Anti human iga hrp

Gamma-irradiated whole cell bacteria Mtb H37Rv (Cat. No. NR-49098) obtained from BEI resources, United States, was resuspended in sterilized Milli Q water and adjusted to OD₆₀₀ of 1.0 for antigen coating of the ELISA plates (Nunc Immuno Maxisorp). Bacterial solution of 20 µL was added, followed by heat fixation at 65 °C for 3 h. Ice cold 70% methanol of 20 µL volume was added and incubated for 2 h at 4 °C. After incubation, the methanol was gently removed and 20 µL of 1X PBS was added to the plates and left overnight at 4 °C. Indirect ELISA was carried out the following day, by blocking with 20 µL of 4% skim milk (Sigma-Aldrich, United States, Cat. No. 70166) in 1X PBS for 4 h, followed by 20 µL of block solution containing 5 µL of B cell culture supernatant for 1 h. This was followed by addition of 20 µL secondary antibody, anti-human IgG-HRP (Thermofisher Scientific, United States, Cat. No. 31413) or anti-human IgA HRP (Thermofisher Scientific, United States, Cat. No. A18781) in 1:5000 added in block solution for 1 h. The plates were read at OD 450 nm after addition of TMB substrate (Thermofisher Scientific, United States, Cat. No. 34029) and stopping the reaction with 1 M concentrated H₂SO₄ after 5 min. All washing steps in between were done with 100 µL volume of 1X PBS thrice (Data availability—Figshare 10.6084/m9.figshare.23549964).

Gamma-irradiated whole cell bacteria Mtb H37Rv (Cat. No. NR-49098) obtained from BEI resources, United States, was resuspended in sterilized Milli Q water and adjusted to OD₆₀₀ of 1.0 for antigen coating of the ELISA plates (Nunc Immuno Maxisorp). Bacterial solution of 20 µL was added, followed by heat fixation at 65 °C for 3 h. Ice cold 70% methanol of 20 µL volume was added and incubated for 2 h at 4 °C. After incubation, the methanol was gently removed and 20 µL of 1X PBS was added to the plates and left overnight at 4 °C. Indirect ELISA was carried out the following day, by blocking with 20 µL of 4% skim milk (Sigma-Aldrich, United States, Cat. No. 70166) in 1X PBS for 4 h, followed by 20 µL of block solution containing 5 µL of B cell culture supernatant for 1 h. This was followed by addition of 20 µL secondary antibody, anti-human IgG-HRP (Thermofisher Scientific, United States, Cat. No. 31413) or anti-human IgA HRP (Thermofisher Scientific, United States, Cat. No. A18781) in 1:5000 added in block solution for 1 h. The plates were read at OD 450 nm after addition of TMB substrate (Thermofisher Scientific, United States, Cat. No. 34029) and stopping the reaction with 1 M concentrated H₂SO₄ after 5 min. All washing steps in between were done with 100 µL volume of 1X PBS thrice (Data availability—Figshare 10.6084/m9.figshare.23549964).

Gamma-irradiated whole cell bacteria Mtb H37Rv (Cat. No. NR-49098) obtained from BEI resources, United States, was resuspended in sterilized Milli Q water and adjusted to OD₆₀₀ of 1.0 for antigen coating of the ELISA plates (Nunc Immuno Maxisorp). Bacterial solution of 20 µL was added, followed by heat fixation at 65 °C for 3 h. Ice cold 70% methanol of 20 µL volume was added and incubated for 2 h at 4 °C. After incubation, the methanol was gently removed and 20 µL of 1X PBS was added to the plates and left overnight at 4 °C. Indirect ELISA was carried out the following day, by blocking with 20 µL of 4% skim milk (Sigma-Aldrich, United States, Cat. No. 70166) in 1X PBS for 4 h, followed by 20 µL of block solution containing 5 µL of B cell culture supernatant for 1 h. This was followed by addition of 20 µL secondary antibody, anti-human IgG-HRP (Thermofisher Scientific, United States, Cat. No. 31413) or anti-human IgA HRP (Thermofisher Scientific, United States, Cat. No. A18781) in 1:5000 added in block solution for 1 h. The plates were read at OD 450 nm after addition of TMB substrate (Thermofisher Scientific, United States, Cat. No. 34029) and stopping the reaction with 1 M concentrated H₂SO₄ after 5 min. All washing steps in between were done with 100 µL volume of 1X PBS thrice (Data availability—Figshare 10.6084/m9.figshare.23549964).