Bull. Org. mond. Sante ) 1964, Bull. Wid Hlth Org. 31, 799-813

Some Modifications of the Fluorescent Test in Human Bilharziasis*

L. 0. C. COOKSON 1

Although the fluorescent antibody (FA) test for human bilharziasis described by Sadun and colleagues has proved ofgreat value, its use involves certain difficulties which the author of the present paper has attempted to obviate. The first part of the paper de- scribes a cheap and reproducible methodfor producing a cercarial antigen conjugated with rhodamine B 200for use in the indirect FA test. The secondpart deals with a new modifica- tion in which the conjugated cercarial antigen is employed with a bentonite-absorbed FJTC antihuman globulin serum and discusses the advantages ofthis test over the normal FA test. Experience has shown that the use ofrhodamine-albumin-coated cercariae, conjugated cercariae or normal fixed cercariae as antigens does not always give valid results when compared with those obtained with the FA test or the ordinary complement-fixation test in bilharziasis. In the third part of this paper, however, the author describes a modification of the complement-fixation test involving the use of a bentonite-absorbed fluorescent anti- guinea-pig serum and the RB 200 conjugated cercariae described earlier. This test has given reproducible results in known positive control human sera which have been valid when compared with the Sadun FA test, the conjugated cercarial FA test and the bentonitefluo- rescent antibody test described in the second part of this paper. In some instances the test results have also been supported by evidence from standard skin tests.

I. THE RB 200 CONJUGATED CERCARIAL ANTIGEN FA TEST

INTRODUCTION of bovine, ovine or rabbit origin, while the specific staining by subsequently applied antibody conjugated The fluorescent antibody (FA) test in human with fluorescein isothiocyanate (FITC) is retained. bilharziasis has been described by Sadun et al. In Sadun's modification the cercariae fluoresce (1960, 1962). Independent investigations in this orange-red due to the RB 200 albumin coating and laboratory, carried out over the same period, have retain their colour unchanged in negative fluorescent confirmed the specificity of the method (Cookson, Coombs tests using an FITC antihuman globulin 1963). serum. In the 1962 paper by Sadun and co-workers, the In positive tests, however, the orange cercariae are test has been modified by coating the formalin- overlaid by the yellow-green fluorescence of the buffer-fixed cercariae of Schistosoma mansoni with subsequently applied antiglobulin antibody. The a lissaine rhodamine RB 200 conjugated bovine degree and distribution of this specific fluorescence albumin. This work is based on a method devised are employed to score the antibody content of the by Smith et al. (1959), whereby immunologically positive test sera under examination. heterologous components in the FA test are counter- This excellent method has been extensively stained with a rhodamine RB 200 conjugated serum employed at the Public Health Laboratory, Salisbury, and at the Bilharziasis Research * This investigation was supported by the World Health Laboratory, Salis- Organization, and was carried out with the technical assist- bury, and has been brought into use for routine ance of Evelyn Pirie, formerly of the Fluorescence Research serological, examination for bilharziasis. Team, Aberdeen University, Scotland. 1 Formerly, Director, Public Health Laboratory, Federal There have been certain disadvantages, however. Ministry of Health, Salisbury, Southern Rhodesia. These have been the occasional difficulty of obtaining

1535 -799- 4 800 L. 0. C. COOKSON consistent coating of cercariae by the rhodamine with a further modification in respect of the FA serum counterstain and the loss of antigenicity of test, and, of particular importance, with a fluorescent the treated parasites if kept for a few days in the complement-fixation test in this disease. refrigerator. Although in the majority of instances the method has worked perfectly, a few anomalous results have been observed which may be due to MATERIALS AND METHODS weak non-specific fluorescent staining. Conjugation of cercariae The best results have been obtained by counter- staining the fixed cercariae with a commercial S. mansoni cercariae in laboratory-infected and preparation of RB 200 conjugated bovine serum.' -reared Biomphalariapfeifferi were kindly supplied by The quantity to be added to each 100 ml of the 5 % Mr V. de V. Clarke and Mr C. Shiff of the Bilhar- formol FA buffered suspension of cercariae for ziasis Research Laboratory, Salisbury. These were successful coating has, however, varied with each fixed by adding equal volumes of a true 10% formol batch of counterstain and has had to be ascertained in FA buffer to the cercarial water with a final con- largely by trial and error. In addition, the price of centration of 5 % formol. the commercial product is rather high for wide- The fixed cercariae were concentrated by sedi- scale use. mentation overnight in conical siliconed urine glasses Attempts to produce RB 200 conjugated counter- in the refrigerator and were then strained through stains, including ovalbumin, here after the method fine-mesh ladies' stockings to remove snail droppings of Smith et al. (1959) have tended to be disappoint- and other extraneous matter. They were then washed ing. free of formol buffer by centrifuging at 1000 r.p.m. In their method Smith et al. employed phenolized in round-bottomed, 15-mi, centrifuge tubes using sera and it has been found that unless the RB 200 three changes of chilled isotonic saline. The cer- conjugated antisera are completely freed of all cariae were then suspended in a measured quantity traces of phenol by prolonged dialysis against FA of chilled saline at an appropriate concentration of buffer in a refrigerator they soon lose their fluo- 200 or more per ml. rescence. An equal quantity ofchilled carbonate-bicarbonate A further complication in respect of locally buffer was added to the saline concentrate of cer- produced ovine and bovine serum counterstains is cariae and conjugation was effected by adding 0.1 ml the difficulty of obtaining sera from animals which of freshly prepared RB 200 2 sulfonyl chloride per ml can be guaranteed free of cross-antigenic S. matthei of cercarial concentrate after the method of Chad- and Fasciola gigantica (Cookson, 1963). This is a wick et al. (1958). factor of some importance where the Sadun modifica- Conjugation was carried out overnight in 20-ml tion of the FA test is employed in mammalian screw-cap containers affixed to a rotating Matburn bilharziasis involving these species. blood-mixer in the refrigerator. The degree of For these reasons, the possibility of attempting conjugation could be controlled by sampling and to conjugate the cercarial antigen directly to RB 200 examining the washed cercariae from time to time. or other fluorochromes has been investigated. The It was found that the conjugated cercariae could be further aim of this modification has been to attempt stored in the conjugation mixture in the refrigerator to produce a highly fluorescent antigen particle for periods of up to two weeks without loss of anti- whose brightness will be enhanced by the subse- genicity. quently applied FITC antiglobulin serum. The conjugated cercariae showed in the majority The resulting enhanced fluorescence has made the of parasites a brilliant fiery orange-red fluorescence. FA test a feasible procedure involving the use of With prolonged conjugation in excess of 24 hours ordinary routine microscopic laboratory equipment many of the cercariae fluoresced a brilliant cardinal with the requisite filters; it has been described by red. Cookson, Clarke & Pirie (1964). Of some interest was the finding that conjugation These provisional investigations have since been of the cercarial proteins commenced early in the confirmed by repeated tests and are described in cephalic glands, which fluoresced a bright orange this part of the present paper. Parts II and III deal

2 ICI Dyestuffs Division, Hexagon House, Blackley, 1 Difco Laboratories, Detroit, Mich., USA. Manchester, England. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 801 as did occasionally extruded droplets of secretion at Tests. One drop of test serum was added to the the terminal ducts. This feature might be of use in a suitably marked 15-ml conical centrifuge tubes with method of selective staining of these structures. their contained cercariae. For use the conjugated parasites were washed The tubes were then incubated for one hour in a free of the last traces of RB 200 with intermittent 37°C water-bath for optimum results, or left over- slow centrifugation in the 15-ml conical centrifuge night at 4°C in a refrigerator, with equally good tubes using FA buffered isotonic saline. results. FITC antihuman globulin sera Control negative sera. These were set up as above. Conjugation of a purified globulin from rabbit Fluorescent control. One drop of the FITC- antihuman globulin serum procured from the South conjugated rabbit antihuman globulin serum em- African Institute of Medical Research was effected ployed in the final stages of the tests was added in this laboratory after the method described by to a control tube of cercariae in parallel with the Nairn (1962) based on the original description of above. Riggs et al. (1958). After incubation or refrigeration, the test and A chromatographically pure grade of fluorescein control sera were washed in two changes of 15 ml isothiocyanate (FITC) 1 was used. The conjugated of FA buffer with intermittent centrifugation at globulin antibody was further absorbed against 1000 r.p.m. and drained of excess buffer. human group 0 and AB red cells and with activated One drop of the FITC antihuman globulin serum charcoal to remove as much non-specific fluorescent was then added to the tests and negative controls staining as possible (Nairn, 1962). and the tubes were further incubated for one hour The final product, dialysed against FA buffer in in a 37°C water-bath (30 minutes were sufficient for the refrigerator for isotonic balance, had a titre of practical purposes if the tubes were gently agitated 1: 3000 against human group 0 Rh-positive cells from time to time). sensitized with an anti-D serum. The tubes were then washed free of the fluores- The conjugate was extremely powerful and gave an cent antibody with two changes of FTA buffer as exceptionally brilliant yellow-green fluorescence in above. positive tests. In addition, commercially prepared The washed cercarial sediment was then gently sera 2 were also employed successfully in the investig- transferred with wide-bore Pasteur pipettes on to ations though without the same brilliance of results. marked microscope slides and examined micro- This may be due to the very pure (and expensive) scopically under cover-slips, clean glassware being grade of FITC used in the above conjugation pro- used throughout. To guard against evaporation cedure and which has given similar exceptional cover-slips were sealed where necessary by applying results with various other antibody conjugates a quick-drying cement around the periphery. This prepared here. was quicker than resuspending the cercariae in buffered glycerol mountant, and the sealed prepara- Test sera tion could be kept effectively up to three days or more in moist Petri dishes in the refrigerator. These were sera from blood which had been sent in for the normal routine Sadun FA test for bilhar- Fluorescent microscope equipment. Preparations ziasis. In addition known negative sera were were viewed with a Reichert Zetopan microscope employed as controls for each batch of tests exam- with a fluorlux maximum-pressure mercury vapour ined. lamp employing the El pass filter; the Reichert special UV x 10 objective with incorporated Schott Conjugated cercarial FA test procedure and Gen. UV-exclusion filter GG9/lmm and x 10 eyepieces with a Wratten 2B filter were employed to f After the final wash with FA buffer to remove all exclude all but the secondary fluorescent image. traces of RB 200, the cercariae were drained of Examinations were carried out by transmitted excess buffer with a mechanical sucker or pipette. light with a Reichert UV condenser NA 1.4, using analytical grade glycerol as a fluid contact. 1 Baltimore Biological Laboratory Inc., Baltimore, Md., There was no USA. particular advantage to be gained 2 From Difco Laboratories, Detroit, Mich., and Roboz by using ultraviolet dark-ground illumination in Surgical Instrument Co., Washington, D.C., USA. such large fluorescent objects. 802 L. 0. C. COOKSON

RESULTS cercariae, has been repeated many times with The conjugated cercariae alone and those in the reproducible results both as regards the conjugation negative and control tests showed the same picture. of the parasites without loss of antigenicity and as They were of a uniform and extremely brilliant regards the actual testing of known positive schisto- orange to orange-red depending on the degree of somal and control sera. conjugation. It was observed that occasional long It is, in fact, a true conjugation because experi- slender cercariae, which occurred as a minority in mental attempts to enhance fluorescence by the any locally bred cercarial population, tended to addition of a variety of buffered fluorochromes to fluoresce a fiery cardinal-red. the first or final stages of the FA test employing The intensity of fluorescence was far greater than fixed untreated cercariae have failed (Cookson et with the cercariae coated with rhodamine bovine al., 1964). serum. In these experiments it was possible to stain Positive tests showed a picture similar to that of cercariae with various fluorochromes but the sub- the albumin-coated cercariae of Sadun's test but sequent application of the FITC antibody resulted with marked characteristics of its own. either in no antibody staining or in considerable There was, foremost, a greatly enhanced fluo- quenching of the specific antiglobulin antibody rescence. fluorescence. Strongly positive tests gave brilliant yellow-green A further confirmatory point of some interest cercariae with an underlying orange-yellow glow has been the successful conjugation of cercariae to condensed at the centre of the head and with a FITC and DANS (1-dimethylamino-naphthalene-5- yellow-green halo around the periphery of the sulfonic acid) 1 using standard methods; this has parasite body (Plate 1). In moderately strong reac- included their successful use in the modified FA test tions the cercariae were of a more yellow colour with a contrasting RB 200 conjugated rabbit anti- with a yellow peripheral halo and a strong orange- globulin antibody. red underlying fluorescence. In weak positives the In the case of the DANS-conjugated cercariae, cercariae assumed a yellow-orange hue with a fine provided that the process is carefully controlled the peripheral yellow halo and with a central orange- positive tests show bright yellow cercariae with a red fluorescence involving both the head and tails. marginal halo of a duller orange-red antibody, and The results with all sera tested were quite unmis- the negatives and controls retain their bright yellow takable. fluorescence unchanged. FITC-conjugated cerca- In 30positive sera tested independentlybytheBilhar- riae give a very much brighter picture with this ziasis ResearchLaboratory using Sadun et al. 'smethod combination of antibody, but since this fluoro- (1960, 1962) and subsequently tested by the present chrome is generally employed for antibody conjuga- method in this laboratory there was close agreement. tion, these investigations were not pursued further. Apart from repeated successful application of the One of the objects of the enhanced fluorescence conjugation method to locally bred cercariae it was test was to fit it to ordinary routine laboratory possible to test the method on two separate batches microscopes using a 30-watt, overrun, tungsten- of S. mansoni cercariae kindly supplied by Professor filament lamp and this has been more fully des- R. Gonnert of Farbenfabriken Bayer A.G., Ger- cribed elsewhere by Cookson et al. (1964). The low many. These cercariae were a West African strain spectral energy of this type of lamp allowed this of S. mansoni raised in laboratory-bred Australorbis goal to be attained with both RB 200 and FITC, glabratus, and the concentrates suspended in 5 % which were sufficiently stimulated to emit a readily formol FA-buffered saline were sent by air. It was recognizable fluorescent image with the requisite found that the parasites gave very superior and secondary filters. The energy emission of such a uniform results with the normal FA test, and that lamp was, however, insufficient to produce a work- they conjugated equally well with RB 200, retaining able fluorescence with the DANS-conjugated cer- their full antigenic properties in the modified test cariae though the modification might find some use described above. with the usual high-pressure mercury-vapour illum- inants. DISCUSSION The above findings suggest a further field of The modified fluorescent antiglobulin test in bilharziasis here described, using RB 200 conjugated 1 Fluka A.G., Chemische Fabrik, Buchs, Switzerland. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 803

investigation in the use of fluorochromes to con- added advantage of completely swamping any jugate antigens with a contrasting colour to the anomalous results which may occur owing to non- subsequently applied antibody. It might make it specific fluorescent staining effects of the subse- possible for fluorochromes which have been found quently applied FITC antihuman globulin sera, and unsatisfactory for antibody conjugation because of which might otherwise be read as false marginal- either colour or lack of intensity to be used for positive results. contrasting staining of antigens by conjugation. It is also possible that these same properties might Reverting to the RB 200 conjugated cercariae tend to give obscure results with very weak anti- and the modified FA test based thereon, the main bodies, but if a first-class FITC-conjugated anti- advantages of the test are in the characteristic globulin serum is used, as has been described above, fluorescence of the antigen particles. This intensity this type of error should be no greater than with and hue of the fluorescence can largely be controlled Sadun's RB 200 coated cercarial antigen. by varying at will the conjugation procedure either There is no doubt that this modified technique in the time taken or in the quantity of conjugate gives intensely bright microscopic fluorescent results which is added. and should be of some value for the unequivocal The production of the fluorescent cercarial antigen serological diagnosis of bilharziasis by the FA is extremely easy and costs but a few pence as against technique, and possibly in bringing the application the much higher cost of both the purchased and of that technique within the reach of many labor- the laboratory-produced RB 200 conjugated bovine atories without the use of expensive specialized counterstains. The exceedingly bright fluorescence ultraviolet microscopic equipment or the complica- of the antigen particles gives an equally bright and tions of using or making RB 200 conjugated protein specific picture in positive tests with any appreciable counterstains. It is also hoped that the principles levels of antibody. involved may be extended to other antigens and other This very powerful antigen fluorescence has the diseases.

II. THE BENTONITE FLUORESCENT ANTIBODY (BFA) TEST

INTRODUCTION All these instances are manifestations of the Rieckenberg phenomenon and are probably due to In the Rieckenberg adhesion phenomenon certain the absorption by electrostatic or other forces of the finely particulate matter, when incubated with immune antibody on the extraneous particulate relatively large antigenic objects in the presence of matter in question. The particles so coated are thus any immune serum, tends to coat such objects in turn immunologically attracted by the homolo- specifically. This reaction has been used as a diag- gous antigens. nostic immunological method in trypanosomiasis, The fact that the adhesion phenomenon has been relapsing fever and leptospirosis disease (Davis, used with success as a specific test in trypanoso- Broom & Brown, quoted by Manson-Bahr, 1954). miasis and treponemal diseases, as noted above, In the application of the adhesion phenomenon suggests that the principle could equally well be to these diseases the particulate material has con- applied to the immunodiagnosis of bilharziasis. sisted variously of platelets, blood cells and bacteria, It was thought that if carefully graded particles and in some cases India ink carbon particles have could be employed selectively to absorb fluoro- been used. chrome-tagged antihuman globulin antibody they It has been a common observation in applying could be applied for bilharziasis in the final stages the FA test to bilharziasis that, where the cercarial of the fluorescent . Furthermore, it suspension contains a considerable degree of extra- was felt that with uniform, minute, FITC-antibody- neous matter-snail droppings, bacteria, unicellular saturated particles, the distribution of these on algae, lint threads, etc., from dust-these adhere the cercarial antigen in the Rieckenberg pheno- strongly to the cercariae in the tests with positive menon could be employed as a means of assisting sera and constitute a serious nuisance unless special the strength of antibody present in any given test precautions are taken. serum. 804 L. 0. C. COOKSON

This section describes the successful application MATERIALS AND METHODS of this idea to the FA test, employing both the Preparation of bentonite absorbates for fluorescent RB 200 bovine serum-coated cercariae and the antibody tests RB 200 (and other) conjugated cercariae described in the foregoing section. The successful application A 0.1 % aqueous suspension of laboratory grade of the principle to a fluorescent complement-fixation bentonite was made by adding 1 g of the substance test will be described in the final part of this com- slowly to 1 litre of deionized water with gentle munication. stirring by a magnetic or other stirrer. (Smaller Preliminary experiments were made to find a proportions could be used.) suitable carrier of fine particle size which would The suspension was kept in a stoppered cylinder strongly absorb and retain the fluorescent globulin or flask for 24 hours with intermittent shaking to antibody. ensure complete wetting and separation of the It was found that bentonite, a natural aluminium silicaceous earth particles. These particles, which silicate clay earth of the montmorillonite group were dissimilar in size, were graded by differential which has ion-exchange properties, gave very good centrifugation in order to obtain a suspension of results. It was very cheap, and easier to handle than fairly uniform particles of the smallest size. The polystyrene latex particles, which were also tried. procedure was as follows. The uptake to saturation point by the bentonite A quantity of the working suspension was poured of globulin antibody conjugated with FITC, RB 200, into siliconed, 1 5-ml, round-bottomed centrifuge or DANS was excellent in FA buffered saline and tubes and spun at 2000 r.p.m. for 15 minutes. this was retained by the particles after repeated The supernatant, containing a suspension of the washing to remove excess unabsorbed antibody. finest particles, was then spun at 4000 r.p.m. for It was found that a uniformly small particle of 15 minutes and the deposit was retained. the earth suitable for the adhesion phenomenon The sediment from two 1 5-ml tubes was pooled by could be easily procured by differential centrifuga- resuspending in 15 ml of deionized water and re- tion and that these particles readily absorbed the centrifuged at 4000 r.p.m. for 15 minutes. conjugated globulin antibody. The bentonite sediment was then freed of water The particles with their absorbed antibody, by gentle draining and to this were added 10 drops suspended in FA buffer, could also be stored at of the fluorescent antiglobulin serum or a potent 4°C for a few days, and probably longer, without fluorescent anti-guinea-pig complement serum de- loss of antibody property. pending on the use to which the bentonite absorbate was to be put. The most important finding, however, was that The when a drop of the bentonite antiglobulin-antibody bentonite was resuspended in the added absorbate was applied in the final stages of the serum by sucking and expelling with a fine-bore FA Coombs test in place of the usual conjugated Pasteur pipette and the suspension was left overnight antihuman it in a refrigerator for maximum absorption of the globulin antibody, selectively adhered the bentonite to the cercarial antigen and in proportion to the antibody by particles. The particles were then freed of excess serum by schistosomal antibody content of the positive test with sera under investigation. washing two changes of 15 ml of FA buffered saline, pH 7.2, with intermittent centrifugation at Negative test and negative control sera and the 4000 r.p.m. bentonite absorbate alone did not adhere to the The resulting sediment was drained free of buffer cercarial antigen. The test, which was tried repeat- and resuspended in 10 drops of FA buffered saline edly on routine sera previously tested by the FA test equivalent to the original volume of added fluo- described by Sadun et al. (1960, 1962) and the con- rescent antibody. A greater volume of buffer could jugated cercarial test mentioned above, gave com- be added provided a sufficient concentration of plete agreement and was reproducible. particles making an opaque solution was retained. The technique of this test, which it is proposed to This constituted the test suspension and was kept call the BFA test for short, is described below refrigerated at 4°C for use. together with the typical appearances which were observed with a series of sera tested whose antibody Cercarial antigen content varied from very strong to weak as judged S. mansoni cercariae, either coated with RB 200 by the two versions of the FA test mentioned above. bovine serum counterstain after the method of MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 805

Sadun et al. (1960, 1962) or conjugated to RB 200 to clean microscope slides with cover-slips and as described above, were used. In addition a limited examined as described for the fluorescent conjugated number of normal, washed, formol-buffer-fixed cercarial antibody technique. cercariae were used in a comparison test. Test and control sera RESULTS The sera used were from suspected S. mansoni Provided the technique was properly carried out and S. haematobium infections which had been sent and especially if the final wash with FA buffer was in and found positive with routine examination by correctly judged to leave most of the excess bentonite the FA test and upon which the conjugated cercarial antibody particles in suspension, the results were FA test had also been employed with similar results. dramatically clear-cut. These sera, totalling more than 30 over the period of the investigations, varied in their antibody con- Conjugated cercarial antigen tent from very strong to weak as adjudged by the In negative results (Fig. 1) and negative controls above tests. the conjugated cercariae were a brilliant orange-red Control negative sera were from European set against a background of very fine highly fluo- immigrant staff who had not been exposed to bil- rescent yellow-green bentonite particles with their harziasis and whose sera were negative to the above absorbed FITC antiglobulin antibody. There was tests. no true adhesion of the bentonite antibody to the cercarial antigen except occasional randomly and Technique ofBFA test mechanically deposited particles. These were in a The technique of the first part of the test was as minority; in the majority of cases the cercariae stood described above for the FA and conjugated cercarial out sharply with a peripheral clear zone against the antigen tests. yellow-green bentonite particles. Furthermore, the After the cercariae with their respective test and occasional superimposed bentonite particles were control sera in 15-ml, conical centrifuge tubes had easily disturbed by gently tapping the cover-glass on been incubated for one hour or left overnight at the microscopic preparations. 4°C in the refrigerator, they were gently washed In very strongly positive cases the cercariae were with one change of 15 ml FA buffered saline, and completely ensheathed with the FITC bentonite the excess buffer was removed by suction. antibody and gave the appearance of being petrified. One drop of the bentonite absorbates of the FITC This is clearly seen in Fig. 2. This appearance was antihuman globulin was then applied to the test scored as + + + +. and control preparations and to a further tube of In strongly positives the underlying fluorescent cercarial antigen as a bentonite reagent control. cercarial body could be seen, but there was a regular The tubes were further incubated at 37°C for moderately dense adherence of the bentonite one hour with gentle intermittent manual agitation. particles to the whole parasite body, including the The contents of the respective tubes were then surface, which had to be suitably focussed. This washed once by adding 15 ml of FA buffer from a constituted a + + + result (Fig. 3). beaker with a minimum of agitation. In positive results, scored as + +, the particles The tests and controls were then centrifuged adhered as a regular fine peripheral coating (Fig. 4). slowly at 500 r.p.m. for 10-15 minutes. In weak positives, scored as +, the adhesion of Gentle handling was essential in these stages so particles was more in random clumps, occasional as not to disturb unduly the adherence of bentonite areas of the parasite body being unaffected. antihuman globulin particles in the positive tests. Weak marginal results showed only minor The final single wash with slow centrifugation was occasional aggregates of bentonite antibody par- important in order to obtain optimum sedimenta- ticles, and these could not be detached to any degree tion of cercariae and at the same time to allow any by tapping the cover-slips of the preparations as unreacted bentonite antibody to remain in suspension could be done with the occasional mechanically in the supernatant FA buffer. deposited particles in the negative tests described The tubes were then sucked free of supernatant above. buffer and suspended bentonite and the deposit was The bentonite reagent control results were the gently removed with a wide-bore Pasteur pipette on same as described for the negative and control tests. 806 L. 0. C. COOKSON

RB 200 bovine-serum-coated cercariae cent staining effects which may occasionally be found Except for the enhanced brilliant fluorescence of in conjugated sera. the conjugated cercariae, the coated cercariae of Because the bentonite particles are saturated with Sadun et al.'s FA test showed much the same the conjugated antibody, this will largely overcome appearance as those described above and the various the former disadvantage. Similarly, the positive sera tested with both types of cercarial antigen gave results with the bentonite technique are true positives identical results. because there is no free antibody in solution with a possible potentially non-specific fluorescent 5 % Formol-FA-buffer-fixed cercariae staining with the RB 200 conjugated or protein- In these the cercariae showed a pale grey-blue coated cercariae. autofluorescence with no attached bentonite anti- It is also possible to use an RB 200 antiglobulin body in negative tests, but in positives there was antibody with either DANS- or FITC-conjugated the same degree of adhesion of antibody particles or bovine-serum-coated cercariae as a contrasting in relation to the antibody content of the test sera antigen. employed as for the tests described above. The experimental basis of the technique indeed The appearance was not so dramatic or clear-cut allows for a considerable variation on this score and because of the absence of the exceedingly bright further latitude for investigation including the use contrasting fluorescence of the yellow-green bento- of fluorochromes hitherto considered unsuitable nite antibody and the orange-red cercarial antigen. for conjugation to protein in the normal accepted fluorescent antibody techniques. Lastly, some mention of the sensitivity and DISCUSSION specificity of the Rieckenberg adhesion phenomenon The above version of the FA test coupled to the on which the above technique is based should be conjugated cercarial antigen modification has given made. According to Davis, Brown & Broom, as consistently excellent results. quoted by Manson-Bahr (1954), the adhesion The microscopic picture shows an exceptionally phenomenon is highly specific and sensitive. brilliant fluorescent contrasting image between the In relapsing fever, for example, the borreliae of FITC antihuman globulin bentonite particulate the first and second attack of a given infection antibody and the orange-red cercarial antigen. This can be differentiated. In leptospirosis it is not only enables the adhesion of the antibody particles to be specific, but it is a means of differentiating between dramatically seen, and gives a very clear-cut picture various strains of leptospirae. The above authors for all positive tests which is quite unmistakable have also noted that the same specificity exists even to the most inexperienced observer. between various strains of trypanosomes and that In the marginal results, the possibility of confu- the immune antibody exhibits a high degree of sion between the occasional random mechanical thermostability. deposition of bentonite antibody particles on the It is felt therefore that the technique, which is cercariae in negative tests can easily be resolved by patently extremely sensitive in detecting antibodies gently disturbing the cercariae. The bentonite and which as a fluorescent antibody test shows antibody particles adhere quite strongly with the quite dramatically clear-cut and unmistakable weak positive tests but are easily detached in the results, should receive further consideration in the doubtful negatives. routine serological diagnosis of bilharziasis. It is felt that this is one of the principal values of Its outstanding value, however, is in the effective the technique because it removes all risks of false modification of the fluorescent antibody method to a marginal positives which may occur with the usual highly sensitive and reproducible complement- FA test either through a weak FITC antihuman fixation test which is described in detail in the final globulin serum or through the non-specific fluores- part of this communication. PLATE I NEGATIVE (LEFT) AND ++ POSITIVE (RIGHT) REACTIONS IN THE RB 200 CONJUGATED CERCARIAL ANTIGEN FA TEST FIG. 1 NEGATIVE RESULT IN BFA AND BFCF TESTS FIG. 2 VERY STRONGLY POSITIVE (++++) RESULT IN BFA AND BFCF TESTS FIG. 3 STRONGLY POSITIVE (+++) RESULT IN BFA AND BFCF TESTS FIG. 4 POSITIVE (++) RESULT IN BFA AND BFCF TESTS

4*

."W

I"

.f.: PLATE 2 STRONGLY POSITIVE (+++) REACTION IN THE BFCF TEST a

.. ;- I'll ip

a A comparable picture would be seen with the BFA technique. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 807

III. THE BENTONITE FLUORESCENT COMPLEMENT-FIXATION (BFCF) TEST

INTRODUCTION The staining by a fluorescent anti-guinea-pig complement serum at this ultimate dilution of anti- Successful complement-fixation tests in human body will therefore be ineffective and will give a bilharziasis have been described by various authors, negative test as well as indicating the end titre of the a recent comprehensive account including a full antibody in question. description of the technique and the various anti- A further advantage of the technique is that one genic extracts investigated from adult S. mansoni fluorescent anti-guinea-pig complement serum is being that of Eliakim & Davies (1954, 1955). sufficient for identifying antigen or antibody, Fluorescent complement-fixation tests have also including its measurement, in practically any animal been described by a number of investigators. Among species, the proviso being that the antibody-antigen the earliest descriptions of applications of the reaction is dependent on complement and that the staining of complement by a rabbit fluorescent anti- naturally occurring species complement of the test guinea-pig serum was that of Goldwasser & Shepard serum antibody can be replaced with complement (1958), who employed the method for the immuno- of guinea-pig or other origin. logical localization of virus and rickettsial particles. Notwithstanding the apparent versatility of the It is not the purpose of this introduction to review technique, it does appear to have come short of the fairly considerable literature that has since unqualified success in many antibody-antigen accumulated on the subject. reactions where success would be expected. Perhaps The basic technical concept is to inactivate given on a superficial judgement most advances have been test serum antibodies for their own complement and in the realms of virology and rickettsial diseases. then to test them in the presence of a known constant, One cause for this could be the necessity of using adequate, quantity of substituted guinea-pig com- very powerful, high-titre, anti-guinea-pig comple- plement with their homologous or other antigen ment fluorescent sera (Tyrell & Hers, 1960). under investigation. Where there is an immuno- between logical combination antibody and antigen, PRELIMINARY INVESTIGATIONS and provided complement is necessary for such a reaction, the resulting antibody reaction complex Attempts to apply the fluorescent guinea-pig will in addition contain a quantity of " bound " complement staining or fixation test to human complement which was added from the guinea-pig bilharziasis in this laboratory have been made on source. There will be in effect an immunological many occasions in the past two years. " sandwich " of antibody, guinea-pig complement, S. mansoni cercariae have been used either freshly and antigen. killed, or by quick cooling to below 4°C, or by fixing In theory, the subsequent application of a power- with 1 % acetic acid in absolute alcohol or 5 %° formol ful anti-guinea-pig complement serum to this FA buffer. The cercariae have also been used complex should stain the complement and hence coated with RB 200 bovine serum after Sadun et al. define by the presence of both (1960, 1962) or in their RB 200 conjugated form antibody and its homologous antigen. already described. The virtue of this immunological mechanism is The results in all cases have been most disappoint- that one fluorescent anti-guinea-pig complement ing. The only encouragement was found in using the serum can be employed to identify an antigen using RB 200 protein-coated or conjugated cercariae. a known antibody, and vice versa where the antigen Some 10 known positive sera and known negative is the known factor. Furthermore, provided the controls were put up with these cercariae and in antigen and guinea-pig complement in a given test doubling solutions for the fluorescent Coombs test system are kept constant, the antibody can be using a potent FITC-conjugated rabbit antihuman applied in serial dilutions up to a point where it no globulin serum. The same sera inactivated for longer exists in sufficient strength to combine with complement were put up in similar doubling dilu- its homologous antigen through the intermediary tions of guinea-pig complement diluted in FA of complement. buffer to contain a final concentration of 5 minimal 808 L. 0. C. COOKSON

haemagglutinating doses (MHD) of complement. was absorbed on to graded bentonite particles as in After the usual rinsing procedures they were stained the bentonite fluorescent anti-globulin (Coombs) with a potent commercial horse FITC anti-guinea- test described in the previous section. pig complement serum with a tested titre in excess This serum was tested against fresh guinea-pig of 1: 30 000 against guinea-pig serum. serum by capillary and had a titre in The comparison of the two series of tests showed excess of 1: 32 000. a fairly uniform pattern of diminishing antibody staining for the diluted fluorescent Coombs test. Test and control sera The fluorescent complement test, however, showed A series of known positive schistosomal sera only an approximate agreement with, and in many which had been previously graded in routine testing cases complete variance at one point or another in from + + + + to + by the local Bilharziasis the serial dilutions used against, its parallel fluores- Research Laboratory using Sadun et al.'s (1960, cent Coombs counterparts. In addition many of 1962) FA test were employed as well as negative the known negative sera gave recognizable positive control sera similarly tested. results by the complement-staining method. It was concluded, therefore, that this highly desir- Techniques of the BFCF test able technique, in the usually accepted form could not be used with certainty as a means of serological An initial dilution of the sera at 1: 10 was made diagnosis in bilharziasis or perhaps most important in FA buffered saline and the diluted sera were of all, in estimating antibody levels in the disease. inactivated in a 56°C water-bath for 30 minutes to In view of the high success of the normal comple- remove all traces of autogenous complement. ment-fixation test in bilharziasis these results were From this initial strength of inactivated sera, not only disappointing but unexpected. doubling dilutions were made by Pasteur pipette The problem was, however, re-examined after the with freshly reconstituted lyophilized commercial successful application of the Rieckenberg adhesion complement 2 diluted with FA buffer to contain phenomenon to the FA technique, which has been 10 MHD. An equivalent strength of diluted com- described in the foregoing section as the BFA test. plement from fresh guinea-pig serum was also used Preliminary experiments with a bentonite absor- and found effective. bate of the previously mentioned commercial horse The final doubling dilutions of serum in com- FITC anti-guinea-pig complement serum were made, plement varied from 1: 20 to 1: 160 except in one the bentonite antibody particles being prepared in case where the dilutions were taken to 1: 640. the same way as described for the FITC antihuman The sera were then incubated with previously globulin absorbate. Known positive human bil- prepared RB 200 conjugated S. mansoni cercariae in harziasis sera and known negative control sera, most 15-ml conical centrifuge tubes for one hour in a of which had been used for the abortive fluorescent 37°C water-bath, or left overnight in a refrigerator. complement-fixation experiments mentioned above, They were then washed once with 15 ml of FA were used. buffered saline with centrifugation at 1000 r.p.m. A similar technique as for the BFA test described After removal of the excess buffer, one drop of above was employed. bentonite absorbate of the FITC anti-guinea-pig These initial investigations gave very clear-cut complement was added to each tube of cercarial and dramatic results which agreed closely with the sediment and these were incubated at 37°C for one FA tests for all the sera examined. The tests were hour with intermittent gentle manual agitation. reproducible and the results of subsequent, more The tubes were then gently washed with 15 ml of comprehensive investigations in parallel to the FA buffer with final centrifugation at 500 r.p.m. as fluorescent Coombs test are described below. described above for the BFA test and for the same reasons-namely, to sediment the cercariae without bringing down the excess bentonite in the final MATERIALS AND METHODS parasitic antigen deposits. The potent commercial horse FITC-conjugated The sediment was then removed with clean, wide- anti-guinea-pig complement serum mentioned above1 bore, Pasteur pipettes on to clean slides with cover-

1 Roboz Surgical Instrument Co., Washington, D.C., " Wellcome Research Laboratories, Beckenham, Kent, U.S.A. England. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 809 slips for ultraviolet microscopy as detailed in Part II Results. The strong sera giving a + + + + reac- above. tion with the above test, and with the BFA and fluorescent Coombs techniques, gave a considerable Bentonite reagent control reduction in the adhesion of the bentonite fluorescent One tube of RB 200 conjugated cercarial antigen antibody particles at the commencing dilutions of was treated in the final incubation stage of the test 1: 20, with a fall in scoring from + + + + to + +. with one drop of bentonite fluorescent anti-guinea- At the higher dilutions, from 1: 40, there was pig serum. complete blockage by the control unconjugated anti-guinea-pig complement serum of the subse- RESULTS quently applied homologous bentonite FITC anti- The tests gave the typical clear-cut and vividly body particles. fluorescent picture which has been described for the Sera with a lower titre of antibody and a weaker foregoing bentonite fluorescent antihuman globulin scoring at the commencing 1: 20 dilutions for the test (see Plate 2 and Fig. 1-4). above tests gave a proportionately effective blocking These appearances were commensurate with the test at this starting dilution. This blocking effect original antibody content as judged by the previous was complete for most sera previously scored as screening Sadun FA test carried out on the sera by + + in the 1: 20 dilution by the foregoing tests. the Bilharziasis Research Laboratory. Negative control sera gave uniformly negative As the sera were progressively diluted, so there results throughout. was a parallel diminution in the degree of adherent FITC anti-guinea-pig bentonite particles until a Control experiments with non-specific bentonite final titre was reached where no true adhesion was absorbates perceptible. Although the above blocking test would appear to The negative control sera gave negative results prove the immunological validity of these investiga- throughout the dilutions of serum examined. tions it was thought that a further check should be The occasional random mechanical non-specific made using the immunologically inert fluorescent deposition of particles on the cercariae could be bentonite absorbates. Accordingly attempts were easily detached as mentioned in Part II and at no made to adsorb the pure fluorochrome dye sub- time gave any real doubt as to a possible false- stances RB 200 and soluble fluorescein on to the positive picture. bentonite. It was found that the particles did not bind the CONFIRMATORY TESTS OF SPECIFICITY dyes in the face of repeated washing and they were thus of little use as fluorescent indicators. Blocking test Absorbates were therefore made with RB 200 The sera employed in the above tests, including the conjugated bovine albumin which resisted repeated controls, were incubated in the usual way with their washing with FA buffer. The RB 200 bovine added complement and in serial dilutions with the albumin bentonite suspension was then applied in RB 200 conjugated antigen. the final stages of the above blocking technique for After the first washing in FA buffer and centrifuga- the fluorescent complement-fixation test. In addi- tion at 1000 r.p.m. the parasitic sediment was tion the absorbate was added after the final stages incubated for one hour at 37°C with two drops of of the FA antiglobulin test. There was no specific control unconjugated horse anti-guinea-pig com- adhesion of the RB 200 bovine albumin bentonite plement serum. This serum was homologous to particles in either of these instances. the FITC conjugate used for preparing the bentonite absorbate. The tests were then washed with FA buffer as CONCLUSIONS above and reincubated with one drop of the bentonite It can thus be concluded from these two sets of absorbate of the FITC anti-guinea-pig serum for experiments that the bentonite fluorescent com- one hour at 37°C. plement-fixation (BFCF) test in bilharziasis is a After a final gentle rinsing and centrifugation at specific immunological reaction. 500 r.p.m. for 10 minutes the sediment was removed The results with the various sera and dilutions and examined in the usual way. tested by the BFCF technique are set out in the 810 L. 0. C. COOKSON accompanying table together with the comparable for the usual FA test with known positive sera still findings with the FA test of Sadun et al. (1960, 1962) gave recognizable positive results with the bentonite and the previously described conjugated cercarial complement-fixation tests. FA test. DISCUSSION USE OF RB 200 BOVINE-SERUM-COATED AND seen UNCOATED ANTIGEN It will be from the earlier experiments described above, that the more usual type of fluo- The above BFCF technique gave identical results rescent complement-staining technique does not with those obtained with the coated S. mansoni agree with the results of the FA test for the series of cercariae described by Sadun et al. (1960, 1962) sera examined in parallel. and normal 5 % formol-buffer-fixed parasites. There is no reason to assume that the disparity The appearances were similar to the results is due to the FA test or faulty technique, because described above under the BFA tests with both this test has given consistent and reproducible these types of antigen. results which have additionally been in close agree- One point of interest noticed was that some ment with the modified techniques described in this batches of coated and normal fixed carcariae which paper. had, through long storage, lost their antigenicity The disagreement and lack of reproducibility thus

COMPARISON OF RESULTS OBTAINED WITH DIFFERENT FLUORESCENT ANTIBODY TESTS IN BILHARZIASIS

Sadun FA Conjugated Bentonite test cFActestl FA test (BFA) Bentonite fluorescent CF test (BFCF)

Patient 1:2 1:2 1:2 1:20 1:40 1:80 1:160 1:320 1 +++ +++ +++ +++ ++ 2 ++ ++ ++ ++ + _ _ _ 3 ++ ++ ++ +++ ++ +

a 4 ++-_ _ _ _ _ 5 b +++ ++++ ++++ ++++ +++ ++ ++ _ 6 ++ + + ++ +--- 7 ++ + + +- -- 8 ++ + + + ± - -

9e +------1 0 d +++ ++++ ++++ ++++ +±+ ++ ++ + Control - to------

BFCF blocking tests: No. 10 ++ - - -_ No.33 _ -_ _ No. 5 + -_ _

a Patient 4 was an adult male laboratory worker who was surprised at the +++ result of the Sadun FA test conducted by the Bilharziasis Research Laboratory which he was visiting. He stated categorically that he had never run any risk of contracting bil- harziasis. b Patient 5 gave a + reaction in the Sadun FA test at a serum dilution of 1: 180; this is similar to his reaction in the BFCF test. c Patient 9 was a senior laboratory technician with over 27 years' residence in Southern Rhodesia. He claimed never to have exposed himself to bilharziasis and was surprised at the + result in the Sadun FA test. A skin test with S. mansoni Melcher antigen from WHO was negative, in confirmation of the negative results in the other FA tests shown above. d Patient, a healthy young male laboratory worker with long residence in Southern Rhodesia, denied any known exposure to bilharziasis. In addition to the positive results shown above, he gave a + reaction in the BFCF test at a titre in excess of 1 : 320. The Melcher skin test was strongly positive, in agreement with the above findings. His urine, stool and sigmoidoscope examinations have, however, been negative, and it is assumed that he has unisexual or infertile parasites. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 811 appear to repose in some factor in the complement- BFCF tests described above might be that these tests staining technique in bilharziasis. utilize a conglutinating type of complement rather In the normal complement-fixation technique for than the normally accepted haemolytic complement this disease use is made of a soluble antigen. The in the usual complement-fixation reaction. It is fact that the antigen is in solution here may make it suggested that the fluorescent antibody particles of more efficient as a receptor of antibody through the the bentonite absorbate adhere because of a con- intermediary of the added complement. glutinin in the immune test serum under investiga- Where the fixed cercariae are used in one form or tion and through the intermediary of a conglutinat- another, the complement-binding antigen may be ing complement. only partially available at the surface of the parasite In the case of the BFA technique this type of body and in a capricious way, depending on a series complement would be derived from the patient's of factors. It is possible that the nature and disposi- normal serum. tion of the antigen on the surface of the entire cer- Where the serum is inactivated for the BFCF test carial body may be of a kind which does not com- the source of conglutinating complement would be bine with the immune antibody through the inter- from the added guinea-pig serum. The subsequent mediary ofthe usually accepted forms ofcomplement. addition of horse FITC anti-guinea-pig complement The immune antibody is unlikely to be at fault in bentonite absorbate in the technique would furnish this sense because of the success of the normal type the conglutinating anti-guinea-pig complement anti- of complement-fixation test in this disease utilizing body or heterologous agglutinin to complete the a soluble antigen from adult parasites. reaction. An alternative source of the anomalous results The above explanation may not take into con- between the FA test and the fluorescent complement- sideration all the factors involved, and these are fixation test may be the nature of the complement admittedly subject to further, and possible different, required for the combination of antibody with the exposition within the concept of conglutination. It intact parasite used in these preliminary investiga- is felt, however, that the explanation for the success- tions. The failure of the fluorescent complement- ful results in the above techniques is along these or fixation test and the success of the bentonite comple- similar lines. ment-fixation technique suggest that this may be The procedures are therefore apparently quite the case. sound on immunological grounds, the best proof of It is therefore felt that the Rieckenberg adhesion which is that they have been found to work and to phenomenon, the bentonite fluorescent antibody give consistent and reproducible results. Apart from test, and the bentonite fluorescent anti-guinea-pig the above theoretical consideration, the sensitivity complement or fixation test described above are and specificity of the bentonite fluorescent comple- manifestations of a type of conglutination reaction ment-fixation (BFCF) test is apparent in the results which is not dependent on the normal haemolytic tabulated above. type of complement for antibody-antigen reactions. In general these results show that the technique It is not feasible to discuss this subject of conglutina- gave a higher assessment of antibody levels at a tion here. In the past it has received less attention starting dilution of 1: 20 than the comparable FA than it deserves and for that reason tends to be little test on 1: 2 dilutions of serum. This also applied understood or used in routine . However, to separate experiments (not shown above) with sera the various manifestations of conglutination and the diluted out in parallel for both tests. This increased uses to which they can be put in immunological sensitivity usually extended to one dilution above the serology are clearly set out in a recent monograph comparable level for the FA technique. by Coombs et al. (1961). The subject certainly In two instances at least (tests 4 and 9 in the above deserves more attention in the context of parasitic table) the technique gave unequivocal negative and diagnosis, and from this work it is results where these were expected on clinical and apparent that conglutination reactions are as valid as, personal-history grounds. The FA test gave a and in some cases even more sensitive than, many definite strong positive and a weak positive result comparable immunological methods in serological for these respective sera, and in the latter, a standard diagnosis. skin test confirmed the negative findings of the On the basis ofconglutination, a tentative explana- BFCF test. It was not possible to do a skin test tion for the success of the BFA and in particular the on patient 4. 812 L. 0. C. COOKSON

In patient 10, the technique confirmed the + ++ Cookson et al. (1964), who successfully used routine findings of the FA test and gave a very strong microscope equipment plus a few inexpensive filters positive result. This patient dissented strongly with for performing a modified threshold FA test. the implication of these findings which were in The BFCF test has been tried with the same and contradiction to his history and repeated negative similar equipment and has given very satisfactory investigations for bilharziasis, but a strong positive results. Indeed the two fluorescent antibody tests skin test result in his case also confirmed the above using bentonite absorbates described here have been findings. particularly suited to this equipment. A further important consideration arising from With the modified set-up, the FITC bentonite this technique and the limited findings tabulated antibody particles fluoresce brilliantly. This is be- above is the relation of antibody level to active cause of the high fluorescent index of the fluorescein disease. conjugate, which is readily activated by the low No dogmatic conclusions can be drawn without spectral energy of the tungsten filament lamp applying the BFCF test to a considerable series of employed. The RB 200 albumin-coated or conjug- cases of bilharziasis. These would have to include ated cercarial antigen is also adequately stimulated a significant cross-section of the affected population, to emit its own contrasting characteristic fluorescence. and there would have to be at least an assessment The basis of the modified equipment is worth of the active disease in relation to age and of the recapitulating here because it brings the above treated disease in relation to a time factor. Other techniques within the reach of almost any considerations within these broad lines would be laboratory. the nature of the infecting parasite and the parasite Any standard modern monocular laboratory load involved. microscope should suffice. This should be used Notwithstanding these points, it does appear that with Kohler illumination with a 35-watt, high- some generalizations can be made on the above intensity, low-voltage tungsten filament lamp, findings; these are that in any positive case the overrun. circulating antibody level is above 1: 20 and that it A normal substage condenser NA 1.40, with a probably lies between this level and 1: 80 for the blue UV substage pass filter, BG 12/3 mm is em- average, and very chronic light or long-treated ployed with glycerol contact. A x 10 objective infection. For active infections the antibody level is with incorporated UV exclusion filter, GG 9/1 mm probably in excess of 1: 80. and a x 12 1/2 eyepiece with a blue exclusion The measurement of antibody levels in relation secondary filter GG 9/1 mm+OG 1/1.5 mm is used to the disease state in bilharziasis is of great potential to exclude all but the secondary fluorescent image value and it would seem that the BFCF test does this of the microscopic tests. in a satisfactory and sensitive way. It is easy to All the above filters have Schott and Genossen perform and gives consistent and reproducible numbers. results. With this equipment positive tests showed orange One obstacle to its general use in the majority of to orange-red cercariae with brilliant yellow adherent laboratories might be the false assumption that bentonite antibody particles, against a near-black fluorescent techniques in bilharziasis using cercarial background, while negatives showed the orange antigens require specialized equipment. cercariae alone with no adherence of bentonite That this is not the case has been shown by absorbed antibody.

ACKNOWLEDGEMENTS

I have to thank the Federal Secretary for Health, Public Health Laboratory, Salisbury, and of the Bilhar- Dr D. M. Blair, and the Director of Medical Services, ziasis Research Laboratory and my secretary, Mrs E. Southern Rhodesia, Dr M. Webster, for permission to Griffiths, for their help. I am grateful to Mr W. I. Creser publish this paper, and the various technical staff of for his expert help with the colour reproductions. MODIFICATIONS OF FLUORESCENT ANTIBODY TEST IN BILHARZIASIS 813

RtSUMt

Depuis quelques annees, des epreuves de diagnostic desavantages, en particulier l'instabilit6 du pouvoir de la bilharziose a Schistosoma mansoni par la methode fluorescent lorsque les 6chantillons sont conserves au de fluorescence ont et6 mises en point et sont entrees refrigerateur, et le risque de reactions faussement posi- dans la routine des laboratoires specialises. Cette tech- tives avec des s6rums animaux contenant des anticorps nique est basee sur la double coloration fluorescente de provoquant des reactions croisees avec la bilharziose. l'antigene d'une part et de l'anticorps d'autre part. L'auteur decrit les experiences par lesquelles on a cherche Dans la technique decrite par Sadun en 1962, les cer- ai eviter ces inconvenients, en conjuguant directement les caires fixees par le formol sont enrobees dans de l'albu- cercaires avec la substance fluorescente. On a obtenu mine d'origine bovine, ovine, ou de lapin, coloree A la d'autre part un accroissement de la brillance des particules rhodamine. Apres contact avec la globuline serique fluorescentes qui a permis de generaliser 1'emploi du traitee par l'isothiocyanate de fluoresceine, la fluorescence test. orangee originelle de l'antigene reste inchangee, si le L'auteur decrit ces diverses modifications et leurs test est negatif; elle vire au jaune verdatre s'il est positif. resultats et propose en outre une modification d'une Bien que couramment employee dans les laboratoires importance particu1iRre pour un test de fixation du de diagnostic de la bilharziose, cette methode a quelques complement, en fluorescence.

REFERENCES

Chadwick, C. S., McEntegart, M. G., & Nairn, R. C., Nairn, R. C., ed. (1962) Fluorescent protein tracing, (1958) Lancet, 1, 412-414 Edinburgh & London, Livingstone Cookson, L. 0. C. (1963) Cent. Afr. J. Med., 9, 429-434, Riggs, J. L., Seiwald, R. J.. Burckhalter, J. H., Downs, 469-478 C. M. & Metcalf, T. G. (1958) Amer. J. Path., 34, Cookson, L. 0. C., Clarke, V. de V. & Pirie, E. (1964) 1081-1097 Cent. Afr. J. Med., 10, 12-16 Sadun, E. H., Anderson, R. I. & Williams, J. D. (1960) Coombs, R. R. A., Coombs, A. M. & Ingram, D. G. Proc. Soc. exp. Biol. (N. Y.), 105, 289-291 (1961) The serology ofconglutination, Oxford, Blackwell Sadun, E. H. Anderson, R. I. & Williams, J. E. (1962) Eliakim, M. & Davies, M. (1954) Parasitology, 44,407-13 Bull. Wld Hlth Org., 27, 151-159 Eliakim, M. & Davies, M. (1955) Parasitology, 45, Smith, C. W., Marshall, J. D. & Eveland, W. C. (1959) 189-194 Proc. Soc. exp. Biol. (N.Y.), 102, 179-181 Goldwasser, R. A. & Shepard, C. C. (1958) J. Immunol., Tyrell, D. A. J. & Hers, J. F. P. (1960) Fluorescent anti- 80, 122-131 body technique. In: Hers, J. F. P., ed., Proceedings. Manson-Bahr, P. H. (1954) Manson's tropical diseases, Boerhaave Cursus, Leiden, Part II, pp. 76, 81, appen- 14th ed., London, Cassell, pp. 122, 185, 203 dix 18