passages.id: PMC-2989791-sec-04__text
passages.type: sec
passages.text: Measurement of IgA1 content
  IgA1 content in the supernatant from each culture well was measured in duplicate using enzyme-linked immunosorbent assay (ELISA). All incubations were performed at room temperature except for capture antibody coating. Briefly, 96-well immunoplates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 microg/mL of F(ab')2 fragment goat anti-human IgA antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), in phosphate-buffered saline (PBS) at 4degreesC overnight. After three washes with PBS containing 0.05% Tween-20 (PBST), plates were blocked by adding PBS containing 1% bovine serum albumin (BSA) to the wells for 90 min. Next, 50 microL of supernatant sample or standard human IgA1 (CALBIOCHEM, La Jolla, CA, USA) was added to the reaction wells and then incubated for 90 min. After three washes, 0.75 microg/mL alkaline phosphatase conjugated goat anti-human IgA (Southern Biotechnology Associates, Birmingham, AL, USA) in 1% BSA/PBS was added to the reaction wells and then incubated for 90 min. Plates were washed three times and developed with a substrate solution of 1 mg/mL p-nitrophenyl phosphate disodium salt (SIGMA, St. Louis, MO, USA) in 0.1 M glycine buffer containing 1 mM MgCl2, 1 mM ZnCl2, pH 10.4. The optical density at 405 nm was determined in a microplate reader (Benchmark PlusTM Bio-Rad Laboratories, Hercules, CA, USA). IgA1 concentration in unknown duplicate samples was determined by interpolation of the respective optical density into the appropriate standard curve. IgG and IgM contents in the supernatant were measured using sandwich ELISA (IMMUNOtek(R), ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacturer's instructions.
  