Horseradish peroxidase hrp conjugated goat anti human igg

For screening the antibody-positive sera, the whole AGS cells were lysed in Laemmli's sample buffer, and the proteins were separated in 10% SDS-polyacrylamide gels (SDS-PAGE gels) and then transferred into nitrocellulose (NC) membrane (Osmonics Inc., MA) for western blotting analysis. The membrane with proteins was cut into 0.3 cm wide strips. After blocking with 3% nonfat milk prepared in PBST, the NC membrane strips were individually incubated with human serum samples (including 104 gastric cancer and 54 normal health sera) diluted at 1 : 100 in 3% milk solution. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Thermo Fisher Scientific, Waltham, MA) was used as secondary antibody at 1 : 10,000 dilution. Finally, the immunoreactive bands were detected with Enhanced Chemiluminescence (ECL) kit (Thermo Fisher Scientific, Waltham, MA).

Enzyme-linked immunosorbent assay plates (Maxisorp, NUNC, Roskilde, Denmark) were coated with 2 μg/well of purified recombinant NPC-2 protein antigen in 0.1 M carbonate/bicarbonate buffer pH 9.6 and maintained at 37°C for 45 min. Thereafter, wells were blocked with phosphate buffered saline, pH 7.4, 0.05% (v/v) Tween 20 (PBST), containing 3% (p/v) skimmed milk, for 1 h at room temperature (RT). The plates were incubated with serum samples diluted 1:200 in PBST with 1% skimmed milk, for 2 h at RT. Duplicates of positive (sera from a confirmed COVID 19 patient), and negative controls were included in each plate. Plates were washed three times with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Thermofisher), diluted 1:5,000 in PBST, for 1 h at RT. Finally, after three additional washes with PBST, plates were incubated with peroxidase substrate (TMB-MAX, Neogen Corporation) for 5 min in the dark at RT. Reaction was stopped by adding 0.5 M H₂SO₄. Optical density (OD) was measured in each well at 450 nm in an ELISA microplate reader. Results were valid when OD in negative control wells was below 0.8 OD units and the OD in positive control wells was over 2.50 OD units.