Validation of Indirect ELISA for Quantitative Testing of Rabies Antibodies During Production of Antirabies Serum using Equines

Rabies is still a cause of global concern, particularly in the developing countries. The treatment for post exposure prophylaxis is a combination of administration of vaccine and specific antiserum. Although equine antirabies immunoglobulin is available, the only approved method by World Health Organization for its testing is the Virus Neutralization Test (VNT), performed using mice. Since a large number of in-process samples are generated during antirabies immunoglobulin F(ab)2 production., VNT is time consuming, utilizes a large number of mice, results are prone to errors; therefore cumbersome and costly for routine testing. Hence, there is an urgent need to develop an alternative test for screening large number of in-process samples.In the present study an attempt has been made to evaluate the possibility of applying Enzyme Linked Immunosorbent Assay (ELISA) for quantifying the samples, viz: Equine sera, Plasma, Purified bulk sera and Batch. A total of 4,946 samples of sixty equines were monitored by indirect ELISA at an interval of two weeks for a period of five years. When seven equines of the above sixty were compared for VNT and indirect ELISA during their primary phase of immunization, a good correlation was observed (r = 1.0). Based on this, the equines could be segregated into ‘Low’ responders (<50 IU/ml), ‘Medium’ responders (between 50-100 IU/ml) and ‘High’ responders (>100 IU/ml). This segregation of equines based on their antibody titres proved to be very useful for initiating corrective remedial measures. On comparison of 14 Plasma samples, 31 Purified bulk sera samples and 17 Batch samples for VNT and indirect ELISA, a good correlation (r = 0.82, 0.923 & 0.874 respectively) was observed. The sensitivity values observed were 100%, 100%, 90.7% & 90.9% and specificity values were 100%, 100%, 94.7% & 100% for Equine sera, Plasma, Purified bulk sera and Batch samples respectively. The Cohen's Kappa index reflected a good agreement between the indirect ELISA test and the VNT test for Equine sera samples (1.0), Plasma samples (1.0), Purified bulk sera samples (0.931) and Batch samples (0.874).Thus, we conclude that indirect ELISA, which eliminates the use of a large number of mice, gives fast, yet reliable and reproducible results with less labor than the VNT test could be used as an optimum and ethical method for screening of antibody titers of all the in-process samples during manufacturing of antirabies serum.