 7 %. A reduc- tion in the concentration of ammonium sulphate to 37 %, as used by Preter (1962) in studies on cholera toxin,yielded results within the 99 % fiducial limits. Most sera could also be used at 1:10 dilution. The substitution of goat or sheep antihuman serum for ammonium sulphate gave equally useful results. The initial incubation time had then to be extended to 2 hr and the incubation after the addition of the antihuman serum required 3 to 4 days at +2 to +4°C. Free labelled exotoxin seldom co-precipitated.

The measurement of bactericidal activity of Shigella antibodies in terms of the release of incorporated radionuclides from labelled bacteria has not yet been developed (but see II.A.3(c)). (c) Results Results obtained with the membrane procedure have been published by the authors (Felsenfeld and Parrott, 1969). Results obtained with the tube radioinmmunoprecipitation procedure with antihuman serum as a co-precipitant have also been published (Felsenfeld et al., 1971). The retention of radioactivity wasl10 % until 6 to 8 weeks after infection, whereas the results of haemagglutination tests (using formolized sheep blood cells) and bactericidal tests fell below the level considered positive within 4 to 5 weeks. Table VI shows addi- tional results demonstrating the usefulness but the lack of species- specificity of these tests.

- 23 - The cross-reactions, as in salmonellosis, are probably due to common antigenic factors which exceed the limits of the sub-groups of shigellae. It is not easy to establish a "gray zone" in labelled antigen tests with these antigens. Further studies of these reactions are indicated, particularly to explore whether the "tissue penetrating" and "not tissue penetrating" strains of the shigellae induce qualitatively and quantitatively different antibody responses. The common endotoxin may also influence the outcome of the tests.

3. Escherichia coli The primary concern here was the group of E. coli which are able to produce diarrhoea. (a) Culture, labelling and test procedures

Known enteropathogenic strains of serotypes 04, 055, 075, and 0111 were studied. The first was isolated from an epidemic in pigs, and others were cultured from man. The micro-organisms were first labelled with 5 1 Cr by a modification of the method of Braude et al. (1955). The strains were grown in Tryptose broth for 48 hr, the bacteria sedimented by centrifugation at 1 200 x E, the sediment suspended in Tryptose phosphate broth (Baltimore Biological Laboratories) and 1 mCi 51Cr as sodium chromate added per litre of suspension. After 12 to 14 hr incubation at +37°C the bacteria were again sedimented by centrifugation at 1,200 x E, successively washed with 70, 95, and 99 % ethyl alcohol, acetone, and diethyl ether, then dried in vacuo at +40°C. The bacterial bodies were suspended in 5 volumes of distilled water and disrupted by ultrasonic vibration in a vessel immersed in an ice bath. An equal volume of 0.25 M (4.09 %) trichloroacetic acid was added, and the mixture shaken for 3 hr at +2°C, then centrifuged at 15 000 x g and 0°C for 30 to 45 min. The supernatant was dialyzed against distilled water until it was free of unbound 5 1 Cr and the dialysate concentrated by means of polyvinylpyrrolidone. The labelled antigen was then precipitated by the addition of ethyl alcohol to give a final concentration of 68 % ethyl alcohol (w/w), and the precipitate dried in vacuo. Approximately 20 mg of labelled material containing principally endotoxin was recovered from the growth in 1 litre of medium. The counting rate of the material in

- 24 - the thallium-activated sodium iodide crystal scintillation counter system was about 6 000 c/min per mg according to the bacterial strain. When labelled antigen (radioimmunoprecipitation) tests were performed with such preparations of homologous and heterologous antigens on sera from groups of 10 children infected with E. coli 055, E. coli 0111, S. wien or Sh. sonnei, as well as a group of 20 healthy children, cross-reactions wJere observed in 53 % of the investigated sera, but no positive tests were observed with 19 of the 20 sera from healthy children. The cross-reactions may have been due to the common enterobacteriaceal endotoxic factor (Felsenfeld and Parrott, 1968). Internal labelling with 14C was therefore preferred. The method of labelling and the test procedures were then identical to those employed for salmonellae and shigellae. Only 1 4 C-labelled amino acid mixture was in fact used for labelling, since 1 4 C-labelled yeast extract was not available at that time.

(b) Results Table VII shows the results obtained with 1 4 C-labelled antigen preparations. A retention of more than 20 % of the radioactivity appeared to be a sign of a positive reaction in E. coli, S. wien, and Sh. sonnei infections. Recalling Table VI, Sh. sonnei showed cross-reactions with other shigellae. The haemagglutinin titres observed in these studies confirmed that individual variations have to be expected in serological tests. Therefore the inclusion of positive and negative controls in each series is a prerequisite, There were few cross-reactions among the E. coli strains tested, but further study of this species will certainly reveal such phenomena. (c) Measurement of bactericidal activity

Rowley (1956) and Spitznagel (1966) suggested the measurement of bactericidal activity in terms of the release of 32 from labelled E. coli, Blachman et al. (1973) the measurement of vibriocidal activity in terms of -Cr release and Fierer and Braude (1972) the use of the same radionuclide for other micro-organisms. Fierer et al. (3974) modified this method using a syringe equipped with a membrane to isolate the released 5 1 Cr. They recommended measurement of the radionuclide after 5, 10, 20 and 30 min. Their results demonstrated that the incubation time and microbial concentration had to be varied according to the species of the micro-organism. - 25 - O. .- t- "-c. t" r-_- 0-0 0 m: " k.oI I. ' 7- r'"\c i ' cdO' c\J C4 C. ~~~~~______vv v 0 *, O-0O(' . tlIC9 ~m'0o~ ICM ct~ncc" -,I . -O'-4-0 0 (Mc oI0 i 0o0 ( c F. 'r ol 0OH HfocOM CcOO C n CD uCD (M Cd~C. 4> 0> o a) ic r-l C,, *r-C" , Cd 0 O'~ CoO COO 6 '0 o . t- Co 4 FF-4 *ir4 -1' 0 *,1 C4- -p : Cd C) (r-D rN-l,-\- O.C'd or-C",. Co P 0 o nc +11 *H~ Cdtz~*~~~~~~~~cC a Cr~~~~r'C 0 0

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- 26 - The authors have used the 5 1 Cr release method to measure the bactericidal activity of sera, particularly for E. coli, by labelling the live micro-organisms with 5 1 Cr as described above, adding 0.9 to 1.2 x 106 labelled micro-organisms to 0.1 ml serum diluted with PBS to a total volume of 0.5 ml, incubating the suspension for 30 min at +37°C, then filtering through a membrane with an average pore size of 0.45 Wm. The membrane was used to determine the retained 51Cr, the filtrate to determine the released 51 Cr. The reaction is complement-dependent, therefore unheated serum and PBS containing 1 miSl calcium and 0.5 mBi magnesium ions had to be used. The authors have used the same method successfully to determine colicidal serum antibodies, but observed numerous (27 %) cross-reactions (Felsenfeld and Brannon, 1973). The method is elegant, but not always appropriate to serological epidemiology.

B. VIBRIOS

1. Vibrio cholerae Studies with V. cholerae were initiated because of the need for improved serological methods in cholera surveillance. Since 1960, cholera has been spreading in Asia and Africa, with various Governments taking different measures to control the disease. Vaccination has been the most common means used to attempt to check the spreading of cholera. Therefore tests not only for antibodies formed after natural infection but also for those produced after immunization were investigated. Cholera is a disease in which the causative micro-organism seldom passes into the blood stream. V. cholerae and its biotype El Tor usually remain in the intestines, adhering to the mucosal surface, and only their toxins penetrate into the gut wall. Therefore antibodies to the vibrios and their toxins in both the serum and the gut (coproantibodies) have to be determined. (a) Culture and labelling procedures For the internal labelling of V. cholerae with 1 4 C, preference is now given to incubation in 3 % peptone broth (pH 8.0) containing either 1 4 C-labelled yeast extract or 14C-labelled amino acid mixture,

- 27 - instead of the earlier recommended method of incubation in Eagle's minimal essential medium (MEM) without lysine (Grand Island Biological) to which a mixture of unlabelled lysine and DL-lysine- 1-14C (specific activity 5 mCi/mM) was added to give a total lysine concentration of 28 mg lysine and a 14C content of 1 mCi per litre of medium (Felsenfeld and Parrott, 1969). The bacterial growth was heaviest when 1 4 C-labelled yeast extract (specific activity 1 mCi/mg) was used. Labelled bacterial antigen was prepared by the procedure described in II.A.l(a). The labelled enterotoxin was separated from the disintegrated vibrios by centrifugation of the emulsion and fractionation of the supernate by elution through a CM-Sephadex column with 0.01 M phosphate buffer containing sodium chloride at concentrations increasing stepwise by 0.1 M from 0 to 0.5 M. The peak eluate fractions were concentrated and tested for enterotoxicity in the ligated rat intestine. Specific activities of 0.15 to 0.21 pCi per mg toxin were obtained (Felsenfeld et al., 1972). Enterotoxin already labelled with 1 C-DL-lysine was also received from Dr. Wm. Burrows, Department of Bacteriology, University of Chicago. The enterotoxin was also externally labelled with 125I by the method of Gruber and Wright (1967) as described in II.A.2(a).

(b) Test procedures Labelled antigen tests on sera were carried out with labelled bacterial antigens and labelled enterotoxins by the usual procedures. Sera were also examined by the 5 1 Cr vibriolytic assay of Blachman et al. (1973) in which 106 vibrios labelled with 5 1 Cr, 0.1 ml guinea pig complement diluted 1:20 and 0.1 ml 5 mg/ml lysozyme solution are added to 0.1 ml serum and the radioactivity released in 30 min measured. For coproantibody determination, the stools were at once blended with 10 parts (v/w) PBS (pH 7.6) at +2 to +4°C and 10 mg soyabean trypsin inhibitor (Sigma: 1 mg inhibitor/l mg trypsin) per ml of suspension and centrifuged at 12 000 x A. The supernate was dialyzed against distilled water and filtered through a Gelman GA-8 or Millipore GS membrane (average pore size 0.22 pm); the filtrate was then concentrated with Lyphogel (Gelman). Fifty j1 aliquots were mixed with 50 xl aliquots of the labelled antigen preparation, the mixtures incubated for 2 hr at +50°C, left overnight at +2 to +4°C

- 28 - and then co-precipitated with 50 1il antihuman goat or sheep serum. The tubes were left overnight at +4°C, then centrifuged at 10 000 x g and 0°C. The counting rates of 25 |1 aliquots of the supernates were determined and compared with those of controls containing either PBS instead of the stool extract or stool extract alone. The percent- age radioactivity retained in the precipitate was then calculated. If the stools could not be examined immediately, they were kept at -20°C for not longer than 3 days. (c) Results

The first results published (Pelsenfeld and Parrott, 1969; Felsenfeld et al., 1972; Felsenfeld and Dutta, 1972a) were assembled by examining sera from approximately 1 100 persons. The results of labelled antigen tests expressed as counting rates or percent radio- activity retained paralleled those of vibriocidal or enterotoxin- neutralization tests of a percent radioactivity retained of >10 %, a serum vibriocidal reciprocal titre of > 200 anc a serum anti-entero- toxin reciprocal titre of > 100 were considered the lower limits of positive. However, vibriocidal tests gave approximately 7 % more positive results in sera from non-vaccinated subjects from cholera- free areas.

Table VIII shows the results of examinations carried out on 100 additional sera of cholera patients by means of labelled antigen tests with both labelled bacterial antigens and labelled enterotoxins, 5 1 Cr vibriolytic assays and vibriocidal tests. Not only V. cholerae strain V41 which is of the Ogawa serotype but also strain 869 B which is of the Inaba serotype were used, the latter adapted to rabbits by Dutta. Labelled bacterial antigens of both strains were prepared by internal labelling with 1 4 C-labelled yeast extract. Strain V41 forms less enterotoxin than strain 869 B, and because the enterotoxins of both Ogawa and Inaba serotypes are identical, only the enterotoxin of the latter strain was used, labelled either internally with 14C or externally with 125I.

Neither labelled antigen tests nor conventional serological tests permitted distinction between infections with the two serotypes. There was a "gray zone" corresponding to radioactivity retained between 10 and 15 %. The results of the labelled antigen tests paralleled those of the other tests, but reached their highest levels

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- 30 sooner and remained elevated for a longer time. Therefore such tests appear to provide a sensitive method for the serodiagnosis of cholera.

2. Vibrio parahaemolyticus

V. parahaemolyticus is a species of vibrio isolated from patients with seafood poisoning. Because often numerous strains are isolated from the same outbreak and the disease is usually of short duration, the use of their common antigen, antigen A, is mandatory in serological tests and such tests have not yet been described that could aid in post-infection diagnosis. Some strains, regardless of their sero- logical classification, cause bloody diarrhoea, others do not. The paucity of autopsy examinations does not permit drawing far-reaching conclusions from this observation, but it is probable that the bloody diarrhoea is a consequence of tissue damage. This indicates that strains that cause tissue damage probably penetrate the surface of the intestinal mucosa. The distinction of "penetrating" and "non- penetrating strains was found important in the evaluation of shigellosis. The importance of V. parahaemolyticus is not great enough to indicate large-scale epidemiological surveillance opera- tions by serological methods, particularly because bacteriological examinations of seafood are carried out with greater ease than population surveys. Nevertheless, the authors have made studies to establish the value of labelled antigen tests in man after V. parahaemolyticus infections. (a) Culture and labelling procedures Antigen A, and its purified form, PPA, were prepared according to a modification of the method of Miwatani et al. (1969). V. para- haemolyticus was grown in heart infusion broth containing 3 % sodium chloride. If internal labelling was indicated, 1 mCi 1 4C-labelled yeast extract or DL-lysine-l- 4C was added. After 18 to 24 hr incubation at +37°C, the cells were centrifuged at 2 000 x E and +2°C for 15 min. The cell suspension was taken up in 4 volumes of 3 % sodium chloride and disintegrated in the bacterial mill. The emulsion was centrifuged at 18 000 x g for 1 hr. The supernate was re-centrifuged at 90 000 x & for 1 hr. Ammonium sulphate was added to the second supernate to 80 % saturation. The mixture was kept overnight at +2 to +4°C, the precipitate dissolved in 0.01 M

- 31 - 0

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- 32 - phosphate buffer (pH 7.0) and the solution dialysed against the same buffer for 20 to 22 hr at +2 to +4°C. The remnant, antigen A, could be further concentrated by means of polyvinylpyrrolidone, refined sucrose, or Lyphogel (Gelman). Its nitrogen content and radioactivity were determined by routine methods. Fraction A could be further purified by adding ammonium sulphate to 30 % saturation at pH 7.0. The mixture was allowed to stand for 3 hr at +2 to +4°C, then centrifuged at 16 000 x g for 30 min. The supernate was again precipitated by adding ammonium sulphate to 45 % saturation. The mixture was kept overnight at +2 to +4°C, then centrifuged at 16 000 x A, and the precipitate suspended in 0.01 M phosphate buffer (pH 7.0), then dialysed for 20 to 22 hr against the same buffer at +2 to +4°C. This gave the purified common antigen (PPA). Additional purification could be carried out by elution through a DEAE-Sephadex column with 0.01 M phosphate buffer (pH 7,0) containing sodium chloride at concentrations increasing stepwise by 0.05 M from 0 to 0.5 M. Most of the purified antigen was present in the peak eluted with 0.3 to 0.35 M (0.155 to 0.185 %) sodium chloride. External labelling of the antigen with 125I was also carried out by the method of Gruber and Wright (1967) as described in II.A.2(a).

(b) Test procedures

Labelled antigen (membrane) tests were performed as previously described, Haemagglutination tests were carried out by the micro- plate method with the antigen adsorbed on human 0 D+ cells. The haemagglutinin titres of the sera of two rabbits immunized with antigen A were 1:160; for two others immunized with PPA the titres were 1:320 and 1:640, respectively. Membranes treated with undiluted immune rabbit serum and labelled antigen retained 50 ± 6 % of the radioactivity according to measurements both with the liquid scintil- lation counter system and the low-background gas-flow Geiger-Muller counter system. Additional studies of these procedures are thus indicated.

(c) Results

Tests could only be performed on 15 human sera. Table IX shows the results. These were positive in 7 patients who had bloody

- 33 - diarrhoea and negative in 8 who did not, indicating that a stronger immunological response sets in when tissue damage takes place.

C. STAPHYLOCOCCI The serological classification of staphylococci is still. in its infancy. About thirty antigens, frequently overlapping, have been described. Haemagglutination tests on the sera of infected persons seldom give higher titres than 1:20 and the results may be positive also because of the patient's previous exposure to staphylococci. Phage typing is helpful in the study of staphylococci isolated from the population, permitting one to trace the spread of infection in small communities, as in a hospital. Staphylococci that cause food poisoning are of special interest. The disease is caused by their enterotoxins. Their culture from the intestinal tract is often of little value because the enterotoxins are developed in the food and the staphylococci may be present in the intestines without causirg disease. Therefore the study of staphylococcal food poisoning has to be directed at the detection of the micro-organisms and their enterotoxins in the suspected food and at the detection of the serological responses to their enterotoxins in the sera and the stools of the afflicted persons. (a) Culture and labelling procedures The authors have been concerned with staphylococca] enterotoxins A, B, and C. The type cultures from which these enterotoxins have been produced were strairs ATCC 13565 for enterotoxin A, ATCC 14458 for B, and 137 for C. The labelled enterotoxins were first prepared according to the method of Bowden (1968). This procedure consisted of growing the micro-organisms in a mediuir (pH 6.8) containing 4 % NZ-Amine A peptone (Sheffield Chemical) and 0.4 % yeast extract (Difco) to which, after autoclaving, sufficient 1 4 C-labelled yeast extract was added to give 1 mCi per litre of medium. The culture was incubated for 22 to 24 hr at +37°C with shaking. Air was drawn from the culture into a cylinder containing 0.5 MI (2.8 %) potassium hydroxide solution to trap the escaping 1C-carbon dioxide. The pH of the culture was adjusted to 7.0. It was then filtered through a membrane

- 34 - with an average pore size of 0.45 Am. The filtrate was dialysed against 6 litres of distilled water for 6 hr, and diluted with 2 vol distilled water, and the pH adjusted to 6.4. Then 1.2 g Amberlite CG-50 resin (sodium form) was equilibrated with 0.05 M phosphate buffer (pH 6.4) and the dialysed filtrate added. The mixture was stirred for 30 min, transferred to a 1 x 10 cm glass column, and washed with an equal volume of distilled water. The column was eluted with 0.15 M phosphate buffer (pH 6.8). The fractions were monitored for protein content by UV spectrophotometry, combined, dialysed against 0.02 M phosphate buffer (pH 7.0) and concentrated by freeze-drying. Further purification by dialysis and Amberlite- chromatography gave higher yields but was not necessary. The specific activity of the product was 0.08 to 0.15 iCi/mg. Later the method of Hallander (1963) was used, with the addition of 1 4 C-labelled yeast extract to the growth medium and subsequent cultivation, filtration, and dialysis as in the method of Bowden (1968). Amberlite adsorption was eliminated. The dialysate was concentrated by means of polyvinylpyrrolidone or Lyphogel (Gelman), then eluted through a Sephadex G-100 column with 0.05 M phosphate buffer (pH 7.2) containing 0.5 M sodium chloride. The enterotoxin appeared in the second peak. The fractions were tested against the respective anti-enterotoxin serum by gel irmunoprecipitation, the active portions of the eluate pooled and concentrated as previously, then preserved at -20°C, and used within 2 weeks. The specific activity of the product was 0.2 to 0.3 MCi/mg. Untermann (1972a, 1972b) compared the various methods of culturing staphylococci for enterotoxin production. lie found Enzamin- NAK (Serva) broth to be suitable for dialysis cultures. Our experience with NZ-Case hydrolysate was equally satisfactory, also for cellophane-over-agar cultures. In the latter method a modified procedure of Minor and Marth (1972) was used. Cellophane discs were soaked in several changes of distilled water and sterilized in an autoclave for 15 min. They were put on the surface of plates consisting of NZ-Case hydrolysate 1.5 %, HY Soy B (Sheffield) 0.5 %, sodium chloride 0.5 %, and agar 1.5 % (pH 7.2), autoclaved, and poured into Petri dishes or Kolle flasks. An 18 to 20 hr culture of the staphylococcal strain was heavily

- 35 - streaked on the cellophane with a bent glass rod. The plates were incubated in an upright position at +37°C for 24 to 26 hr. The micro-organisms and their toxins were harvested by washing the cellophane discs twice with PBS. The washings were centrifuged at 2 000 x F for 10 min and the supernate suspended in 0.01 M borate buffer (pH 8.2) containing 0.85 % sodium chloride and 0.07 % bovine serum albumin. The supernate contained 15 to 50 pg enterotoxin per celophane disc of 9 cm diameter, according to the strain and the size of the inoculum. This supernate was used for external labelling with 125I. 125I labelling was carried out by the method of Gruber and Wright (1967), as described in II.A.2(a). The results agreed with the observation of Minor and Marth (1972) that labelling was satis- factory when 0.2 ml 0.002 M potassium iodide, 50 to 100 4Ci 125I (as sodium iodide) and 0.2 ml 0.014 M sodium dichromate in 1.8 N sulphuric acid were used with 1 ml of an antigen solution containing 1 to 2 mg protein. (b) Test procedures Labelled antigen (membrane) tests with 1 4 C-labelled enterotoxins were carried out by mixing aliquots of the test serum and the labelled enterotoxin preparation on a membrane with an average pore size of 0.22 Wm, incubating the membrane for 3 to 4 hr at +52°C, washing with a few ml of PBS and measuring the retained radioactivity in the liquid scintillation counter system or low-background gas-flow Geiger-MAller counter system. Sera were used undiluted or at 1:10 dilution with PBS. In tests with 1 2 5 I-labelled enterotoxins, usually 0.3 ml aliquots of the test serum and the labelled antigen preparation were mixed and the mixture incubated for 30 min; 0.6 ml of 3.2 M (42.32 %) ammonium sulphate was then added. The mixture was allowed to stand overnight at +2 to +4°C and the tubes centrifuged at 2 000 x f for 30 min. The counting rates of 0.3 ml of the supernate were then measured in the thallium-activated sodium iodide crystal scintillation counter system and the percent radioactivity retained in the precipitate calculated. Stool specimens were prepared for tests for coproantibodies by mixing with one part (v/w) PBS containing 10 mg soyabean trypsin

- 36 - inhibitor, filtering through gauze and centrifuging the filtrate at 1 000 x g and 0°C. The supernate was filtered through a membrane with an average pore size of 0.47 jm and the globulins precipitated by adding either sodium sulphate to 16 % saturation, or ammonium sulphate to 47 % saturation. The precipitate was dialysed against distilled water for 3 to 5 days in a cellophane bag, then concentrated or lyophilized. The results of tests with labelled antigens were compared with those of the latex agglutination test of Salomon and Tew (1968), and the micro-haemagglutirati on-inhibition test in the modification of ?torse and Mah (1967).

(c) Results Felsenfeld et al. (1972) reported that the percent retained radioactivity in labelled afti.gen tests for serum antibodies ad coproantibodies on 7 subjects with staphylococcal. food poisoning averaged more than. 80 %, Results of a further study are presented in Table X. Only 15 sera from patients wit.h stalhylococcal food poisoning were available for the latter. Further samples were collected 2 to 4 weeks after the outbreaks or camre from "susnected" cases, or were collected from persons without history of recent diatrrhoea. Only the haemagglutination-inhibitio!,. test couldc be carried out on all sera. It gave positive resultls, but could make no distinction between the causative strains. Labelled antigen tests, particularly 2 to 4 weeks after infection, gave significantly (P<0.01) higher specific reactions. The resul..ts obtained during the acute phase fall within the "gray zone" for demonstrable serum anti- bodies. Therefore this procedure should be further investigated with special regard to diagnosis after the acute phase of the infection and when specimens of the suspected food are not available. Sera from patients with food poisoning caused by staphyloerlterotoxin C were not available. This also underlines the need for additional studies,

D. MENINGOCOCCI

Meningococcal meningitis is one of the most pressing problems of epidemiology, particularly in Africa,

- 37 - 000 000 0 0 0 0 0 0 ,00 0O 000 0 00 cN 0 0 +1 fv § 050 0

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- 38 (a) Culture and labelling procedures Neisseria meningitidis polysaccharide was successfully labelled with 14C by Brandt et al. (1972) using sodium acetate-l- C as an internal label and isolating the polysaccharide as described by Gotschlich et al. (1969). The authors employed a similar method, using the medium of Prantz (1942) containing four times the usual phosphate concentration and the commercially available Muller-Hinton broth. One-half mCi of sodium acetate-l-14C (specific activity 2 mCi/mM) was added, the medium was inoculated with a 24 hr-old growth of meningococcion Muller-Hinton agar, and incubated at +37°C for 16 to 18 hr on a rotatory shaker operating at 15 to 20 rotations per min. The culture was centrifuged at 2 000 x g and +2 to +4°C for 30 min. The supernate was mixed with 0.1 % cetyl methyl ammonium bromide, Cetavlon (Eastman Kodak). The precipitate was extracted with 0.09 M (10 %) calcium chloride. To the extract, 25 % (v/v) absolute ethanol was first added to precipitate nucleic acids, then the polysaccharide itself was precipitated at 75 % (v/v) absolute ethanol. The second precipitate was dissolved in 0.1 M tris-(hydroxymethyl) amino-ethane hydrochloride buffer (pH 7.4) containing 0.15 M sodium chloride. The polysaccharide antigen was further purified by elution through a Sepharose 4B column with the same "tris" buffer. The fractions were tested for antigenicity against normal known positive and negative human sera. They were also standardized according to their radio- activity and their phosphorus (group A polysaccharide) or sialic acid (group B and C polysaccharides) content (Brandt et al., 1973). (b) Test procedures Labelled antigen (tube radioimmunoprecipitation) tests were carried out with 50 to 150 4l of the labelled antigen preparation The optimal amount was established by mixing 0.1 ml serum with labelled antigen preparation diluted in 0.1 M borate buffer (pH 8.3) with 0.15 M sodium chloride to make 0.3 ml. After 16 to 18 hr at +2 to +4°C, an equal volume of saturated ammonium sulphate (767 g/l, dissolved by moderate warming and kept at +2 to +4°C) was added. After 30 min, the mixture was centrifuged at 1 000 x E and 0°C for 15 min and the precipitate washed with half-saturated ammonium sulphate, air-dried for 1 hr, dissolved in 4 to 6 volumes of NBS solubilizer (Amersham-Searle) and transferred into a counting vial,

- 39 - TABLE XI Results of Labelled Antigen and Haemagglutination Tests in Meningococcal Infections

LA Weeks . Serum from after se Group A Group B Group C HA infection sera LS GM LS GM LS GM 0.5 1 15 10 47 67 39 28 80 Patient No. , I 1 21 20 71 40 30 19 160 fgroupe 2 1 19 11 60 31 19 18 320 4 1 18 17 23 21 18 11 oa

Patient No. 2, 0.5 1 21 18 14 11 10 13 0 garouiep Bio. ,1 I 19 21 39 30 14 17 80 group B 2 1 22 21 41 40 16 15 160 infection 4 1 28 26 35 36 17 16 40 Pat N. 3,0.5 1 16 14 52 44 17 9 0 Patient No. 3, 1 1 17 17 78 68 16 10 80 groupB 2 1 9 10 62 59 10 10 80 4 1 11 13 37 31 9 8 80

Patt . 4,0.5 1 9 10 27 22 11 15 0 Patient CNo. , 1 1 18 11 26 28 78 61 80 grinfet 2 1 16 10 29 21 69 60 160 4 1 10 9 21 28 46 38 80 Patient No. 5, 0.5 1 27 28 25 24 27 29 40 group C 1 1 38 37 31 33 29 39 160 infection 3 1 31 29 33 28 27 20 80

b Carries, 11 l l 1 8b 58b 42b 1 6b ll 0 Carroup. 5 11 9 8 7 6 10 46 + 11 group B .534 10 11 12 9 8 7 0 Contr238 40 21 23 22 15 0 Control subjects,5 10 9 10 7 8 5 32 + 7 infectiofn* 3 21 27 31 27 21 24 28 ± 9 40 9 8 7 8 6 8 0

LA : Labelled antigen (tube radioimmunoprecipitation) test with antigen of group specified. LS : Measurements in liquid scintillation counter (Beckman LS-150C) system. Percent radioactivity retained. GM : Measurements in low-background gas-flow Geiger-Muller counter system. Percent radioactivity retained. HA : Haemagglutination test with mixed antigens of groups A, B and C. Reciprocal titre (a) c 20 (b) Mean. Standard deviation 4± 20 % (c) Mean * standard deviation

- 40 - the tubes being rinsed with 0.9 ml toluene. A toluene-based liquid scintillator (Liquifluor, New England Nuclear or Spectrafluor, Amersham- Searle) was then added for radioactivity measurement in the liquid scintillation counter system. Alternatively, 0.1 ml of the supernate after ammonium sulphate precipitation was pipetted on to a planchet and air-dried for radioactivity measurement in the low-background gas-flow Geiger-Muller counter system. (c) Results Table XI shows the results of tests on sera from patients with systemic meningococcal infection, 50 meningococcal carriers, and 50 persons without a history of meningococcal disease and negative nose and throat swabs. The tests were specific for 4 of the 5 sera of the patierts. Antibodies were detected in the sera of 11 of the 50 carriers. Two of the 50 control sera gave positive results. Therefore the method does not appear feasible for the detection of meningococcal carriers, but seems to be helpful in the diagnosis of meningococcal infections. The indirect haemagglutination test with meningococcal polysaccharide-coated human,group 0 D+ red blood cells gave similar results.

E. ",SMALL GRAM NEGATIVE BACTERIA" The genera Brucella, Haemophilus, Bordetella, Pasteurella, Yersinia, and Francisella are often grouped under the designation of "small gram negative bacteria" in medical bacteriological textbooks. zany of them require some additional factors and reduced oxygen tension for growth. The authors could not work with Prancisella tularensis and Yersinia pestis in their laboratory. (a) Culture and labelling procedures Eugonagar (Baltimore Biological), a semi-solid medium with 0.25 % agar, 0.07 % cystine, 0.4 % sodium chloride, 0.02 % sodium sulphate, 0.055 % dextrose and high (2 %) refined peptone content was the basic maintenance medium. The addition of the V and Z factors, in the combination called IsoVitolex (Baltimore Biological) or Supplement B (Difco) and 10 % sterile defibrinated blood permitted maintaining a variety of fastidious micro-organisms in this medium.

- 41 - The Bordet-Gergou medium supplemented with 1 % peptone and 15 % (v/v) defibrinated blood had to be used with 0.25 % agar for the growth of the Bordetella pertussis strain initially used in these experiments. Subsequently, after several transfers, the micro- organism was adapted to grow in Eugonagar. The internal labelling of the micro-organisms was carried out by adding 400 to 500 PCi of 1 4 C-labelled amino acid mixture (specific activity 1 mCi/mJN) or the equivalent of sodium acetate-l- 1 4 C (specific activity 3 mCi/mnf) or uniformly labelled 1 4 C-glucose (specific activity 2 mCi/nM) per litre of medium without agar. The micro-organisms were cultured in loosely capped bottles in an atrros- phere containing 5 % carbon dioxide. After 48 hr incubation at +37°C, the bacteria were harvested and the polysaccharide separated from the supernate by the modification of the method of Brandt et al, (1972) described in II.D(a). The micro-organisms in the sediment were disintegrated in the bacterial mill, the emulsion centrifuged at 5 000 x g and the supernate chromatographed through Sepharose 4B. The fractions were tested for antigenicity in radioimmunodiffusion tests with homologous rabbit antisera and also for radioactivity. As Table XII shows, both the bacterial protein and the poly- saccharide antigen fractions retained radioactivity, the retention varying with the micro-organisms and their antigenic composition. (b) Test procedures

Both membrane and radioimmunoprecipitation tests were performed. Membrane tests were carried out with 0.1 ml undiluted serum and O.1 ml labelled antigen preparation on membranes with an average pore size of 0.22 pm; counting rates of 1 000 to 3 000 c/min were achieved in the liquid scintillatior counter system. Radioimmuno- precipitation tests were carried out as described in II.D(b).

(c) Results Preliminary results obtained with 14C-labelled Brucella and Bordetella antigens have been published (Felsenfeld et al., 1972; Felsenfeld, 1973). Lack of sera from patients with pertu.sis and disease carried by Haemophilus influenzae delayed investigation of these infections. However, sera from 50 human brucellosis patients and 50 rodents with pseudotuberculosi,2 were available. The antigens

- 42 - TABLE XII Retention of Radioactivity by Antigenic Fractions of Brucella abortus, Haemophilus influenzae, Bordetella pertussis and Yersinia pseudotuberculosis in Internal Labelling with 14C with Various Agents

Radioactivity retained

Amino acid Sodium G icro-organism mixture li cro-organi sm acetate. Glucose IA Bact. Poly- Bact. Poly- Bact. Poly- protein sacch. protein sacch. protein sacch. Brucella abortus 1.2 1.9 0.6 0.8 0.5 0.7 160 strain 544

Haemophilus influenza 0.8 1.2 0.5 0.9 0.6 0.9 320 strain 4560 Bordetella pertussis 0.8 1.9 1.0 1.9 0.5 0.9 640 strain 10380 Yersinia tuberculosiss culoss 1.2 2.1 1.7 1.1 1.1 2.2 320 strain 48

Radioactivity retained : Percent radioactivity retained by fractions of bacterial protein and polysaccharide giving positive results in radioimmunodiffusion test after internal labelling with 14C- labelled amino acid mixture, sodium acetate-l-14C or uniformly labelled 1 4 C-glucose. IHA : Indirect haemagglutination test. Reciprocal titre of unlabelled micro-organisms against homologous antiserum.

were labelled with 1 4 C-labelled amino acid mixture. Six replicate membrane tests were carried out on each serum, three being evaluated in the liquid scintillation counter system and three in the low- background gas-flow Geiger-Muller counter system. An additional three replicate tests were carried out by tube radioimmunoprecipita- tion. The results are presented in Table XIII and show that, under the experimental conditions applying, the labelled antigen tests were superior to the indirect h'emagglutination test.

- 43 - TABLE XIII Results of Labelled Antigen and Indirect Haemagglutination Tests in Brucella and Yersinia Infections

Months T LA Serum from after Of. ,s IHA infection ea A B 0

an patiet21 41 ± 7 58 ± 4 73 + 9 68+ 18 Human patients 3 -6 6 31+ 6 55+ 5 71 ±7 O(a) with brucellosis 20 + 8 27 4 11 + 4 20 ± 4 Humans with 192 11 2 + 14 3 13 + 2 0 6 21 ± 6 33 ± 5 18 ± 2 0 no brucellosis 2 10 + 2 12 ± 2 14 ± 3 26 ± 7

Rodents with 41 51 t 7 63 + 5 66 ± 4 178 ± 21 Yersinia pseudo- 41 510 66 8 71 ± 5 1 tuberculosis Rodents with 2 17 + 4 18 + 5 15 + 4 46 + 18 no disease 20 10 ± 3 14 + 2 14 + 3 0

LA : Labelled antigen tests with 4C-labelled homologous antigen. (A) Membrane test. Measurements in low-background gas-flow Geiger-Muller counter (Beckman Lowbeta) system. Percent radioactivity retained. rEean ± standard deviation. (B) Membrane test. Measurements in liquid scintillation counter (Beckman LS-150C) system. Percent radioactivity retained. Mean i standard deviation. (C) Tube radioimmunoprecipitation test. Measurements in liquid scintillation counter (Beckman LS-150C) system. Percent radioactivity retained. Mean ± standard deviation. IHA : Indirect haemagglutination test with homologous antigen. Reciprocal titre. (a) Mean c 20

F. TETANUS AKD DIPHT-HERIA TOXINS

The labelling of Sh. dyrsenteriae exotoxin withl14 or 125I and its use thus labelled was described in II.A.2(a) and that of staphylococcal enterotoxins in II.C(a). Among the other toxins produced by bacteria, those derived from tetanus and diphtheria bacilli have been studied.

- 44 - (a) Culture and labelling procedures The labelled tetanus and diphtheria antigens used were formol- toxoids (anatoxins) with a long period of antigenic reactivity prepared by treating the respective toxins with 14C-formaldehyde by the method of Rice and 1Means (1971). The amount of 1 4 C-formaldehyde necessary to label each batch of toxin varied. It was feasible to attempt to produce a toxoid giving a counting rate of 5 000 c/min per ml. Unlabelled carrier formaldehyde was added to the 14C- formaldehyde (specific activity 10 mCi/mM) when necessary to ensure complete conversion of toxin to toxoid. The production of tetanus toxin is not recommended except in laboratories that are specially equipped for this task. However, diphtheria toxin can be produced in any well-equipped bacteriological laboratory and titrated against antitoxin standards that can be obtained from vaccine manufacturers and national health institutes.

(b) Test procedures Labelled antigen (membrane tests were carried out using 0.1 ml serum and 0.1 ml 1 4 C-labelled toxoid preparation on Gelman GS-8 membranes (average.pore size 0.22 mn). Incubation was for 2 hr in a moist chamber at +37°C. The membranes were then carefully washed with PBS under mild suction, dried with warm air, immersed in liquid scintillator in counting vials and the retained radioactivity measured in the liquid scintillation counter system.

(c) Results A preliminary report (Felsenfeld and Dutta, 1972a) showed that the percent radioactivity retained in labelled antigen tests for both diphtheria and tetanus antitoxins paralleled the serum antitoxin level determined in haemogglutination tests. Fig.2 shows the results of tests on sera collected in Louisiana, USA, India, Colombia and Jamaica. For comparison, the IIA test with formolized sheep cells and refined diphtheria toxoid or tetanus toxoid, 700 Lf/ml, obtained through the courtesy of Drs. A. Bernstein and R.L. Makison, Wyeth Laboratories, was used. Only 3+ and 4+ reactions were considered positive. The serum antitoxin levels in antitoxin units (AU) were

- 45 - 80! t ! I I

-60

g> TeansTetanus.

, /, I , 20

0 <0.01 0.01 0.025 0,05 0 1 0.5 >0.5 Antltoxin Level Per ml Serum

Fig. 2. Variation of percentage radioactivity retained an labelied antigen tests for diphteria and tetanus antitoxins with setum antitoxin level detremined in haemagglutlnation teast.

determined by comparison with standards of the respective antitoxin. One-hundredth AU/ml was considered the lower limit that can be expected to protect the individual against either tetanus or diphtheria. The critical level in the results appears to be at 20 % retention of radioactivity. Therefore, this method permits dividing the population into "protected" (>20 % retention) and "unprotected" (>20 % retention) groups. However, because the curves flatten out at higher AU concentrations, it does not warn against overdoses of toxoid, which may set in at serum levels >1 AU/ml.

The authors' experiments with tetanus were hampered by lack of pure tetanus toxin. However, they were able to confirm the results of Habermann and Wiegand (1973), who used tetanus toxin labelled with 125I as described by Habermann (1970). In these studies, the serum or other sample to be tested was permitted to react with an excess of labelled toxin and the residual unbound labelled toxin isolated by reaction with antitoxin covalently bound to cellulose and its radio- activity determined, this being inversely related to the antitoxin level in the sample. This method also proved highly effective; it did, however, require strict adherence to technique. Human antibodies against tetanus toxin (Tetagam, Behringwerke) were necessary for standardization.

- 46 - The authors have also carried out studies with Clostridium botulinum toxins, but only on a limited scale. External labelling of the toxins with 125I could be effected using the method of Gruber and Wright (1967) in the cold to ensure stability of the toxin, as described by Habermann (1970). Attempts at internal labelling with 1C have not yet been successful. The six toxigenic types of this species give cross-reactions because of their common alpha-toxin.

III. EXPERIMENTS WITH PROTOZOA AND METAZOA

A. ENTAMOEBA HISTOLYTICA (a) Culture and labelling procedures Entamoeba histolytica strain 200 was cultured monoxenically in the presence of trypanosomids as described by Diamond (1968). The amoebae were maintained in TTY medium consisting of Tryptose 1 g, Trypticase 1 g, yeast extract 1 g, glucose 0.5 g, L-cystine hydro- chloride 0.1 g, ascorbic acid 0.02 g, sodium chloride 0.5 g, monobasic potassium phosphate 0.08 g, dibasic potassium phosphate 0.08 g, glass- distilled water to 95 ml. The pH was adjusted to 7.2. The complete medium contained TTY 95 ml, horse serum 5 ml, and defibrinated rabbit blood 0.25 ml. The medium was inoculated with the amoebae and with T. cruzi maintained in TTY-6 medium consisting of Tryptose 0.5 g, yeast extract 0.5 g, sodium chloride 0.5 g, monobasic potassium phosphate 0.08 g, dibasic potassium phosphate 0.08 g, glass-distilled water to 80 ml. On the day before harvesting, 0.1 mCi uniformly labelled 1 4 C-glucose (specific activity 2 to 3 mCi/mM) was added to each 100 ml of culture medium. The separation of the labelled E. histolytica antigen was carried out by the method of Ali Khan and Meerovitch (1970), slightly modified. The cultures were pooled and the amoebae separated from the trypanosomids by differential centri- fugation in the cold. When microscopic examination disclosed only a negligible (< 1 %) proportion of trypanosomids in the sediment, four volumes of tris-(hydroxymethyl)-aminomethane-hydrochloride buffer (pH1 8.0) containing 0.2 M sodium chloride were added. This buffer was prepared by dissolving 12.11 g "tris" and 11.68 g sodium chloride in 1 litre of distilled water, then adding sufficient 1 N hydrochloric acid (approximately 60 ml per litre) to bring the pH to 8.0.

- 47 - Filtration through a sintered glass filter is recommended to assure a long life of the column to which the buffer will be applied. The amoebae, suspended in this buffer, were disrupted by ultrasonic vibration or in the bacterial mill and the emulsion then centrifuged at 20 000 x E for 30 min. The supernate was concentrated by means of refined sucrose or polyvinylpyrrolidone. It was then preserved at -20°C. Fractionation was carried out by elution through Sephadex G-200 columns with 0.15 M phosphate buffer (pH 7.2). The first two peaks gave the highest titres in the haemagglutination and quantitative immunodiffusion tests, and were used for labelled antigen tests in pools containing 30 to 50 ig/ml, adjusted to give counting rates of at least 500 c/min per 0.1 ml in the liquid scintillation counter system. Labelling with 0.1 pCi 1 4 C-labelled yeast (specific activity 1 mCi/mg) per 100 ml of culture also gave satisfactory results.

(b) Test procedures Labelled antigen (tube radioimmunoprecipitation) tests were carried out by adding 0.5 ml serum to 0.1 ml labelled antigen preparation. The tubes were kept at +37°C for 3 hr, 0.1 ml antihuman goat or sheep serum was added and the tubes were kept at +2 to +4°C for 24 hr. They were then centrifuged at 300 to 400 x A. For radioactivity measurements in the liquid scintillation counter system, 0.1 ml of each supernate was transferred to a counting vial and a suitable liquid scintillator (Aquasol, Aquafluor: New England Nuclear) added. For measurements in the low-background gas-flow Geiger-I.filler counter system, 0.1 ml of each supernate was soaked into a filter paper disc, which was then dried and pasted to the bottom of a planchet. (c) Results Table XIV compares the results of labelled antigen tests on sera from 15 patients with amoebiasis, as well as 100 control subjects, with those of haemagglutination tests performed according to Krupp (1969). These results are preliminary, but appear to indicate that labelled antigen tests may be useful in serological surveys for E. hystolytica.

- 48 - TABLE XIV Results of Labelled Antigen and Other Tests in Amoebiasis

Serum from No. of recordedrecordd LA patient with sera for HA

ii5 weeks 28 4 100 ± 43 Acutoeamoebiasis 2

Chronic 5 6 to 12 4 + 4 610 + 76 amoebiasis months Chronic 51 c 80 amoebiasisaChronic s 1 7 months Chronic Chronic 1 8 months l0 640 amoebiasis

Small-type 8 to 14 E. histolytica 3 lmonhs 80 infections

No history or symptoms of 100 18 ± 2 42 ± 3 amoebiasis

LA : Labelled antigen (tube radioimmunoprecipita- tion) test. Percent radioactivity retained. Mean + standard deviation. HA : Haemagglutination test. Reciprocal titre. Mean _ standard deviation.

The authors had no opportunity to test strain DKB of E. hystolytica, which they maintained in TTY medium with Crithidia before chromatographic separation of the antigen according to Ali Khan and Meerovitch (1970), against other than rabbit antisera. This strain, cultured in the presence of PPLO micro-organisrrs, has been used successfully in the haemagglutination test, e.g. by Healy and Kraft (1970), and required further investigation before the most feasible antigen can be chosen. No additional amoeba strains have been investigated to date.

- 49 - B. SCHISTOSOMA MANSONI

The number of serological tests proposed for the diagnosis of schistosomiasis is legion. Recent reviews (Fife, 1971; McCarten et al., 1975) favour the fluorescent antibody, complement fixation, and slide flocculation tests. However, cross-reactions have been observed between Schistosoma antigens and sera of patients with Ascaris, Enterobius, Trichuris, Trichinella, Strongyloides and hookworm infections. It seems that soluble cercarial antigens have a greater specificity, even though the differential diagnosis of the Schistosoma species may not be possible by serological methods. (a) Culture and labelling procedures In the studies reported here, labelled cercarial antigens were used. Cercariae of Sch. mansoni were kindly supplied by Dr. Sharon File, Department of Parasitology, Tulane University Delta Regional Primate Research Center.

The cercariae were treated according to Chaffee et al, (1954). They were first desiccated, then disintegrated and extracted in anhydrous diethyl ether in a grinder, with chilling of all apparatus and reagents. The disintegration and extraction were carried out using 10 ml cold ether per 100 mg desiccated cercariae, the grinder being immersed in a calcium chloride - ice water bath at -17 to -18°C. The supernate was decanted and kept refrigerated at +3 to +4°C. A second extraction with 5 ml cold ether per 100 mg desiccated cercariae followed. The two extracts were pooled and centrifuged at 900 x E and 0°C for 30 min. The ether was decanted off and the residues in the centrifuge tube and the grinder were dried by suction. They were then extracted with veronal-buffered saline (pH 7.3 to 7.4) containing sodium chloride 8.38 g, sodium bicarbonate 0.52 g, sodium diethyl barbiturate 0.3 g and diethyl barbituric acid 0.46 g dissolved in 1 litre of hot distilled water. For this opera- tion the combined residues were transferred into the grinder with 15 ml of the buffer, ground at room temperature, and kept at +3 to +4°C for 4 hr. The suspension was centrifuged at 900 x E at +2 to +3°C for 30 min and the supernate distributed into ampoules, shell- frozen, and preserved at -20°C. The antigenic reactivity did not change within 3 months.

- 50 - The lipid-free antigen was purified by a modification of the method described by Williams et al. (1971), involving elution through a Sephadex G-200 column with PBS (pH 7.2). The first fraction, antigenically highly reactive and containing approximately 30 % of the protein, was concentrated by dialysis and labelled with 125I by the method of Gruber and Wright (1967) or with 14C by that of Rice and Means (1971). The labelled preparation was standardized to give counting rates of 1 000 to 5 000 c/min per ml.

(b) Test procedures

Labelled antigen (tube radioimmunoprecipitation) tests were carried out by mixing 0.1 ml serum, 0.4 ml buffer and 30 to 50 gl labelled antigen preparation in 0.1 ml buffer. After 4 - 6 hr incubation at ambient temperature, 0.6 ml saturated ammonium sulphate solution was added. After a further 24 hr at +2 to +4°C, the tubes were centrifuged at 8 000 x a for 5 min. The radioactivity of 0.1 ml of the supernate was determined and the percent radioactivity retained in the precipitate calculated.

Shorter incubation at +37°C or +50°C gave fewer positive results. Membrane tests required the same incubation time. (c) Results Labelled antigen tests were carried out with labelled Sch. mansoni cercarial antigen preparations on sera from patients with Sch. mansoni, Sch. japonibum and Sch. haematobium infections obtained from Colombia, Egypt, Japan and the Sudan. Sera of healthy individuals collected in Lafourche Parish, Louisiana, USA served as controls.

The results, shown in Table XV, demonstrate the efficacy of the method, but indicate that cross-reactions with other helminthic infections occur, as in other serological tests not involving labelled antigens.

C. TRYPANOSOMA CRUZI

(a) Culture and labelling procedures

T. cruzi strain Y, received through the courtesy of Dr. R. Yaeger, Department of Parasitology, Tulane University Medical School was grown in Yaeger's liquid medium at +28°C. When approximately 109 - 51 - 0 O bO + .p 4-p a)4U) cO 1 0'r C) CO 0) 0<^ 0 cot +z 0-

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- 52 - micro-organisms per ml medium were present, the growth was centri- fuged at 1 000 x g, washed twice with PBS and resuspended in an equal volume of PBS, then disintegrated in the bacterial mill. The supernate was subjected to preparative polyacrylamide gel electro- phoresis in a Shandon instrument, using 7.5 % gel. The eluate was monitored for protein content by UV spectrophotometry. The fractions were concentrated by dialysis and tested according to the Ouchterlony method for antigenicity against specific anti-T. cruzi rabbit serum. The first two peaks showed the highest reactivity. These were pooled and used for labelling. Labelling with 15I could be carried out by the method of Gruber and Wright (1967) or rcConahey and Dixon (1966) without loss of antigenicity. Labelling with 14C by the procedure of Rice and Means (1971) was successful, but required exhaustive dialysis before and after labelling.

(b) Test procedures Labelled antigen (tube radioimmlunoprecipitation) tests with 125I- or 14C-labelled antigens were carried out with 0.25 ml serum and 0.25 ml labelled antigen preparation adjusted to give counting rates of 5 000 to 10 000 c/min. After 2 hr incubation at + 37 C, an equal volume of saturated ammonium sulphate was added. The mixture was left overnight at +2 to +4°C, then centrifuged at 900 to 1 000 x g. For 125I, the radioactivity of 0.1 ml of the supernate was measured in the thallium-activated sodium iodide crystal scinti'- lation counter system. For 14 C, the same quantity of supernate could be soaked into a filter paper disc that was then dried and either pasted to a planchet for measurement in the low-background gas-flow Geiger-ruller counter system or immersed in liquid scintillator in a counting vial for measurement in the liquid scintillation counter system. Membrane tests gave consistent results only when no large particles of antigen were present. For this purpose, centrifugation of the labelled antigen preparation at 25 000 to 50 000 x g was necessary. The optimal proportions of antigen and antibody had to be scrupulously ascertained, because antigen surplus more frequently led to dissolution of the antigen-antibody complex than in experiments with bacteria. The antigen-antibody bonds were also more easily disrupted by careless washing of the precipitate in the filter apparatus. - 53 - ., 00

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- 54 - (c) Results Table XVI presents a comparison of the results of labelled antigen tests for T. cruzi and L. brazeliensis antibodies with those of fluorescent antibody tests carried out according to the method of Sadun et al. (1963). It appears that the labelled antigen tests for T. cruzi are more sensitive, but that cross- reactions with sera of patients infected with Treponema pallidum and Leishmania,already reported, are more frequent with these tests. Ten out of 30 sera from patients with syphilis gave positive results in labelled antigen tests with labelled T. cruzi antigen, 6 in fluorescent antibody tests with T. cruzi and L. braziliensis. All of 10 sera from patients with L. braziliensis and 30 sera from patients with T. cruzi gave positive results in labelled antigen tests with labelled antigen prepared from the other micro-organisms, whereas only 8 of the L. braziliensis sera and 26 of the T. clrzi sera gave titres l:4 in fluorescent antibody tests with the other. Crossereactions could be eliminated by prior absorption of the serum to be tested with the cross-reacting micro-organism. Thus, incubation for 1 hr with 3 to 5 mg L. braziliensis per ml serum diluted 1:10 in PBS eliminated cross-reactions in the fluorescent antibody tests with T. cruzi.

D. PLASI1MODIA Human plasmodia, comprising P. vivax, P. falciparum, P. malariae and P. ovale, cross-react with several other species of the genus Plasmodium, particularly with P. knowlesi, a simian malarial parasite. The microhaemagglutination test of Farshy and Kagan (1972, 1973) gave a higher proportion of positive results when P. vivax and P. falciparum antigens were used. A combination of P. falciparum and P. knowlesi antigens also increased the sensitivity of the test. However, since P. vivax, P. falciparum, P. malariae and P. ovale were not available in the authors' laboratory, the experiments were carried out with P, knowlesi. (a) Culture and labelling procedures The antigen used was an aqueous extract of the parasites prepared either according to the method of Rogers et al. (1968) or by that

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- 56 of D'Antonio et al. (1966). In the latter method, P. knowlesi- infected monkey erythrocytes were disrupted in a French pressure cell under critically controlled pressure and the intact parasites were washed with PBS. Separation of the erythrocyte fragments by differential centrifugation may have been necessary. After final centrifugation of the parasites at 2 000 to 3 000 x g, they were ruptured by freeze-thawing, then suspended in the supernate. The centrifugation was repeated at 10 000 x E and the supernate used as the antigen. Fractionation by means of Sephadex G-200, DEAE-cellulose, poly- acrylamide gell or Biogel has not yet yielded highly reactive antigens in sufficient amounts. Therefore only results with total PBS extracts are reported. External labelling had to be employed and gave the best results when carried out with 125I by the method of Gruber and Wright (1967).

(b) Test procedures Labelled antigen (tube radioimmunoprecipitation) tests were performed by mixing 0.3 ml serum and 0.1 ml labelled antigen prepara- tion, the latter standardized to give a maximum reaction with known positive human sera but a negative reaction with known negative sera, The counting rate was usually 2 000 to 3 000 c/min per ml of labelled antigen preparation. The tubes were incubated at +37°C for 2 hr, 0.1 ml antihuman goat or sheep serum was added and the tubes were then kept overnight at +2 to +4°C. The mixture was then centrifuged at 1 000 x & for 5 to 10 min and the radioactivity of 0.1 ml of the supernate measured. The results were expressed as percent radio- activity retained in the precipitate. Retention of <20 % was considered positive. (c) Results Table XVII shows the results to be similar to those of the indirect haemagglutination test, in which a titre of 8 was considered positive. The method requires further study.

- 57 - E. MISCELLANEOUS PARASITES The radioactive labelling of pathogenic protozoa and metazoa requires the selection of a parasitic developmental stage at which antigenically reactive components eliciting demonstrable antibodies are present. Unfortunately such antigens are often non-specific. Exo- and endotoxins may be demonstrated in several parasites, but their production in vitro is usually laborious and not feasible on a large scale. In other instances, the amounts of sera available from infected persons have not yet been sufficient for the elabora- tion of feasible and simple methods of labelling. Leishmania and Anaplasma can be labelled by the same procedures as Trypanosoma and Plasmodium, respectively, particularly when they are cultivable in tissue cell cultures, but the results, whether with internal or external labelling, have not yet been uniform. Clonorchis sinensis, Opisthorchis, Filaria, Strongyloides,and hookworm larval forms, echinococcal fluid, and Toxoplasma yield a number of antigens which can be separated by various methods (reviewed by Fife, 1971), of which gel filtration and colunn chroma- tography are giving the best yields in the authors' hands. In none of these cases, however, has a procedure applicable in large-scale epidemiological surveys yet been elaborated. The results of efforts to produce such procedures, if successful, will be published in extenso.

IV. EXPERIMENTS WITH VIRUSES A. VACCINIA VIRUS In a preliminary publication, Felsenfeld et al. (1971) reported that 1 4 C-labelled antigens are helpful in the determination of anti- bodies formed as a response to immunization with vaccinia. The authors have since revised and extended these findings.

(a) Culture and labelling procedures The Copenhagen strain of vaccinia virus was used. It was grown in monkey kidney tissue cultured as cell monolayers in a mixture of equal volumes of M199 and Eagle's MEM with 0.5 % lactalbumin hydro- lysate and 10 % foetal calf serum. After 3 days incubation, the

- 58 - cultures were washed with PBS. Then 2 [Ci 1 4 C-labelled amino acid mixture (specific activity 1 mCi/mg) was added per ml medium and the tubes seeded the next day with 103 TCID5 0 vaccinia virus in M199-MEMi medium without serum. After 3 more days or when optimal cytopathic effect (CPE) was observed, the cells were scraped off and disrupted in the pressure cell. The suspension was stirred for 4 hr with 10 % (v/v) veronal buffer (pH 7.9) containing 0.14 1.sodium chloride, the suspension centrifuged at 1,000 x E and 0 to +2°C for 30 min and the supernate clarified by filtration through a membrane with an average pore size of 0.45 pmand then dialysed against distilled water until the dialysing fluid gave a net counting rate of less than 10 c/min per 0.1 ml in the liquid scintillation counter system. The labelled antigen was concentrated by means of Lyphogel (Gelman) or refined sucrose. It was then preserved at -60°C. The final product usually contained 0.4 to 0.9 Cii 4C per mg of protein and was tested against homologous rabbit antiserum.

(b) Test procedures All sera for test were first absorbed with 100 mg unlabelled tissue culture cells per 0.5 ml of serum, the mixture being incubated at room temperature for 2 hr, and the cells then centrifuged off at 1 000 x g. The labelled antigen preparations were standardized to give counting rates of 100 to 500 c/min in the liquid scintillation counter system at the optimal dilution giving a full haemagglutina- tion-inhibition titre with the standard serum. The test proper was carried out by mixing 0.1 ml labelled antigen preparation with 0.3 ml serum, incubating for 4 hr at +37°C, adding 0.4 ml saturated ammonium sulphate solution, letting the mixture stand overnight at +2 to +4°C, centrifuging at 1 000 x g for 10 min, and measuring in the liquid scintillation counter system the radioactivity of 0.1 ml of the supernate absorbed in a 20 to 23 mm diameter membrane immersed in liquid scintillator. Controls were set up with unlabelled non-infected tissue culture cells and with non-infected tissue culture cells, plus labelled antigen prepara- tion, in each case both with and without the addition of sera with known negative and positive titres in the neutralization test. The percent radioactivity retained in the precipitate from the serum under

- 59 - 6 0 r \- q14

,--- 4o -3 a: o 0 \r~~~ \-2I z 2 'v5 20Dh \I J

* I

_ I * I X ;F_ I 2 3 4 12 24 36 48+ Months After Immunizo tion

Fig. 3. variation of percentage radioactivity retained in labelled antigen test (solid line) and mean titre in neutralization test (broken line) for vaccinia antibodies with time after immunization.

test was estimated in terms of the difference between the net counting rate of the corresponding supernate and that of the control with labelled antigen preparation but without serum ("percent difference").

(c) Results

Fig.3 shows the variation with time after immunization in the mean percent radioactivity retained (as "percent difference") in the labelled antigen test and the mean titre in the neutralization test in groups of 20 persons who received primary immunization with 108

to 109 TCID 5 0 per ml stardard vaccine by the multiple pressure method and showed a major response ("take").

The result of the labelled antigen test (solid line) remained positive (percent radioactivity retained >10 %) for nearly 4 years, whereas that of the neutralization test (broken line) became negative in 2 years. Epidemiological observations have proved that smallpox vaccination is effective for at least 3 years. Therefore a retention of >12 % in the labelled antigen test may be considered a reliable indication of immunity against smallpox.

- 60 - Labelled antigen tests on sera from 20 non-vaccinated children gave a positive reaction (with retention 21 %) in only one instance. Cross-reactions with related poxviruses may be expected, but' probably not with varicella-herpes zoster virus, since tests on 5 sera collected from children 4 to 6 weeks after an attack of chickenpox all gave negative results.

B. ADENOVIRUSES (a) Culture and labelling procedures Adenovirus type 4 was' used as a model. KB cell monolayers were cultured in Eagle's IMEM containing 5 % foetal bovine serum. After 1 or 2 days incubation, the cultures were inoculated with virus in MEM containing 2 % foetal calf serum and 1 pCi 14 C-labelled amino acid mixture (specific activity 2 mCi/mg) added per ml medium. After 3 more days, the cells were harvested, then disrupted by freezing and thawing. They were disintegrated by ultrasonic vibration under cooling and the emulsion treated with 0.2 % sodium deoxycholate (pH 9). The pH was restored to 7.2 and the mixture centrifuged at 10,000 x g to sediment contaminating particles. Elution through a Sepharose B2 column with 20 mML sodium phosphate buffer (pH 7.'2) containing 0.14 M sodium chloride followed. The eluate was monitored for protein content and radioactivity. Alternatively, polyacrylamide gel electrophoresis was used to separate the antigenically active protein. Further purification was carried out on Sephadex G-200. Concentration of the antigen was accomplished with Bio-fibres No. 80. The reactive fractions could be stored at -20°C for 2 weeks. The results of preliminary titration with homologous rabbit antiserum showed that at least two antigenic fractions took up the 14 C label to an extent that was easily demonstrable in the liquid scintillation counter system at optimal antigen-antibody proportions. When these fractions were pooled, 0.05 ml of the mixture precipitated 0.25 ml of homologous rabbit antiserum at dilutions of 1:10 to 1:160. The counting rates of 5 different batches were 4,000 to 5,000 c/min per -' (b) Test procedures All sera for test were first absorbed with 100 mg unlabelled KB cells per 0.5 ml of serum. Labelled antigen (tube radioimmuno- precipitation) tests were set up with 0.05 ml labelled antigen prepcra

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- 62 tion and 0.25 ml of serum diluted 1:10 in 0.2 % bovine albumin con- taining PBS. The mixture was incubated at +37°C for 2 hr. An equal volume of saturated anmonium sulphate solution was added and the tubc- were then kept overnight at +2 to +4°C. After centrifugation at 500 x g for 5 min, the radioactivity of 0.05 ml of the supernate was measured in the liquid scintillation counter system. Controls were set up with unlabelled KB cells alone and with unlabelled cells with labelled antigen preparation but without serum. Sera with known positive and negative titres in haemagglutination tests were also tested.

(c) Results The results are shown in Table ]XVIII. The haemaggl.utinaition test carried out accordirg to Norrby (1969) was used for corparison. Since these experiments were only preliminary, the results vere expressed in terms of the counting rate of -the supernate, not in terms of percent radioactivity retained in the precipitate. However, it appears that an approximately 50 % reduction in counting rate is indicative of adenovirus infection. Both the labelled antigen art the haemagglutination teslt gave cross-reacions, ;which are expected with adenoviruses. It is believed that better results can be achieved by a judicious choice of the artigeric fractions.

C. POLIOVIRUSES

(a) Culture and labelling procedures Poliomyelitis virus type Sabin 1 was used. It vas grown in VERO cells. The medium was Eagle's MEI, with 10 % f'oeLal calf serum and 0.05 % glutamine. After the cell sheets had developed, the cultures were inoculated with the virus suspended in MEM with 2 % foetal calf serum and 1 pCi 1 C-labelled amino acid mixture (specific activity 1 mCi/mg) added per ml medium. The next day, the fluid was exchanged for radionuclide-free maintenance medium. The cells were harvested at optimal growth and the labelled antigen isolated, then fractioned by elution through a Sepharose B2 column as described in IV.B(a). The virus-containing part of the filtrate was frozen and tnawed at least 3 times, then disintegrated by ultrasonic vibration. The debris was centrifu.,ed off at 20 000 x Z and the eupernate fractioned

- 63 - by elution through a DEAE-cellulose column. Careful elimination of entire or fragmented virus particles is of great importance in the labelled antigen (membrane) tests because such particles, uncomplexed by antibody, may adhere to filter membranes even of larger (0.45 nm) average pore size, giving spuriously high countinp rates. Concentration of the antigen was effected by means of Lyphogel (Gelman) and with Bio-fibres To. 80. The concentrates reacting in the Ouchterlony test with homologous rabbit antiserum diluted 1:10 gave counting rates of 1 000 to 2 000 c/min per 0.1 ml in the liquid scintillation counter system.

The labelling of polioviruses with 125I has been described by Gerloff et al. (1962), but has not yet gained popularity. (b) Test procedures Labelled antigen (membrane) tests were carried out by depositing 0.1 ml serum and 0.1 ml labelled antigen preparation on a me-.brane with an average pore size of 0.22 pm. After 2 hr incubation at +37°C, the unreacted labelled antigen was eluted with PBS in the filter apparatus, and the membrane dried. The radioactivity retained on the membrane was then measured in the liquid scintillation counter system.

Alternatively, tube radioimmunoprecipitation tests were performed by mixing,0.3 ml serum, diluted 1:10 in PBS, with 0.1 ml antigen, incubating the mixture for 2 hr at +37°C, then adding 0.1 ml anti- human rabbit serum. The tubes were allowed to stand overnight at +2 to +4°C, then centrifuged at 500 x g for 10 min. The radio- activity of 0.1 ml of the supernate was then measured in the liquid scintillation counter system. (c) Results

Table XIX presents results obtained with sera collected from children who had been vaccinated orally with one dose of a type- specific Sabin vaccine. Labelled antigen tests were carried out with labelled antigen preparations from all three types of virus. The complement fixation test was used for comparison. All sera were negative in neutralization tests and complement fixation tests with all three types of virus before the experiment. The labelled antigen test appears to be specific. Further refining of the procedures may improve the result's.

- 64 - TABLE XIX

Results of Labelled Antigen and Complement Fixation Tests with Poliovirus Antigens after Poliomyelitis Vaccination

Subjects N. f Time LA CF vaccinated . o of with sera study A B Polio 1 Polio 2 Polio 3 Before 41 vaccinationBefore20 15 c8 8 8

36 aweeks 51 + 7 63 + 8 L8 <8 r8 Polio 1 after_ 4 4afteer9after <20 <15 <8 48 '8

1 4after wees 20 c15 32 48 8

Before 20 5 vaccination

Polio 2 after ___

2 4weeksafter 31 37 8 8 - 64 8

1 afterwees 20 <20 <8 <8 d8

Polio 3 4after weeks 20 c20 8 48 128

LA : Labelled antigen test with 1 C-labelled type 1 antigen. Percent radioactivity retained. Mean ± standard deviation. A : Membrane test. B : Tube radioimmunoprecipitation test. CF : Complement fixation test with antigen specified. Reciprocal titre. Range.

D. INFPLUENZA VIRUSES (a) Culture and labelling procedures Influenza virus A2/Taiwan/64 was used. It was cultured in human embryonic lung cells in a medium consisting of equal parts of 1i199 and Eagle's MVEM with 10 % foetal calf serum. After the cell growth had reached its maximum, the cultures were inoculated with the virus. One day later, maintenance medium with 1 uCi 1 4 C-labelled amino acid mixture (specific activity 1 mCi/mg) was added per ml medium. After 2 days, radionuclide-free maintenance medium was substituted and the cultures incubated in roller drums and examined daily for CPE and haemabsorption. The culture was harvested at optimal grovrth and treated as described in IV.C(a). The 1 4 C-l~belling was less effective

- 65 - than for the poliovirus, approximately 23 %, of tie protein becoming labelled. In polyacrylamide gel electrophoresis, the labelled fraction behaved like the S-antigen. The concentrated antigenic fraction gave counting rates of 500 to 700 c/min per 0.1 ml in the liquid scintillation counter system. (b) Test procedures

All sera for test were first absorbed with 100 mg unlabelled human embryonic lung cells per 0.5 ml of serum. Labelled antigen (tube radioimmunoprecipitation) tests were performed by mixing 0.1 ml of serum diluted 1:10 with PBS with 0.1 ml labelled antigen preparation and incubating the mixture for 2 hr at +32°C. Then 0.1 ml antihuman rabbit serum was added and after overnight incubation at +2 to +40C the tubes were centrifuged at 500 x f for 5 min. The radioactivity of 0.1 ml of the supernate was then measured in the liquid scintillation counter system. (c) Results Tests were performed on 200 sera randomly collected after the 1964 influenza epidemic in the USA. The CP test with S-antigens prepared from the same A2 strain anci from influenza B/Lee/40 strain was used for comparison. Sera wvith knon positive and negative haemagglutination-inhibition test, compl ement fixation test and neutralization test titres v!ere included as controls. Table XX shows the results. Labelled antigen tests gave results that were within the 90 % fiducial limit of those of the complerment fixation test. To sera of sick and convalescent persons were available, but only sera of individuals exposed to influenza during the epidemic. Additional studies are therefore necessary.

E. MISCELLANIEOUS VIRUISES Herpesvirus prepared in a manner similar to that used for adeno- viruses gave non-specific results in more than 20 % of tests. Tests for arboviruses were performed on 60 sera collected after an outbreak of California encephalitis in Louisiana, USA, 120 collected after an epidemic of dengue fever in Jamaica, 57 after an outbreak of Japanese B encephalitis in Taiwan, and 30 from a West Tile virus infested area in lEgypt. The viruses were isolated from

- 66 - TA BLE XX Results of Labelled Antigen and Complement Fixation Tests in Influenza

CP No. of LA F sera A2 B antigen antigen

46 ± 7 115 (25 to 67) 8

21 238 8 (.1 to 51) 8 24 ±4 21 (1224 to 31)4 8 -8 28 ± 4 10 (2 to 44) <8 33 12 '8 <8

LA : Labelled antigen (tube radio- immunoprecipitation) test with 14C-labelled A2 antigen. Percent radioactivity retained. Mean ± standard deviation (range). CF : Complement fixation test with antigen specified. Reciprocal titre.

infected mouse brain, rurified by centrifugation, extracted with 0.2 % deoxycholate, disinte;.rated by ultrasonic vibration, and purified by elution through Sephadex G-200 columns, then through either DEAE or CM cellulose. The concentrated antigen extracts were labelled with 14C by the method of Rice and Ieans (1971) or with 1 2 5 I by the method of Gruber and WVright (1967). None of the labelled anti-ens proved specific wher the results of tests with them were comps.red with those of neutralization, haemagglutination and complement fixation tests.

Tests with rubella virus yielded non-specific results in >20 % of cases.

- 67 - V. ",I11FEG

Moulds and yeasts causing systemic infections are neither numerous nor frequent in the areas from which serum samples were available to the authors. Therefore their experiments have been restricted to a few species. Some genera, as Actinom.yces and Nocardia, are listed with bacteria in modern taxonomic literature. However, the majority of medical workers prefer to regard then as causative agents of mycotic infections. Studies with labelled ]Tocardia antigens have been reported (Fel.oenfeld and Dutta, 1972). Cross-reactions with related mi cro-organisms, as .'ycobacteria, have been observed. Such cross- reactions are rather the rule than the exception. in t.he serology of mycotic infections.

(a) Culture and labellinrg procedures Li~yccotic agents do rot have uniform and equally distributed antigens. Dimorphic fungi yielded artiFens -hich appeared to be more stable in their yeast phase. Spores were more dif-ficult to di sintegrnte than mycelia. Therefore attem't-s were made to worl viith the yeast phase acd to collect tfhe g'rovth of sporeformers before the spores beca.re too numerouts. Both internals ard external labc'llin.: w-ere found. possible. F'or internal labellin;, the addition of 0.2 mCi 14 C-l.abelled yeast extract (specific activity 1 to 1.5 mCi/mng) per 100 ml li.uid culture medium gave satisfacLsry results. Hloever, nol all l.iouid culture media for mycotic ag;cnts yielded sat-isfactory growthl of every strain. Actinomyces broth (Baltimore JBiological) containing a salt mixture, Trypticase 4 g, cysteine 1 g, yeast extract 5 g, soluble starch 5 g and glucose 5 g (pH 6.9), permitted mairntenance of Actinomyces and INocardia. ?Vycophil broth (of' the sa me manufac- tulrer) containing; Polypeptore 10 g/1 and glucose 40 g/l (pH 7.0) supported the development of the yeast-like phase of biphasic (dimorphic) mycotic agents such as li.stoplasma, Sporotrichumn and Coccidioides. Yeast-like fungi, as Candida, grew well in both media, particularly when the plI was lowered to 4.4 or 4.8. Fila- mentous fungi, as Aspergillus, freqlnently multiplied better in

- 68 - Sabouraud's glucose medium (pH 5.4 to 5.6). The antigen was extracted after centrifugation of the growth at 1 000 x L and washing the sediment with PBS. This procedure was repeated at least three times. The last sediment was suspended in an equal volume of PBS and disintegrated in the bacterial. mill under carbon dioxide cooling. The glass beads used in the container were centrifuged off at 900 x g for 5 min. The supernate was transferred to ultracentrif..ge bottles and certrifuged at 100 000 x g and +2 0 C for 3 to 4 hr. If several layers were formed, the water-clear layer was collected, dialysed, and concentrated by the addition of Lypho-:el (Gelman) or by lyophilization. Approim-tely 5 to 7 % of the antigenic protein was found to be lbelled.

For external labelling, the mycotic agent v;were grownm in radionuclide-free media, but disintegrabted and concentrated by the same method. The protein content of the concentrated supernate was determined. The protein was then labelled with 125I by the method of Gruber and 'right (1967), labellin with 14,14C-formaldehyde by the method of Rice and M,,eans (1971) being not always successful. Each labelled antigen preparatior was titrated with homologous rabbit antiserum and the amount necessary to give optimal precipita- tion combined with a sufficiently high counting rate in radioi'muno- precipitation tests established. Rabbit scra free from antibodies against the respective antigen and absorbed with the other (heterologous) antigens were used as controls.

(b) Test procedures

Tube radioimmunoprecipitation tests were carried out by mixing 0.1 ml serum and 0.1 ml labelled antigen preparation diluted to give optimal precipitation and counting rates of at least 100 c/min in the liquid scintillation counter system if internal labelling with 14C was used, and 1 000 to 2 000 c/min in the thallium-activated sodium iodide crystal scintillation counter system if external labelling with 125I was used. The mixture was incubated at +37°C for 3 to 4 hr, then precipitated with an equal volume of half-saturated ammonium sulphate. The tubes were allowed to stand overnight at room temperature then centrifuged at 1 000 x g for 3 min and 0.1 ml of the supernate was collected and its radioactivity measured.

- 69 - 1 I '0 L.- CO +1 +1 cOHF- '.o C\) +1 00 -r-)(^( r-' H- ms o . H 0 4<-P0a- p l +>4' 0 r-f --4 - h4> 4Q rt- h> h:> S 0a- - b1- a 1S 01 +! +1 + eHl 0 0 << HI -- O n r-l rHl dg0 . " o ' H Jm Hi0l C t_ 'W C4 CM *r4 5 >a 4 -'* 10 0 0 '.D r-- OJ +1 +1 +1 LUO r-I r-1 r- r-l 0H-) 4 4 *H 5O r- Cr O'~ t- td a) e o n2 . H-1 r-l mq 5 3s*-H *+- 'Og 3'- 0o aCO 0 0

QO0 H O' o E" cr> 0cO ri*- F '0 t'

m , COCM iH +-lH +1 +1 0 k t t } o o P0 C O cl o .H ,0r\- C4N Ln H) 03 O ' 0 *H 1H +14C 0o 0) _ .l _~ 0 H H0 , n ..o- ) -c -I O C) *rl q 0 0 EH o d L .r. C) CM3Lr~- " +f +1 +1 O' - 'I , o c O, r-l r -- l 03 r' .r4 Q) 0 H m P a*, ., tf >| Ck_ "I + I +1O3 ) 1 %~ 0 ( ·H) *rl' 4 rc0 0 a1 + 0 -. - '': ; 0 o CtI> a +>S. 4a.i 0

S..I0 +(¶ a a h -H b0 r,4,0 44 T ( 4>'r *l m) P4 4 Hoo bD ri-l NCO' Q I ( ! , --CM. OtH +1 +1 +.1C (- CM-H > , .iH +1 +1 -H o: *,1d 4D 0 pn 0 4 sE- °0 ri i 4 +A +1 3H C t n -1 0- h ( - O0)

_ . ,4 t * OM 543+0 >4' 4ace c o o Co a. r4 $oh-. 4' '4- CVJ ( m' 4*H ' 0*H 4- 4J 4' -p03 s I 0'i a, 'D CuO r cm 4H0H5?-1

U-i H ::'0W lc0- O r-shs. m u-- r4 cM r4 +1 +1 +1 COO 0 CM0, tmn rH cm CM r4 ,-, ?54 5- .034 u ' -pp (14 B ( h Mi0 c1 CM CM E0E!4 40 4'00 w 0 o*- 0 0 0+0 E4 4' F O CM - r. -rj 0 C'O -O - 0 a> o - m° .00 V-- C t--r4 +1 +1 +3 r-4 n^ cRc0 H 0 *4; .04 4 CMO" t, r- r4 r4 a' CJ 0 r4 (M *41 a 0 bn -10 04 (E;a CM 003 *Ha4 " " *H~kt 4

1 ,. t00-,.....oJ, .cO o2i rH rH

H ao +c¶ OQ30P1 h 0 0 0Ha + -H -H OO -H b0 *l e *rlr4 0 > Q)q ^ hl h pi@h gh h CE- , -03 40 - 0.c-o 0 o-00 o : ) c O -1 OHO O H 0- H m H 0'- Cr Mc-H H 41-sOC M-r hd 3 m 4"0T% 3-tD h ¢d

4 U)r- r40 rl 4 r-r- ,4 5_ B -p mrA 4 00 00 403c Pir °ds+ £drlBOP-s Oo Psd o td h- Pda,l + le P, p,4 P4 P -4 Q W

- 70 - (c) Results Table XXI shows hi!;herto unpublished results. For comparison, the complement fixation 'est was used for Coccidioides, the latex flocculation test for Histopla;sma and Candida, and the immunoelectro- precipitation test for Aspergillus infections (in "farmer's lung"), as well as for Actinomyces, Sporotrichium and Nocardia infections. Neither conventional nor labelled antigen tests yielded sufficiently specific reactions. Particularly Candida and Aspergillus antigens gave cross-reactions. Purification of the antigens by chromatography may permit exclusion or reduction of the number of false positive results or increase the differences in the titres of negative and positive sera.

ACKNOWLEDGEMENTS

We are deeply indebted to the International Atomic Energy Agency for Research Contract No. 828/RB and related contracts which made these investigations possible. We are particularly grateful to Dr. E.H. Belcher, sometime Head of the Agency's Medical Applications Section, for his help and advice. Dr. N.K. Dutta was a faithful collaborator until his new assignment took him away from work on this project. We wish to thank Dr. R.B. Brannon, now at the Charity Hospital in.New Orleans, and all technicians who worked on this project, for their co-operation. We are indebted to all scientists who furnished us with sera and strains. Their enumeration would take up another volume. Dr. Addine G. Erskine carefully revised the language of the text. Mrs. Mary LaCroix and Mrs. Hilda Tollison deserve sincere thanks for the typing and re-typing of the manuscript.

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- 75 - 77-0651