Standardization of indirect IgG ELISA
Indirect ELISA for detection of IgG against JEV in horses was standardized using the checker board titration method for determining the ideal blocking agent, optimum dilution of primary antibody (serum) and optimum dilution of HRPO conjugated secondary antibody. The recombinant NS1 protein of JEV earlier expressed in our laboratory (Dhanze et al., 2019) was used as antigen in the ELISA. The set of 24 JE positive and 24 JE negative horse serum samples previously confirmed by virus neutralization test (Kapdi et al., 2017) were used for standardizing the ELISA.
The concentration of recombinant NS1 protein was made to 10 µg/ml in carbonate buffer (pH 9.6) and 50 µl of it was added to each well of 96 well polystyrene maxisorp plate (Nunc, Denmark). The plate was incubated at 4ºC for overnight. The un-adsorbed antigen was removed by washing the plate thrice with PBS-T (0.05 per cent Tween 20) manually. The ideal blocking agent was determined using two buffers including 5 per cent skimmed milk powder (Difco, US) in PBS-T and 1 per cent bovine serum albumin (SRL, India) in PBS-T. The blocked plate was incubated for 2 h at 37ºC. After incubation, the blocking buffer was removed by washing thrice with PBS-T. The two dilutions of 1:100 and 1:200 of positive and negative sera in different diluent buffers including 5% SMP in PBS-T and 1% BSA in PBS-T were prepared and 50 µl of each was added to the wells of plate. After incubation at 37ºC for 1 h, plate was washed three times with PBS-T. The goat anti-horse horse radish peroxidase (HRPO) conjugate (Novus, US) was diluted in two dilutions of 1:5000 and 1:10,000 in different diluent buffers including 5% SMP in PBS-T and 1% BSA in PBS-T and 50 µl of each dilution was added to the wells of plate. It was incubated at 37ºC for 1 h and then plate was washed four times using PBS-T and pat dried. Further, 100 µl of freshly prepared substrate solution (10 ml citrate buffer, 10 mg OPD and 8 µl H2O2; pH 4.5) was added to each well and the color was developed by incubating the plate in dark at room temperature for 5 minutes. The reaction was stopped using 50 µl of stop solution and the absorbance was read at 492 nm using ELISA reader (Thermo Scientific, Multiskan Ex). The conditions that resulted in highest absorbance ratio between positive and negative sera (P/N value) were considered as optimal for further screening of field samples by ELISA. The field sample was considered positive if S/N ratio is more than 2 where S stands for absorbance of test sample while N stands for absorbance of negative control. The positive and negative sera were included as controls while screening the field samples.
Evaluation of diagnostic efficacy of ELISA
The diagnostic efficacy of rNS1 protein based IgG ELISA in terms of sensitivity and specificity in comparison with virus neutralization test (VNT) was calculated as per Thrusfield, (2005). The VNT was carried out using porcine stable (PS) kidney cells, 300 TCID50 of the JEV and heat treated test serum using the protocol described by Gulati et al. (2011). A total of 522 horse serum samples were screened using VNT. The reciprocal of the highest serum dilution that completely inhibited cytopathic effect (CPE) was expressed as the neutralization titer. The serum samples showing titer of 8 and above were considered as positive for JEV antibodies.
Validation of Equine IgG ELISA kit for the detection of JE
A batch of 26 coded samples including 17 positive serum samples and 9 negative serum samples was selected for validation. The equine IgG ELISA kit was validated according to OIE guidelines (OIE, 2014) in 3 different laboratories of our institute and in 3 different institutes across India. The Cohen’s Kappa value was estimated to know the agreement between results of our laboratory and the other laboratories/institutes as per Thrusfield (2005).
Field Sample screening
A total of 2069 horse serum samples were screened for JE from 13 states of India including 1566 samples from North zone, 373 samples from West zone, 86 samples from North-east zone, 40 samples from East zone and 4 samples from central zone during April 2017 to May 2020, using in-house developed IgG ELISA.