Arch Virol DOI 10.1007/s00705-014-2033-3
Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment Ebenezer Angel Jemima • Seeralan Manoharan Kathaperumal Kumanan
Received: 27 August 2013 / Accepted: 18 February 2014 Ó Springer-Verlag Wien 2014
Abstract The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/ concordance was found to be 97.39 %. A rapid field test—a latex agglutination test (LAT)—was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.
E. A. Jemima S. Manoharan (&) K. Kumanan Department of Animal Biotechnology, Madras Veterinary College, Chennai 600 007, India e-mail: [email protected]
Introduction Rabies is one of the most feared zoonotic diseases in the world. Development of rabies can be prevented by prophylactic as well as post-exposure vaccination to people in risk groups and victims of rabid animal bites. Assessment of rabies virus antibody is important for testing vaccination efficacy and also for assessing the immune status of animals and humans. The mouse inoculation test (MIT) was first used for confirmatory diagnosis as well as assessment of protection by rabies vaccines in 1935 , and a modification of the MIT, the mouse virus neutralization test (MNT), was also widely used . A WHO-approved rapid fluorescent focus inhibition test (RFFIT), which measures the amount of virus-neutralizing antibody in serum, is the most widely used method . A micro test, the fluorescent antibody virus neutralization test (FAVN), which is an adaptation of the RFFIT, has been developed and evaluated for quantifying neutralizing antibodies against rabies virus . An ELISA was first developed for the titration of antirabies antibodies in vaccinated animals or humans but later modified for detecting rabies virus antigens in brain tissue . A competitive ELISA was designed and evaluated for the estimation of rabies-virus-neutralizing antibodies for post-exposure rabies vaccination in humans . The rapid neutralizing antibody (RAPINA) test was developed to determine the presence of neutralizing antibodies in serum and could be a substitute for neutralization tests that use live viruses, cultured cells, and fluorescence microscopy . An immunochromatographic test strip (ICTS) for detecting rabies virus antibodies was developed, using colloidal gold particles labeled with rabies virus glycoprotein as the tracer .
E. A. Jemima et al.
The latex agglutination test (LAT) is simple and rapid and does not involve the use of live virus. It will be useful to test many samples in a very short time. A LAT has been reported to successfully detect rabies viral antigens in the saliva and brains of infected dogs . A modified LAT method has also been developed using rabies virus glycoprotein purified from whole virus particles for coating latex beads to detect antibodies from immunized animals or humans and was found to have high specificity and sensitivity . This article describes the development of a latex agglutination test using recombinant rabies virus glycoprotein for rabies virus antibody detection and evaluation by the RFFIT method.
Materials and methods Bacterial strains and plasmids A recombinant plasmid containing the rabies virus glycoprotein (RVG) gene, pTriEx 1.1 Neo RVG, which was available at the Dept. of Animal Biotechnology, Madras Veterinary College, Chennai, India, was propagated in the E. coli pLac I strain and the plasmid was isolated using a kit (Bio Basic Inc., Canada). Restriction enzyme digestion was done with NcoI and XhoI to confirm the recombinant glycoprotein gene insert (1595 bp) by agarose gel electrophoresis. The recombinant plasmids were also used for amplification with glycoprotein-gene-specific primers in PCR.
serum samples were stored at -30 °C until further use. All of the samples were heated at 56 °C for 30 min before testing for complement inactivation. International reference serum (69 IU/ml) obtained from Indian Immunologicals Limited, Hyderabad, India, was diluted to 2 IU/ml and used as a standard positive control in RFFIT. A known negative serum from an unvaccinated dog with 0.08 IU/ml was used as a negative control in LAT. Production and characterization of recombinant rabies virus glycoprotein Bulk recombinant RVG protein was produced in Sf9 cells by harvesting on the fifth day of infection at an MOI of 2– the time at which maximum protein expression was obtained. The recombinant RVG protein was purified from the Sf9 cell lysate using an Ni-NTA purification system (Invitrogen, USA) as per the kit protocol under denaturing conditions. The purified recombinant RVG protein was further confirmed by SDS-PAGE and western blotting using a known rabies-positive serum. RFFIT A standard RFFIT protocol was followed in this study using N2A cells. Each well containing 20 microscopic fields was observed at 2009 magnification and the fluorescent foci in each field were counted. Using the method of Reed and Muench , the difference between the logarithm of the starting dilution and the logarithm of endpoint dilution and the titre were calculated.
Cell lines, viral vector, and plaque confirmation Sensitization of latex beads Neuro-2a cells (ATCCÒ CCL-131TM) and Sf9 cells (ATCCÒ CRL-1711TM) were used in this study. A Bac vector (1000 Triple Cut Virus DNA kit, Novagen, USA) was used to transfect Sf9 cells with pTriEx 1.1 Neo RVG for producing recombinant baculovirus. The plaques were visualized by the neutral red staining method as per the kit protocol. The RVG gene was amplified from the plaques by PCR, gel purified (Invitrogen, USA), and sequenced. The recombinant RVG gene sequence was compared to other sequences available in the GenBank database by BLAST analysis. Subsequently, the recombinant baculovirus with RVG was confirmed in indirect FAT by infecting Sf9 cells at a multiplicity of infection (MOI) of 2. Virus strain and serum samples The CVS-11 strain, maintained in N2a cells, was used in RFFIT. A total of 228 blood samples were collected from rabies-vaccinated and unvaccinated dogs from Madras Veterinary College Hospital, Chennai, India, and separated
For the preparation of latex beads and performance of the latex agglutination test, the method of Lengyel et al.  was followed with few modifications. Latex beads (0.9 lm, Sigma, USA) were washed twice in 0.06 M carbonatebicarbonate buffer (pH 9.6) with centrifugation at 8000 rpm for 3 min each time, and a 1 % suspension of latex beads was made in carbonate-bicarbonate buffer. The latex beads were mixed with pooled recombinant RVG antigen at 0.1, 0.25, 0.50, 0.75 and 1.0 mg/ml for sensitization. The sensitized beads were clarified by centrifugation at 60109g for 3 min, and the pellet was resuspended in PBS containing 0.05 mg/ml of BSA and 0.1 % sodium azide as a 0.25 % suspension. The sensitized latex beads were stored at 4 °C until use. Latex agglutination test and grading The LAT was performed in a VDRL plate by mixing an equal volume of serum and sensitized latex beads (20 ll
Latex agglutination test for rabies virus antibodies
each). The plate was gently rocked for a maximum of 5 min. Positive and negative control serum samples were included in the LAT for testing 228 serum samples. The grading of LAT was done with 10, 5, 2.5, 1.25, 0.625 IU/ ml of standard reference serum with known antibody titers, and the results obtained with these dilutions were defined as ????, ???, ??, ? and -, respectively.
there was no amplification in plaque 1 or in the non-template control. Further PCR amplification was done with plaque 10 for sequencing, and the sequence obtained showed 96 % identity to that of the glycoprotein gene of rabies virus strain CVS in the GenBank database.
Recombinant RVG protein was purified from 700 ml of culture supernatant from recombinant-baculovirus-infected insect cells using Ni-NTA anti-histidine antibody-tagged affinity columns, and the protein concentration, determined by the Bradford method, was found to be 13.0 mg/ml. Insect cells on cover slips infected with recombinant baculovirus were processed for indirect FAT using a rabiespositive serum from a dog, and these cells showed positive cytoplasmic fluorescence when compared to an uninfected control (Fig. 2a, b). SDS-PAGE and staining with Coomassie brilliant blue showed that the recombinant glycoprotein was *70 kDa when compared to a standard protein marker (Fig. 3), and this was confirmed by immunoblotting with a rabies-positive serum and anti-histidine monoclonal antibodies (Fig. 4).
Confirmation of recombinant plasmid pTriEx 1.1 Neo RVG The pTriEx 1.1 Neo RVG plasmid propagated in E. coli pLac I strain was isolated. The purity was good, and the concentration was found to be 122 ng/ll. Digestion of the purified recombinant plasmid with NcoI and XhoI resulted in release of the insert, as shown by agarose gel electrophoresis (Fig. 1). The recombinant plasmid was used as template to amplify the 1595-bp glycoprotein gene by PCR. Transfection of insect cells with pTriEx 1.1 neo RVG The recombinant RVG gene was successfully introduced into Sf9 insect cells by transfection using a baculoviral vector. A total of 11 different recombinant baculoviral plaques were identified at different dilutions by neutral red staining. PCR screening and sequence confirmation of recombinant baculovirus The plaques extracted with insect cell medium were screened for the presence of the RVG gene by PCR using gene-specific primers. Of the 11 plaques screened, 10 showed amplification, and the intensity of the amplicon was high in plaque 10 as well as in the positive control, but
Recombinant RVG protein production and characterization
Standardization of the latex agglutination test In this study, 0.9-lm latex beads were used for coating with the recombinant protein, with an optimal concentration of 0.5 mg/ml in LAT for testing 228 dog serum samples based on the intensity of agglutinated particles/ clumps, and also based on the time taken for agglutination. The grading of LAT results was done with 10, 5, 2.5, 1.25, 0.625 IU/ml of standard reference serum with known antibody titers, as ????, ???, ??, ? and - for uniform suspension of the beads (Table 1; Fig. 5a). An agglutination reaction was observed within 5 min. Large clumps forming within 1–2 min were graded as ????, medium clumps forming within 2–3 min were graded as
Fig. 1 Agarose gel electrophoresis (1 %) of restriction-enzyme-digested plasmid and PCR product for confirmation of the rabies virus glycoprotein gene insert. Lane 1, recombinant pTriEx 1.1 neo RVG plasmid, (*8.2 kb); lane 2, pTriEx 1.1 Neo RVG digested with NcoI and XhoI (G gene, *1595 bp); Lane 3, 1-kb ladder; lane 4, PCR amplicon (G gene, *1.6 kb)
E. A. Jemima et al. Fig. 2 Indirect FAT done on Sf9 cells infected with recombinant baculovirus plaque TCF showing cytoplasmic fluorescence (4009). A, uninfected Sf9 cells; B, recombinant baculovirus containing RVG gene showing protein expression as cytoplasmic fluorescence
Fig. 3 SDS-PAGE (12 %) analysis of insect cell expression of RVG in crude lysate as well as in purified form. Lane 1, pre-stained ColorBurst protein marker (8–220 kDa); lane 2, purified RVG protein (*70 kDa)
???, small clumps forming within 3–4 min were graded as ??, and very small clumps forming within 4–5 min and barely visible to the naked eye were graded as ?. Samples in which agglutination did not occur within 5 min were considered negative (-). Out of 228 dog serum samples tested by LAT, 145 were positive with different levels of agglutination. The following LAT results were obtained: ????, 13 samples; ???, 55 samples; ??, 32 samples; ?, 45 samples; -, 83 samples (Table 2; Fig. 5b)
Fig. 4 Immunoblotting confirmation of recombinant RVG using antihistidine monoclonal antibodies. Lane 1, Pre-stained ColorBurst protein marker (8–220 kDa); lane 2, NCM showing a single band of recombinant RVG protein in western blotting using anti-histidine monoclonal antibodies (*70 kDa)
Table 1 Grading of LAT results with standard anti-rabies antibodies of known titer Sample no.
Antibody concentration (IU/ml)
Grade for agglutination
Measurement of rabies virus neutralizing antibody titers by RFFIT RFFIT was performed with the 228 serum samples using a known standard serum, the CVS–11 strain, and N2a cells. An antibody titer of 0.5 IU/ml was considered protective and was taken as positive for comparison to LAT. Out of
228 samples, 151 were positive in both rabies-vaccinated and unvaccinated dog serum samples, and serum samples from unvaccinated animals were used as control. The titers of the sera used as standards to evaluate LAT are given in Table 2.
Latex agglutination test for rabies virus antibodies
Fig. 5 A. Grading of LAT results with titer standard anti rabies antibodies of known B. VDRL plate showing LAT results using dog serum samples
Table 2 Summary of the results of LAT and RFFIT testing of 228 dog serum samples
Table 4 Statistical analysis of the evaluation of LAT in comparison with RFFIT
Total no. of samples
RFFIT titer ranges IU/ml
Formula for calculation
LAT with RFFIT
a/a ? c
3.2 \ 6.75
d/b ? d
1.61 \ 3.10
a ? d/(a ? b ? c ? d)
0.88 \ 1.59
K = (a ? d - P)/(1 – P), where
P = (a ? b) (a ? c) ? (c ? d) (b ? d) v2-value
Table 3 Summary of LAT and RFFIT testing results for 228 dog serum samples LAT
Total number of samples tested
Number of positive percentage
** Highly significant [ 2.56 (P [ 0.01)
samples was[7 IU/ml; for the ??? grade, it was between 3.2 and 6.75 IU/ml; for the ??grade, it was between 1.61 and 3.10 IU/ml; for the ?grade, it was between 0.88 and 1.59 IU/ml, and for negative results, it was below 0.88 IU/ ml (Table 2).
Evaluation of LAT in comparison with RFFIT
A known standard serum was used as a positive control for both assays. Out of 228 samples tested, 145 were positive in both RFFIT and LAT, and the specificity was found to be 100 %. Of the 151 RFFIT-positive samples, 145 were positive in LAT (Table 3). The sensitivity was 96.03 %, and the accuracy was 97.39 %. The kappa value was calculated to measure the strength of the agreement between the two methods, and the results are shown in Table 4. The RFFIT titer range obtained for ???? LAT-positive
The latex agglutination test is simple, rapid test which has an advantage over other available techniques because of its specificity, efficiency, suitability, economy and rapidity in testing many samples in a very short time. Glycoprotein is a major antigenic stimulant of the host immune system during infection and vaccination, specifically inducing neutralizing antibodies . An insect cell system was chosen as the host for expression of recombinant RVG to increase its antigenicity and yield, and to allow proper
E. A. Jemima et al.
posttranslational modifications to occur in the recombinant protein . In this study, although fewer plaques were obtained, all but one were positive, which was sufficient for protein expression analysis. In this work, western blot analysis was done with rabies-positive serum as well as with anti-histidine monoclonal antibodies as a double confirmation of the recombinant protein, which showed the presence of conformational epitopes in the recombinant protein. Latex beads have been used extensively as a carrier in agglutination assays because it is not itself antigenic and binds strongly to proteins . The sensitivity of the reaction is influenced by the amount of protein antigens or antibodies bound to the latex particles . LAT was used to detect neutralizing antibodies against rabies virus by coating with the optimal concentration of 0.5 mg/ml onto 0.9-lm-diameter plain latex beads. Previously, 0.8-lmsized latex beads were coated with 1 mg/ml rabies virus glycoprotein purified from whole viral particles . The lower concentration used in this work was mainly due to the high purity of the recombinant glycoprotein, which was purified using an anti-histidine-antibody-tagged Ni-NTA agarose affinity column and was reflected in the sensitivity and specificity of the LAT compared to RFFIT. Also, the larger size of latex beads used in protein coating was responsible for the better sensitivity in this work. Any variation, such as the color of the latex beads, did not alter the results of LAT, as reported previously . In the conventional RFFIT, fixed strains of rabies virus such as CVS and PV [1, 9] have been used. However OIE, recommends the use of the CVS strain , and in this study also, the CVS-11 strain was used to determine the anti-rabies neutralizing antibody level in neuroblastoma cells, and scoring of the fluorescent foci was done as described , using 20 microscopic fields at 2009 magnification. An antibody level of 0.5 IU/ml  is the recommended minimum protective level in RFFIT testing. However, no antibody level has been prescribed for in vitro tests such as ELISA or LAT, as discussed by earlier investigators . In the LAT grading system using reference rabies virus immunoglobulin, B0.625 IU was considered negative. Moreover, when comparing the LAT results with RFFIT for the 228 serum samples, the samples graded as ? had a titre range between 0.88 and 1.59 I.U. Hence, it is suggested that this LAT could be used as a screening test for rabies virus neutralizing antibody at the field level, as it does not involve the use of any infectious virus. If the need arises, samples that are negative by LAT may be tested by the RFFIT method. In the screening of 228 serum samples by RFFIT and LAT, it was found that many vaccinated dogs did not have antibodies at all or has insufficient levels of antibodies. This could be attributed mainly to poor cold storage of
vaccine by the distributors, poor storage conditions in the pet clinic, or to the quality of the vaccines. Also, some of the unvaccinated dogs—mostly stray dogs—had more than the minimum protective level of antibodies, which could be due to some indirect exposure to rabies virus from the environment. Evaluation of LAT was done by the RFFIT method, which is the WHO- and OIE-recommended gold-standard methods for measuring rabies virus neutralizing antibodies, and a suitable comparison was made earlier [9, 19]. The issue of specificity, sensitivity and reproducibility must be considered when diagnostic tests are compared. The results obtained in this study were correlated with earlier results [9, 19]. The LAT in this study showed 100 % specificity, 96.03 % sensitivity, and 97.39 % accuracy. It has been stated that an LAT with 94 % sensitivity is sufficient for anti-rabies antibody screening purposes , but with a sensitivity of 96.03 %, it was possible to decrease the detection limit to 0.88 IU/ml from 2 IU/ml, as has been reported earlier . This may be the first report of the development of an LAT using recombinant glycoprotein of rabies virus with a very good detection limit. In this study, an attempt was also made to correlate the RFFIT titer to the intensity of the LAT clumps/agglutination formed, but a large number of serum samples need to be tested and compared with RFFIT before assessing the antibody levels against rabies based on LAT results. However, this LAT is best suited for field tests for screening of large numbers of serum samples for rabies virus antibody. Acknowledgment We wish to thank the Vice Chancellor and Registrar of TANUVAS, Chennai, South India, for financial support.
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