Evaluation of rHyC based dot-ELISA
The dot ELISA was performed with rHyC under optimized conditions to
determine the assay specificity and cross reactivity using known
positive sera against different important parasitic and bacterial
diseases of goats. The diagnostic specificity (DSp) and diagnostic
sensitivity (DSn) of the dot ELISA were determined by, using a two-sided
contingency table (Table 1.) . Known positive serum samples with
brown spots compared to nil in negative serum were reported as positive.
The sensitivity and specificity were calculated by using the following
formula: Sensitivity = (TP/TP+FN) × 100; Specificity = (TN/TN+FP) × 100;
where, TP-true positive; TN-true negative; FP-false positive and
FN-false negative. The positive predictive value (PPV) and negative
predictive value (NPV) were also calculated based on the optimized assay
as shown in Table 1 .
Also, the inter-rater reliability of dot-ELISA was evaluated with
Cohen’s Kappa test, taking the rHyC based microtitre plate
indirect-ELISA as reference standard.18, 29 A set of
149 sera samples (69 positive and 80 negative) from ICAR-NF laboratory
were tested for evaluating the kappa index between both assays for the
diagnosis of GWFI.
In a separate experiment, the optimized dot ELISA was tested with neat
samples (blood, plasma and serum) and lower dilutions at lower
incubation temperature (18 °C) and time (30 min) to check the
suitability of the test at field conditions under sub-tropical and
temperate climates of GWFI prevalent regions. Samples of a known GWFI
positive goat were used for blood and plasma whereas a GWFI negative
goat was used to derive negative control samples of blood, plasma and
serum. The positive samples of GWFI (blood, plasma and weak positive
serum) were tested as neat, 1:2 and 1:10 dilutions and control negative
samples for neat blood, plasma and serum. A weak positive serum was
taken as positive control to ensure reactivity of weak titre at lower
time and temperature of incubation.
Serum samples (n = 274) from different regions of Union territory of
Jammu and Kashmir that were available in the ICAR-NF laboratory were
tested with the optimized dot-ELISA.