Validation of haemagglutination, haemagglutination inhibition and antibody neutralization assays used to determine the titre of CPV -2 virus and anti CVP – 2 egg yolk antibodies

Abstract

The safety and efficacy of a biological product is determined by the highly variable in-vivo or in-vitro assays. Hence the manufacturers have to minimize the test variabilities to an acceptable limit through optimization of test conditions and validation as per the regulatory guidelines. In this study the test variables were optimized and validated the methods HA, HI and antibody neutralisation assays. During optimisation of HA method, it was observed that no change in the HA titre either due to incubation temperatures (5 ± 3°C, 25 ± 2°C and 37 ± 2°C) or % of RBC used (1% and 0.5%). But the difference was observed for species of RBC used and time taken for HA reaction. CPV – 2 isolate 1 did not show HA activity with chicken RBC and showed agglutination with porcine RBC, whereas isolate 2 was showing HA reaction with both chicken and porcine RBC. During validation studies of HA, HI, CCID50 and antibody neutralization assays the % CV observed was 31, 31, 1.1 and 2.12 respectively which are less than the expected variability of > 50%.