IgG antibodies were detected by indirect ELISA as previously described (18 (link)). Briefly, each purified recombinant antigen or polypeptide was diluted to their optimal concentration with coating buffer (0.05 M Na2CO3-NaHCO3, pH 9.6) and 100 µL was used to coat each well of 96-well Immunosorp plates (Nunc, Denmark) overnight at 4°C. Plates were washed three times with 375 µL PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween-20), and then blocked in 0.2 mL 5% skimmed milk for 2 h at room temperature. After three washes with 375 μL PBST, test serum samples (diluted 1:50 in blocking buffer) were added and incubated at 37°C for 1 h. After three washes, 100 µL horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) (Promega, USA) was added for 0.5 h at 37°C. After five washes, the color reaction was developed by 100 µL TMB (3, 3’, 5, 5’-tetramethylbenzidine) substrate solution (eBioscience, USA) and stopped by the addition of 50 µL 2 N H2SO4 (eBioscience, USA). The optical density (OD) at 450 nm was measured using a microplate reader (ELX50, Bio-Tek Instruments, USA). A novel evolved immunoglobulin-binding molecule (NEIBM)-ELISA method, which was designed to detect human IgG, IgM, and IgA, was also used to detect the antibody level against the polyprotein (27 (link)–29 (link)).
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