Horseradish peroxidase hrp conjugated streptavidin

We measured serum TFF1, TFF2, and TFF3 by enzyme-linked immunosorbent assay (ELISA). These ELISA systems were constructed in our laboratory, and the precise information was reported in our previous publication¹⁸ . Briefly, polyclonal antibodies TFF1, TFF2, and TFF3 coated onto 96-well microtiter plates were blocked with 0.1% Bovine serum albumin (BSA) in phosphate buffered saline (PBS). Then the blocking solution was removed, and 100 μl of assay buffer (1 M NaCl, 0.1%BSA, PBS) was added to each well. And then 50 μl of the samples or standard human TFF1, TFF2, and TFF3 were added to the wells. After incubation overnight at room temperature, the plates were washed, and diluted Biotin labeled anti-TFF polyclonal antibodies were added to each appropriate well. After incubation for 2 hours, the plate was washed, and diluted Horseradish peroxidase (HRP) conjugated Streptavidin (Vector) was added to each well. After incubation for 2 hours at room temperature, the plates were washed, and 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Scytek Laboratories, INC.) was added. After incubation for 10 minutes at room temperature, Stop solution (H₂SO₄) was added, and the absorbance at 450 nm was measured. Concentrations of human TFF1, TFF2, and TFF3 in the samples were calculated from the standard curves of recombinant human TFF1, TFF2, and TFF3.

Total and antigen-specific plasmablasts in peripheral blood or plasma cell responses in bone marrow were determined by ELISPOT assay as previously described (42 (link)). Briefly, 96-well multiscreen HTS filter plates (Millipore) were coated overnight at 4°C with 100 μl/well of 5 μg/ml of goat anti-monkey IgG or IgA antibodies (Rockland) or of 5 μg/ml of HIV gp120 (Immune Tech) or Ebola GP protein (IBT Bioservices), respectively. Plates were washed with PBS-0.05% Tween 20 (PBS-T) and blocked with complete medium at 37°C for 2 h. Freshly isolated cells were plated in duplicates in serial 3-fold dilutions and incubated overnight in a 5% CO₂ incubator at 37°C. Plates were washed with PBS-T and incubated with biotin-conjugated anti-monkey IgG or IgA antibodies (Rockland) diluted 1:1,000 for 1 h at 37°C. After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated streptavidin diluted 1:1,000 (Vector labs) at room temperature and developed using the AEC substrate kit (BD Biosciences). To stop the reaction, plates were washed extensively with water followed by air drying. Spots were imaged and counted using the Immunospot ELISPOT Analyzer (Cellular Technology Limited). The number of spots specific for each Ig isotype was reported as the number of either total or antigen-specific antibody producing cells per million.