Optimization of rHyC based Dot-ELISA
Dot ELISA for simple and rapid diagnosis of GWFI was standardized on strips of Nitrocellulose membrane (NCM) with pore size of 0.45 µm using positive and negative reference serum samples for GWFI. NCM were cut into 0.8 cm x 2.5 cm strips and rHyC protein was dotted at three different places on to membrane maintaining the gap of 0.5 cm to allow separation between the dots. The working concentration of recombinant antigen, serum antibodies and conjugate for dot-ELISA were determined by standard checkerboard titration. Two different concentrations of purified rHyC antigen 94 ng/dot (1 µL) and
188 ng/dot (2 µL) were titrated against three dilutions of serum (1:400, 1:800 and 1:1200) and two dilutions (1:4000 and 1:8000) of anti-goat IgG HRP conjugate in dot ELISA format using 5% skimmed milk powder (SMP) in PBS as blocking buffer. The antigen concentration, serum and conjugate dilution that showed the development of well-defined dots on the membrane with positive serum and none with negative serum were selected. The optimal conditions included NCM strips dotted with rHyC proteins 2 µL dots. The dotted strips were air dried for 15 min at room temperature and blocked with 5% SMP in PBS (pH 7.4). After incubation at 37 °C for 1 h, strips were washed two times with PBS-T 0.075% and one time with PBS. After washing, strips were soaked in 1 mL of known positive and negative serum sample diluted in PBS (1:800) and incubated at 37 °C for 1 h. After wash, the strips were incubated with 1 mL of anti-goat IgG HRP-conjugate (Sigma Aldrich, USA) at 1:4000 dilution in PBS (pH 7.4) at 37 °C for 1 h. Following three washing, ELISA dots were developed by incubating the strips with DAB substrate (VWR-Amresco Life Science, 5 mg/10 mL) solution for 5 min at room temp in dark. The reaction was stopped by washing the strips in distilled water. The development of brown dots on strips indicated that serum samples were positive for GWFI while negative sera showed no dots. All the strips were handled with forceps.