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Table 4 Optimised incubation time of the mixture containing chicken sera and NDV-Nb5-HRP fusions incubated with the antigen and optimal time for the colorimetric reaction after adding TMB using a checkerboard assay with cELISA

From: Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay

Times (min) of color reaction Sera type Incubation times (min) of chicken sera, NDV-Nb5-HRP fusions and antigens
5 10 15 20 25 30 45 60
10 Positive 0.69 0.86 0.52 0.54 0.48 0.24 0.24 0.29
Negative 0.75 0.89 0.94 0.97 1.02 0.99 1.01 1.11
P/N 0.92 0.97 0.55 0.56 0.47 0.24 0.23 0.26
15 Positive 0.70 0.63 0.50 0.47 0.39 0.25 0.26 0.29
Negative 0.80 0.75 0.87 1.00 0.97 1.02 1.15 1.12
P/N 0.86 0.84 0.57 0.47 0.40 0.25 0.23 0.26