Para nitro phenyl phosphate pnpp substrate

The protocol was adapted from Nkando et al., [7 (link)] as follows: 96-well Nunc-Immuno-plate MaxiSorp; Thermo Fisher Scientific, USA were coated with 100 μl/well-containing 1 μg/ml of antigen in Na₂CO₃: NaHCO₃ buffer (3.03 g Na₂CO₃; 6.0 g NaHCO₃ in1000 ml dH₂O for 100 mM at pH 9.6, diluted to 1:10 with dH₂O before use) and incubated at 4⁰ C overnight. The following day, plates were washed five times with 300 μl wash buffer {PBS with 0.05% Tween 20 (PBST)} followed by blocking with 200 μl blocking buffer (PBST + 0.5% Horse serum from Gibco Life Technologies™, New Zealand) for 1 h in a shaker incubator at 37⁰ C. The wells were washed five times with 300 μl wash buffer before adding 100 μl serum samples (diluted 4-fold 1:100 in row A to 1: 409,600 in row G) followed by 1 h incubation at 37⁰ C and five washes as described above. In total, 100 μl alkaline phosphatase-conjugated rabbit-anti-bovine IgG antibody (KPL151–12-06, Sera Care, USA), diluted 1: 5000 in PBST, was added to each well before incubation for 1 h at 37⁰ C. Following five washes as above, 100 μl/well of para-Nitro phenyl phosphate (pNPP substrate, Sigma Aldrich, USA) was added. The reaction mixture was incubated for 45 min in the dark and the absorbance (OD) measured at 405 nm in a BioTek Synergy HT, USA plate reader.