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Radionuclide-Labelled Antigens in Serological

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Radionuclide-Labelled Antigens in Serological 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
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