Journal of Clinical Virology 27 (2003) 129 /135 www.elsevier.

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Development and evaluation of a latex agglutination test for rabies antibodies

S.N. Madhusudana *, S. Saraswati
Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), PB No. 2900, Hosur Road, Bangalore 560029, India Accepted 6 August 2002

Abstract Background: The presently recommended tests for estimation of rabies neutralising antibodies like Rapid Fluorescent Focus Inhibition test (RFFIT) and Mouse Neutralisation test (MNT) are laborious, time consuming and not costeffective for routine use. Simple, rapid and economical tests need to be developed for routine use. Objectives: The main objective of the present study was to develop and evaluate a rapid Latex Agglutination Test (LAT) to detect rabies specific antibodies. Methods: Latex beads were coated with purified rabies glycoprotein at a concentration of 1 mg/ml followed by coating with 0.3% bovine serum albumin (BSA). These sensitised beads were used to detect antiglycoprotein antibodies in sera of 152 people who had taken a course of post exposure rabies vaccination with different cell culture vaccines and whose antibody titers were pre determined by MNT. Sera from 52 normal healthy people without any detectable levels of rabies antibodies were included as controls. The test was carried out on glass slides by mixing 20 ml of sensitised beads and 20 ml serum. Results: Preliminary evaluation with rabbit serum of known potency indicated that for clear agglutination of sensitised beads, a minimum of 2 IU/ml of rabies antibody should be present in the serum samples. Visible agglutination was noticed in positive sera with a titer E/2 IU/ml within 3 /5 min after mixing. Seven Sera whose MNT titers were less than 2 IU/ml did not show agglutination. None of the negative control sera showed agglutination. Thus the specificity of the test was 100% and sensitivity was 95.4%. Conclusions: The LAT described here detects rabies specific antibodies E/2 IU/ml and can be used to screen large number of sera from vaccinated people to know the protective status after vaccination. This simple and rapid test may be used routinely in antirabies treatment centres to monitor sero conversion in vaccinated people. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Rabies; Rabies antibodies; Latex agglutination

1. Introduction Rabies is a major public health problem in India and other developing countries. As per the latest World Health Organisation (WHO) estimate, it accounts for about 50 000 human mortality world-

* Corresponding author. Tel.: '/91-80-6995128/129; fax: '/ 91-80-656-4830/2121 E-mail address: mshampur@hotmail.com (S.N. Madhusudana).

1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 3 5 - X

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wide annually with India alone reporting 30 000 human deaths (World Health Organisation, 2002a). Other Asian countries like Thailand, Philippines and Sri Lanka together report around 1000 deaths in a year (World Health Organisation, 2002b) The number of post exposure treatments (PEP) in these countries is also on the rise. In India alone nearly 1.5 /2 million people take PEP out of which nearly 60% are opting for modern cell culture vaccines and remaining 40% are administered with nerve tissue derived Sample vaccine in government hospitals and dispensaries (Ichhpujani et al., 2001). The disease can be effectively prevented if approved prophylactic measures are instituted and post exposure vaccination with potent cell culture vaccine is administered without delay. Three types of cell culture vaccines are currently in use in India and other Asian countries i.e. human diploid cell vaccine (HDCV), purified chick embryo cell vaccine (PCEC, Rabipur) and purified vero cell rabies vaccine (PVRV, Verorab, Verovax). All these vaccines prevent the development of the disease by inducing rabies virus neutralising antibodies (Nicholson, 1996). If the disease is to be effectively prevented, protective levels of antibodies are to be generated as early as possible after the institution of PEP because in many cases of exposures the incubation period of the disease tends to be very short. The antibody response to these vaccines may vary from person to person with occasional non-responders. The levels of antibodies may be less than adequate in malnourished children, immunocompromised people, people taking steroids and antimalarials (World Health Organisation, 1992). Therefore, it is necessary to monitor the level of protection induced by PEP so that additional booster doses may be advised if necessary. As of today only two tests are recommended by WHO (1996) for estimation of rabies antibodies i.e.: Rapid Fluorescent Focus Inhibition test (RFFIT) and Mouse Neutralisation test (MNT). These tests are generally performed in one or two centres in India, are quite expensive and hence majority of people who undergo PEP are not routinely monitored for seroconversion. Recently, a modification of RFFIT called fluorescent antibody virus neutralisation test (FAVN) has been developed which is com-

paratively simpler and more economical (Cliquet et al., 1998; Briggs et al., 1998) Other tests that have been developed to detect rabies specific antibodies include an ELISA based on purified glycoprotein (Perrin et al., 1986) counter immunoelectrophoresis (Diaz and Myer, 1980) and dot blot immuno assays (Herberling and Kalter, 1986). A more specific competitive ELISA for detection of neutralising antibodies has been reported by Elmgren and Wandeler (1996). In India commercially available ELISA kits are to be imported and are very expensive for routine use. Moreover, facilities like ELISA readers may not be available in many centres. Antibody detection based on latex agglutination is an established procedure and commercial kits are available for diagnosis of many bacterial and viral diseases. In a previous study, Perrin et al. (1988) reported the utility of a latex agglutination test (LAT) for the detection of rabies antibodies, using latex particles coated with purified whole virus. Among the various proteins present in rabies virus, it is the glycoprotein (G protein), which induces the formation of neutralising antibodies that offers protection against the disease (Dietzschold et al., 1987). It may be advantageous to detect only antiglycoprotein antibodies following rabies vaccination as it correlates with the extent of virus neutralisation and the degree of protection offered by the vaccines. Keeping this in view, we have improvised the LAT developed by Perrin et al. by coating latex particles with purified rabies glycoprotein. We report here our results obtained with serum of patients vaccinated with two different rabies vaccines.

2. Materials and methods 2.1. Virus propagation and purication Vero cell adapted challenge virus strain (CVS 11 obtained from Central Research Institute, Kasauli, India) was grown in vero cells (ATCC CCL 81, obtained from National Centre for cell sciences, Pune, India) by using established procedures (Wunner, 1985). Viral harvests were collected every third day and pooled. The pooled harvest

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was titrated in mice (Koprowsky, 1996) and the infectivity titer was found to be 106.5 LD50/ml. The pooled viral harvest was concentrated by pelleting at 50 000 g in a Sorvall (Discovery, 100 S, model, made in USA) ultracentrifuge. The concentrated virus was purified by Sucrose gradient (15 /50%, Sigma Chemicals, USA) centrifugation at 100 000 g using the same ultra centrifuge. The band of virus collected was dialysed against PBS for 24 h and stored at (/75 8C.

2.3. Coating of latex beads with glycoprotein Polystyrene latex beads were first coated with rabies glycoprotein followed by bovine serum albumin (BSA). One ml of 10% latex beads (0.8 microns in diameter, Sigma Chemicals, USA Cat No.LB-8) was washed three times in 5 ml of carbonate buffer, pH 9.6. They were then centrifuged at 2500 )/g for 30 min. The sediment was resuspended in 3 ml carbonate buffer and 1.75 ml of glycoprotein and incubated for 3 h at 37 8C and then overnight at 4 8C. The sensitised beads were centrifuged and re suspended in 5 ml carbonate buffer containing 5% sucrose and 0.3% BSA. The beads were then incubated for 30 min at 37 8C, centrifuged and washed twice in PBS. They were then re suspended in 3 ml PBS. 2.4. Preliminary evaluation of latex agglutination with rabies immunoglobulin We did a preliminary evaluation of the test with our in house reference rabies immune globulin (RIG) prepared from rabbits and calibrated against the national reference standard (obtained from Central Research Institute, Kasauli) with a potency of 80 IU/ml. The in house reference RIG had a rabies neutralising antibody titer of 32 IU/ml as tested in MNT. The RIG was diluted to different potencies ranging from 0.5 to 16 IU/ml. Twenty micro litres of each of the dilutions and 20 ml of coated latex particles were mixed on a clean slide and gently rotated for 5 min. The lowest and highest dilution showing clear agglutination was noted. Pre immune serum from the same rabbit was used as negative control. 2.5. Serum samples The main aim of this study was to determine the sensitivity and specificity of the LAT test using defined serum samples obtained from people who had a course of PEP with cell culture vaccines. Therefore, Serum samples from 152 vaccinated people and whose antibody titers were pre-determined by MNT were included in this study. These patients attended antirabies clinics of the National Institute of Mental Health and Neurosciences

2.2. Purication of rabies glycoprotein Rabies virus glycoprotein was purified by affinity column chromatography using concanavalinA CL agarose column as described earlier (Perez et al., 1996). Briefly 0.5 ml of purified virus was incubated with an equal volume 4% octyl /beta-Dglucopyranoside (OGP, Sigma Chemicals, USA cat No. 0-9882) in PBS at room temperature for 1 h in order to solubalise virus glycoprotein. The mixture was then layered on a 4 ml packed Con-A CL agarose column (Bangalore Genei, Bangalore, India Cat. No. LIA 9-S) pre-equilibrated with 250 mM methyl-D-mannopyranoside (Sigma, cat No. M-6882) in PBS and 250 mM methyl-D-glucopyranoside (Sigma Chemicals, USA cat No. M-9376) in PBS. After loading with virus material, the column was washed with PBS and the bound protein was eluted with PBS containing 1 M methyl-D-mannopyranoside and 1 M methyl-Dglucopyranoside. Ten 1 ml fractions were collected at flow rate of 0.1 ml/min. The absorbance of these fractions was read at 280 nm. The fractions with high protein content were pooled and dialysed. The protein content of the pooled fractions was 0.48 mg/ml as determined by Lowrys method. The purity of the glycoprotein was confirmed by SDSPAGE when a single band of 65 kDa was observed. Further, identity of this rabies glycoprotein was established by Western Blot using a anti-glycoprotein poly clonal antibody (obtained from Rabies Unit, Institut Pasteur, Paris). The glycoprotein was aliquoted and stored at (/75 8C

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(NIMHANS), Bangalore, India or Kempegowda Institute of Medical Sciences, Bangalore, India. Out of these, 85 samples were from people who had taken five intramuscular (IM) doses of PCEC vaccine (Rabipur, Chiron vaccines, India) and 57 samples from people who had 5 IM doses of PVRV (Verorab, Cadila Pharmaceuticals, India) Fifty-two serum samples from normal healthy people without history of animal bite or rabies vaccination in the past were included as controls. To further assess the specificity of the test, we also tested 25 serum samples known to be positive in high dilutions for antibodies to Japanese encephalitis (n 0/12), Measles (n 0/5) and Herpes simplex I (n 0/8). 2.6. Mouse neutralisation test (MNT) The samples had been tested for rabies neutralising antibodies by performing standard MNT as advocated by WHO (Atanasiu, 1973) Briefly, doubling dilutions of serum samples were incubated with equal volumes of 100 LD 50 of CVS for 60 min in a water bath at 37 8C and inoculated intracerebrally in to groups of mice (Swiss Albino, 4 weeks old) and kept under observation for 14 days. The antibody titer of the serum was calculated based on survival/mortality in each dilution. The titers were expressed in international units in comparison to in the house standard of RIG calibrated against the national standard with a potency of 80 IU/ml (obtained from Central Research Institute, Kasauli, India). For conduction of MNT, prior permission had been obtained from the Institution Animal Ethics committee and the animals were housed, maintained and experimented according to the institutional regulations in force. 2.7. Latex agglutination test (LAT) This was carried our on clean and grease free microscopic slides. To prevent drying while taking the reading at room temperature, the slides were kept in a moist chamber. To 20 ml of neat serum 20 ml of sensitised latex beads (5% final concentration) were added and gently agitated for 3 /5 min.

3. Results The agglutination seen with positive serum samples was clearly visible to the naked eye in comparison to the homogenous suspension with negative controls. In the preliminary evaluation, done with in the house preparation of rabbit RIG, we observed that no agglutination was observed in RIG dilutions having a titer less than 2 IU/ml. Clear agglutination appeared with in 5 min in lower dilutions containing increasing amounts of rabies antibodies. (Table 1). The agglutination could also be graded from '/'/ to '/'/'/'/'/ depending on size of agglutinated particles and the rapidity with which they appeared. In the grade '/'/ small clumps barely visible to naked eyes appeared after a delay of 3 /5 min whereas in grade '/'/'/'/ to '/'/'/'/'/ large and discrete clumps appeared within 2 min. The grade '/'/'/ '/ or '/'/'/'/'/ corresponded to titers equivalent or greater than 8 IU/ml and grades '/'/ to '/'/'/ corresponded to titers greater than 2 IU/ml (Table 1). None of the dilutions of pre immune sera showed agglutination, confirming the specificity of agglutination. Out of 152 samples positive by MNT, 145 samples were also positive by LAT. However seven Serum samples with MNT titers less than 2 IU/ml by MNT were found negative by LAT. Therefore, the overall sensitivity of this test is 95.4% None of the control samples negative by MNT were found positive by LAT, and agglutination was also not observed with any of serum
Table 1 Results of Preliminary evaluation of latex agglutination with rabbit RIG Dilution of RIG Potency (IU) Grades of agglutination observed '/'/'/'/'/ '/'/'/'/ '/'/'/ '/'/ 0 0 0

1:2 16 1:4 8 1:8 4 1:16 2 1:32 1 1:64 0.5 Pre immune serum RIG, rabies immunoglobulin.

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samples positive for JE, Measles or herpes simplex I. Therefore, the specificity of this test is 100%. As with the reference RIG, the extent of agglutination observed with clinical samples could also be graded with samples having a titer /8 IU/ml showing 4'/ agglutination (Table 2).

4. Discussion Demonstration of adequate sero-conversion following rabies vaccination is necessary to instil confidence to the people exposed to a fatal disease for which there is no specific antiviral therapy. Some of the presently available tests like MNT and RFFIT are time consuming and not cost effective for routine use. Use of antigen coupled latex particles for detecting specific antibodies is a well-established procedure in clinical microbiology. The utility of this procedure has been successfully demonstrated for detection of several viral antibodies like varicella zoster (Steinberg and Gershon, 1991) Rubella (Duverlie et al., 1990) and Cytomegalovirus (Adler et al., 1985). As mentioned earlier antibodies to rabies glycoprotein play an important role in offering protection. In this study we have purified the G protein by a simple affinity chromatography, confirmed its purity and then used it to coat the latex beads so that only anti glycoprotein antibodies are detected. This has the additional advantage of increasing the specificity of the test. As reported by Perrin et al earlier, the LAT was positive with serum samples having a titer greater
Table 2 Comparative results of antibody titers obtained with MNT and degree of agglutination observed in LAT MNT titers (range in IU/ml) (n 0/56) (n 0/64) (n 0/12) (n 0/13) 0.5 /1.5 (n 0/7) Negative controls (n 0/52) /10 IU 7 /10 IU 4 /6 IU 2 /3 Latex agglutination '/'/'/'/'/ '/'/'/'/ '/'/'/ '/'/ 0 0

Figures in parenthesis indicate number of serum samples; MNT, mouse neutralisation test; LAT, latex agglutination test.

than 2.5 IU/ml. But in this study we found that all serum samples including the reference RIG (as in the preliminary evaluation) having a MNT titer of 2 IU/ml and above showed clear agglutination within 3 /5 min. Extending the time beyond 5 min did not increase the sensitivity and resulted in drying. As the test detects only antibodies to glycoprotein, which is the major protein of rabies virus capable of inducing neutralising and protective antibodies, the results of this test are valuable in predicting the level of protection offered by the vaccines. Presently WHO advocates a minimum level of 0.5 IU/ml of neutralising antibody titer as determined by RFFIT or MNT as suggestive of protection. However, there is no such criteria for values obtained with in vitro tests such as ELISA or LAT. In fact, in our laboratory the antibody titers obtained in ELISA based on purified virus were consistently higher than the titers obtained with MNT. If the results of LAT are directly correlated with MNT or RFFIT it is possible that serum samples having a titer less than 2 IU/ml may be read as false negative. However, such a situation is unlikely to occur, provided the patients have taken one of the modern cell culture vaccine like PCEC or PVRV which are known to produce neutralising antibody titers much above 2 IU/ml both by regular intramuscualr (IM) regimen and abbreviated intradermal (ID) regimens. For instance, in our laboratory we generally see a mean titer of 18.5 IU/ml in patients vaccinated with a full IM course of PCEC vaccine and 16.4 IU/ml with PVRV vaccine. The mean titer with ID regimens is also well above 2 IU/ml. The LAT reported here is 100% specific for detecting antiglycoprotein antibodies though it was not as sensitive as MNT in detecting low level of antibodies. However, there is 100% agreement between the two tests with the samples having a titer equal to or greater than 2 IU/ml. Keeping this value as cut off, the LAT described here is comparable in sensitivity to LATs developed for rubella virus (99% sensitivity), Cytomegalovirus (96.6% sensitivity) and varicella zoster (98% sensitivity). Thus, the test may serve to screen large number sera of vaccinated individuals for the presence of rabies specific antibodies and only

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S.N. Madhusudana, S. Saraswati / Journal of Clinical Virology 27 (2003) 129 /135 Briggs DJ, Smith JS, Mueller FC, et al. A comparison of two serological methods for determining immune response after rabies vaccination in dogs and cats being exported to rabies free areas. Biologicals 1998;26:347 /55. Cliquet F, Auber M, Sague L, et al. Development of a Fluorescent antibody virus neutralisation test (FAVN) for the quantitation of rabies neutralising antibodies. J Immunol Methods 1998;212:79 /87. Diaz AM, Myer DM. Determination of serum neutralising antibodies to rabies virus by modied counterimmunoelectrophoresis(CIEP) test. J Clin Microbiol 1980;12:175 /9. Dietzschold B, Tollis M, Lafon M, Wunner WH, Koprowsky H. Mechanism of rabies virus neutralisation by glycoprotein specic monoclonal antibodies. Virology 1987;161(1):29 / 36. Duverlie G, Roussle C, Driencourt M, Orla J. Latex enzyme immunoassay for measuring antibodies to rubella virus. J Clin Pathol 1990;43:766 /7. Elmgren LD, Wandeler AI. Competative ELISA for the detection of rabies neutralising antibodies. In: Meslin F-X, Kaplan MM, Koprowsky H, editors. Laboratory Techniques in Rabies, 4th ed.. Geneva: WHO, 1996:200 /8. Herberling RL, Kalter SS. Rapid dot-immunobinding assay on nitrocellulose for viral Antibodies. J Clin Microbiol 1986;23:109 /13. Ichhpujani RL, Bharadwaj M, Chhabra M, Dutta KK. Rabies In India. In: Proceedings of the Fourth International symposium on rabies control in Asia, 5 /9 March 2001. Merieux Foundation and World Health Organisation, 2001. Koprowsky H. The mouse inoculation test. In: Meslin F-X, Kaplan MM, Koprowsky H, editors. Laboratory Techniques in Rabies, 4th ed.. Geneva: WHO, 1996:80 /7. Nicholson KG. Cell culture vaccines for human use: general considerations. In: Meslin F-X, Kaplan MM, Koprowsky H, editors. Laboratory Techniques in Rabies, 4th ed.. Geneva: WHO, 1996:271 /9. Perez L, Estepa A, Coll JM. Purication of the glycoprotein G from viral hemmorrhagic septecemia virus, a sh rhabdovirus by lectin afnity chromatography. J Virol Methods 1996;76:1 /8. Perrin P, Versmisse P, Delagneau JF, Lucas G, Rollin P, Sureau P. The inuence of the immunosorbent on rabies antibody EIA: advantages of puried glycoprotein. J Biol Stand 1986;14:95 /102. Perrin P, Versmisse P, Sureau P. A rabies agglutination test(RAT) for rabies antibodies. J Biol Stand 1988;16:281 /6. Steinberg SP, Gershon AA. Measurement of antibodies to varicella virus by using latex agglutination test. J Clin Microbiol 1991;29:1527 /9. World Health Organisation. Expert committee meeting on rabies. 8th report WHO Tech Rep Ser 824, Geneva, WHO, 1992. World Health Organisation. Weekly Epidemiological record No14 2002 77, 109 /120.

samples found negative by this test may be subjected MNT or RFFIT. The test can be performed in any routine laboratory or even in physicians chamber if sensitised beads are made available commercially or supplied from a reference laboratory. We have stored the sensitised beads for more than 6 months at 4 8C and this has not affected the sensitivity or specificity of the test. Therefore this test may be found very useful in antirabies clinics, where the sensitised beads can be stored in refrigerators and serum samples tested after a course of vaccination. This will instil confidence in treating doctor as well as exposed patient with the risk of developing fatal rabies encephalitis. The test is also very economical compared to laborious and time consuming MNT and RFFIT which requires constant supply of FITC rabies conjugate, expensive fluorescence microscope and facilities for cell culture. The test is also more rapid and economical than ELISA, as this too requires expensive spectrophotometers. To conclude, we have developed a simple LAT based on purified rabies glycoprotein which promises to be a rapid clinician or doctor test in the peripheral antirabies treatment centres for evaluation of the protective status after rabies postexposure vaccination using cell culture rabies vaccines.

Acknowledgements The authors wish to thank Dr M.K. Sudarshan, Professor and Head, Department of Community Medicine, Kempegowda Institute of Medical Sciences, Bangalore for supplying some of the serum samples for evaluation.

References
Adler SP, McVoy M, Biro VG, Britt WJ, Hider P, Marshall D. Detection of cytomegalovirus antibody with latex agglutination. J Clin Microbiol 1985;22:68 /70. Atanasiu P. Quantitative assay and potency test of antirabies serum and immunoglobulin. In: Kaplan MM, Koprowsky H, editors. Laboratory Techniques in Rabies, 3rd ed.. Geneva: WHO, 1973:314 /8.

S.N. Madhusudana, S. Saraswati / Journal of Clinical Virology 27 (2003) 129 /135 World Health Organisation. World Survey of rabies No. 35. WHO, Geneva, 2002.

135

Wunner WH. Growth, purication and titration of Rhabdoviruses. In: Mahy BWJ, editor. Virology: a Practical Approach. Oxford: CRL Press, 1985:70 /93.