Further studies on an improved haemagglutination inhibition test with higher sensitivity for rabies virus antibody

Bulletin of the World Health Organization, 64 (1): 133-137 (1986) © World Health Organization 1986 Further studies on an improved haemagglutination inhibition test with higher sensitivity for rabies virus antibody K. MIFUNE,' K. MANNEN,' N. MINAMOTO,2 & Y. T. ARAI3 The efficiency of the removal of non-specific inhibitors of rabies virus haemag- glutinin by treatment with colloidal silicic acid, which was proposed in an earlier study, was examined in a number of test samples. The non-specific inhibitors were removed in 289 out of296 normal human sera (97.61%) by this treatment to a level that was undetect- able at the 1:4 starting dilution in the haemagglutination inhibition test. Antigenic differ- ences among three strains of rabies virus were detected in the haemagglutination inhibition test and the antibody titres to the homologous antigens were apparently higher than those to heterologous antigens. In a previous study (1), we demonstrated a method for increasing the sensitivity of the haemag- glutination inhibition (HI) test for rabies virus anti- body in two ways. First we developed a technique for the removal of non-specific inhibitors of rabies virus haemagglutinin by treatment of the sera with colloidal silicic acid (Aerosil) or with acetone plus Aerosil. This reduced the non-specific inhibitors to a level that was undetectable at the 1:4 starting dilution in the HI test. Secondly, using bromelain-treated goose erythrocytes, which are much more susceptible to haemagglutination by rabies virus than the un- treated erythrocytes, we obtained considerably in- creased HI antibody titres. The use of both these techniques increased the sensitivity of the test and led to the detection of low levels of antibodies in sera from immunized human subjects. The following aspects relevant to the practical use of this new HI procedure were examined in the present study: how efficient is the Aerosil treatment of the serum in removing the non-specific inhibitors of rabies virus in a number of samples, and can antigenic differences among the virus strains be detected in the test and what are their effects on the serum HI antibody titres? It has been shown that ' Department of Microbiology, Medical College of Oita, Hazama-cho, Oita 87956. Japan. Requests for reprints should be addressed to Professor K. Mifune. 2 Department of Public Health, Faculty of Agriculture, Gifu University, Gifu, Japan. 3 Department of Virology and Rickettsiology, National Institute of Health, Shinagawaku, Tokyo, Japan. detailed antigenic differences can be determined by a panel of monoclonal antibodies (2)!' METHODS The high egg passage (HEP) Flury strain, the Challenge Virus Standard (CVS) strain, and the Nishigahara (RCEH)b strain of rabies virus were cultured in chick-embryo fibroblast cells in medium 199 without serum. The culture fluid, which was harvested after 7 days of incubation at 36 °C and in- activated by 3-propiolactone after removing cell debris by low-speed centrifugation, was used as the HA antigen. In some experiments, this antigen was concentrated 100-fold by ultracentrifugation and purified by CsCl (caesium chloride) density gradient centrifugation. For each antigen, three guinea pigs were im- munized with five weekly subcutaneous inoculations of the purified antigen. Before each inoculation, an aliquot of blood was taken from the animal by car- a Report of the German Green Cross/WHO Workshop on Monoclonal Antibody in Rabies Diagnosis and Research. Un- published WHO document WHO/Rab.Res./84.20 (1984). b The Nishigahara strain was purportedly derived from the original Pasteur isolate by approximately 90 passages in guinea pigs and rabbits. However, there is no accurate documentation of the passage history. The virus was named Nishigahara strain after the place in 1918 where the veterinary disease institute was located. The RCEH variant was obtained by an additional 296 passages of the Nishigahara strain in chick-embryo cells and is currently a standard strain of rabies vaccine for animal use in Japan. 4647 -133- K. MIFUNE ET AL. diac puncture for assessment of antibody production. Antibodies were determined by the HI test and neutralization test (NT). The HI test was carried out in the usual manner in microtitration plates, as described previously (1). For the HI test, the sera were diluted 1:2 with borate saline (pH 9.0) and treated with an equal volume of 50 g/litre of Aerosil in borate saline for 30 min at room temperature. Human sera were treated with acetone, as described elsewhere (3), before the Aerosil treatment. The virus neutralization test was carried out essentially as described previously by one of us (4). Briefly, the serially diluted sera were mixed with 200 fluorescent focus-forming units (FFU) of virus and incubated for 1 hour at 37 'C. Aliquots of the mixture were inoculated onto chick-embryo fibroblast cells in a 96- multiwell tissue culture plate in triplicate and incubated for 5 days with medium 199 supplemented with 1 g/litre of bovine serum albumin. The mono- layers were fixed with 30%o cold acetone and stained with anti-CVS mouse ascitic fluid and peroxidase- conjugated antimouse IgG goat serum according to the method of Graham & Karnovsky (5). Fifty percent neutralizing titres of the serum were cal- culated by the formula of Reed & Muench (6). RESULTS AND DISCUSSION Table 1 shows the results of the efficiency of the removal of non-specific inhibitors to rabies virus in normal human sera by treatment with acetone plus Aerosil. In 289 out of 296 sera (97.6%), non-specific inhibitors were undetectable at the 1:2 starting dilution of the HI test and low levels (1:8 to 16) of Table 1. The efficiency of the removal of non-specific inhibitors to rabies virus in human sera by treatment with acetone and Aerosil Sera showing Sera with complete removal inhibitors of inhibitors remaining Serum dilution No. % No. % Titre 1:2 289 97.6 7 2.3 8-16 1:4' 3 1.0 4 1.3 8-16 a The seven sera with low levels of remaining inhibitors were freshly diluted 1:4 and treated. inhibitors remained in 7 sera. The inhibitors in 3 of these 7 sera became undetectable when the sera were treated at the dilution of 1:4, but the inhibitors in the remaining 4 sera were not removed in spite of various attempts. Table 2 shows the HI antibody production against each strain of rabies virus in guinea pigs and their cross-reactivity to different antigens at 2 weeks and 6 weeks after immunization. The results of the cross neutralization test are also shown as controls. The HI antibody titres to homologous antigens were apparently higher than those to heterologous anti- gens and the antigenic differences were readily detected by the HI test. The cross HI titres correlate very well with their respective cross NT titres. To examine whether these cross-reactivities of antisera are present in the IgM antibody or the IgG antibody, the antisera were fractionated by adsorp- tion with the Cowan 1 strain of Staphylococcus aureus or by sucrose density gradient centrifugation. Table 2. Cross HI and NT titres of antisera at 2 weeks and 6 weeks after immunization of guinea pigs Antiserum to RCEH Antiserum to CVS Antiserum to HEP Test and virus straina 2 weeksb 6 weeks 2 weeks 6 weeks 2 weeks 6 weeks Haemagglutination inhibition: RCEH 64 128 32 128 16 64 CVS 16 64 64 512 64 128 HEP 16 128 32 128 128 256 Neutralization test: RCEH 503 9051 200 7879 50 985 CVS 200 2599 400 15759 50 900 HEP 50 450 141 4525 1396 9051 0 See text for designation of virus strains. b The serum was bled at 2 weeks after the first immunization doses. 134 HAEMAGGLUTINATION INHIBITION TEST FOR RABIES VIRUS ANTIBODY RCEH 0 0 5 10 20 cvs 0 5 10 20 HEP 0 5 10 20 Incubation time (min) Fig. 1. Kinetic curves of neutralization: 1 06FFU of each strain of rabies virus was mixed with 50 neutralizing doses (50%) of each guinea pig antiserum and incubated at 37 OC. At intervals, an aliquot of the mixture was taken and diluted immediately 1 :10 to 1 :100 with Eagle's minimum essential medium and the surviving virus was measured in chick-embryo fibroblast cells in 96-multiwell microtitration plates. (Bar on the name of the virus strain means the antiserum to that strain.) Table 3 demonstrates the cross-reactivity of the sera adsorbed with S. aureus. Cross-reactivities still re- main after adsorption in parallel with those of the unadsorbed sera. IgM antibody was no longer detected in the sera bled at 6 weeks. Similar results were obtained in the IgM and IgG fractions after sucrose gradient centrifugation (data not shown), indicating that cross-reactivities exist in both the IgM and IgG antibodies. In the next experiment, the kinetics of the neutralization test were examined with 106 FFU of the virus and 50 neutralizing doses (50%) of each anti- serum to discover to what extent antigenic differences were present among these strains. Fig. 1 shows that each strain of the virus was neutralized fastest with the homologous antiserum among the antisera and that the HEP strain appears to be antigenically more distinct from the RCEH strain of the virus than the CVS strain. Another experiment was carried out to examine how these antigenic differences were reflected in the HI antibody titres in human sera after vaccination with rabies vaccine. Forty-eight human sera with various histories of vaccination with the HEP strain of rabies virus were used. In Fig. 2, the ratios (X) of the HEP titre to the RCEH titre were plotted against the ratios (Y) of the HEP titre to the CVS titre. Most of the spots were below the diagonal of Y = X, which means that the HI antibody titres to the homologous HEP strain were highest, then the CVS titres, and lowest the RCEH titres. These results are compatible with the pattern of cross-reactivity of guinea pig antisera and with that of the kinetic curves of virus neutralization. The present study has demonstrated that treatment of human sera with acetone and Aerosil can remove the non-specific inhibitors with high efficiency (97.6%) at the lowest dilution (1:2) of the serum, that 0 2 3' 4 5 135 K. MIFUNE ET AL. Table 3. HI antibody titres of antisera before and after adsorption with Staphylococcus aureus (Cowan 1) HA antigens Strain of antiserum, and treatment HEP RCEH CVS HEP (2 weeks)' None 128 16 64 Cowan 16 <4 8 HEP (6 weeks) None 256 64 128 Cowan <4 <4 <4 RCEH (2 weeks) None 16 64 16 Cowan 8 32 16 RCEH (6 weeks) None 128 128 64 Cowan <4 <4 <4 CVS (6 weeks) None 32 32 64 Cowan <4 <4 4 a The serum was bled at 2 weeks after the first immunization doses. an apparently higher HI antibody titre can be ob- tained to the homologous antigen than to hetero- logous antigens, and that antigenic differences among the strains of rabies virus can be detected by yl-- ii- - 64 32 16 U a. uiw 4 2 I a . . 0 * 0@0 * .0 * *@@ @00.00 0 0 *-: 000Ina* ** 1 2 *0* __ . . 4 8 16 HEP titre/RCEH titre 32 1 64 1X Fig. 2. HI antibody titres against the HEP, CVS and RCEH strains of rabies virus in sera from 48 human subjects vaccinated with HEP strain rabies vaccine. The ratios (X) of the HEP titre to the RCEH titre were plotted against the ratios (Y) of the HEP titre to the CVS titre. Each spot refers to one individual. the HI test. These results have practical implications in the use of new procedures in seroepidemiological studies of rabies by the HI test. ACKNOWLEDGEMENTS This research was supported in part by a grant from Yakult Honsha Co. Ltd, Tokyo, Japan, and a grant from the Japan- United States Cooperative Medical Science Program. RItSUMI TRAVAUX COMPLEMENTAIRES SUR UNE EPREUVE D'INHIBITION DE L'HEMAGGLUTINATION DE SENSIBILITE AMtLIOREE POUR L'ETUDE DES ANTICORPS ANTIRABIQUES Un certain nombre d'6chantillons ont e etudies en vue d'evaluer l'efficacite d'une nouvelle methode pour l'6limi- nation des inhibiteurs non specifiques presents dans les serums humains par traitement au moyen d'acide silicique colloidal et d'ac6tone, methode qui avait ete expos&e dans un pr6cedent article. Le traitement a permis d'6liminer ces inhibiteurs sur 289 des 296 serums humains normaux (97.6%) et de les ramener a un niveau ind6celable a la dilution initiale de 1:4 utilisee dans l'epreuve d'inhibition de l'hemagglutination. Sur trois souches de virus rabique, on a etudie la possibilite de mettre en evidence des differences antigeniques par l'epreuve IH en cherchant a voir si ces differences se manifestaient dans le titre d'anticorps. L'epreuve d'inhibition crois&e sur serum de cobaye a permis de mettre en evidence des differences antigeniques analogues a celles que r6v6lait l'epreuve de neutralisation crois&e. Dans le cas des antigenes homologues, la cin6tique de neutralisation etait visiblement plus rapide que dans le cas des antigenes h6terologues et les titres d'anticorps plus eleves. Une reactivite crois&e du meme genre a e mise en evidence sur des serums provenant de 48 sujets humains ayant subi la vaccination antirabique. _ . _ _ _ T I 136 Yi HAEMAGGLUTINATION INHIBITION TEST FOR RABIES VIRUS ANTIBODY 137 REFERENCES 1. MANNEN, K. ET AL. Methods of increasing the sensitivity of the haemagglutination inhibition test for rabies virus antibody. Bulletin of the World Health Organization, 62: 883-892 (1984). 2 WIKTOR, T. J. & KoPROWSKI, H. Monoclonal anti- bodies against rabies virus produced by somatic cell hybridization. Detection of antigenic variants. Pro- ceedings of the National Academy of Sciences, USA, 75: 3938-3942 (1978). 3. CLARKE, D. H. &. CASALS, J. Techniques for hemag- glutination inhibition with arthropod-borne viruses. American journal of tropical medicine and hygiene, 7: 561-573 (1958). 4. MINAMOTO, N. ET AL. A microassay for the quantitation of rabies virus by an immunoperoxidase technique. Research bulletin of the Faculty of Agriculture, Gifu University, 45: 187-196 (1981). 5. GRAHAM, R. C. JR, & KARNOVSKY, M. J. The early stages of adsorption of injected horseradish peroxidase in the proximal tubules of mouse kidney; ultra- structural cytochemistry by a new technique. Journal oj histochemical cytochemistry, 14: 291-302 (1966). 6. REED, L. J. & MUENCH, H. A simple method of estimating fifty percent end-points. American journal of hygiene, 27: 493497 (1938).