Immunosorp plates

An enzyme-linked immunosorbent assay (ELISA) was used for the detection of anti-Ag85B antibodies in the sera of the immunized animals. Briefly, the purified protein derivative (PPD) or the Ag85B protein was adjusted to 2 μg/mL with coating buffer (0.05 M Na₂CO₃-NaHCO₃, pH 9.6), and 100 µL was added to each well of 96-well Immunosorp plates (Nunc, Weston, FL, USA) for incubation overnight at 4 °C. The 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 3% bovine serum albumin in PBS for 2 h at room temperature. After three washes, the collected serum samples (diluted 1:200 in blocking buffer) were added to each well and incubated at 37 °C for 1 h. After five washes five times, 100 µL of 1:5000-diluted HRP-conjugated anti-human IgG, IgG1, or IgG2 secondary antibody (Promega, Madison, WI, USA) was added for incubation at 37 °C for a further 1 h. After five washes, the color reaction was developed with 100 µL TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate solution (eBioscience, San Diego, CA, USA) and stopped via the addition of 50 µL 2 N H2SO4 (eBioscience, San Diego, CA, USA). The optical density (OD) was measured and recorded at a wavelength of 450 nm using a microplate reader (ELX50, Bio-Tek Instruments, Salem, MA, USA).

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)).

IgG antibodies were detected by indirect ELISA against eight TB serodiagnostic antigens. Briefly, the purified recombinant antigens were diluted (final concentration 0.5–2 μg/ml) in coating buffer (0.05 M Na₂CO₃-NaHCO₃, pH 9.6) and coated on 96-well Immunosorp plates (Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA) overnight at 4°C. Plates were washed thrice with 375mL of PBS-T (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 tested serum samples (diluted 1:50 in blocking buffer) were added and incubated at 37°C for 1 h. Plates were washed thrice and HRP-conjugated secondary antibody (1:10,000) was then added for 0.5 h at 37°C. Each plate well was washed five times before the color reaction was developed by TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution and stopped by the addition of 2 M H₂SO₄. The optical density (OD) at 450 nm was measured using a microplate reader (ELX50, Bio-Tek Instruments, Winooski, VT, USA). A positive antibody test was defined as one with an OD value greater than the cutoff value, i.e., the mean OD value plus three SD from negative (healthy) control serum.