F ab 2 fragment of goat anti human iga

Concentrations of total IgA in serum specimens and cell-culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA), as previously described.10 (link), 11 (link), 12 (link), 13 (link), 14 (link) F(ab’)₂ fragment of goat anti-human IgA (2.5 μg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) was used to coat MaxiSorp ELISA plates (Nunc; Thermo Fisher Scientific, Waltham, MA). Serially diluted samples were applied on the plates and the captured IgA was treated with 10 mU/ml neuraminidase (Roche Diagnostics Corp., Indianapolis, IN) to remove sialic acid. After washing, the samples were probed with biotin-labeled lectin from Helix aspersa (Sigma-Aldrich, St. Louis, MO) that is specific for N-acetylgalactosamine, followed by avidin–horseradish peroxidase conjugate and peroxidase substrate o-phenylenediamine-H₂O₂ (Sigma-Aldrich). Absorbance was measured at 490 nm. The Helix aspersa reactivity of IgA1 in each sample was then calculated as units of Gd-IgA1 per 100 ng of total IgA. A galactose-deficient IgA1 myeloma protein (Ale) purified from plasma of a patient with IgA1 myeloma was used as standard.14 (link), 15 , 16 Optical density at 490 nm for 50 ng neuraminidase-treated IgA1 (Ale) was defined as 100 U of Gd-IgA1.

Circulating IgA1 and Gd-IgA1 were detected by ELISA as previously described.¹⁵ (link) Briefly, high-binding MaxiSorp 96-well plates (Nalge Nunc, Rochester, NY) were coated with F(ab’) 2 fragment of goat antihuman IgA (Jackson Immuno-Research Labs, West Grove, PA). After overnight incubation at 4 °C, the coated plates were washed and blocked with 1% bovine serum albumin (Sigma Chemical Company, St Louis, MO). Then, diluted serum samples and standards were added. After incubating for 1 hour, the level of IgA1 was determined by incubation with horseradish peroxidase–labeled mouse antihuman IgA1 antibody. As for Gd-IgA1, samples were treated with sialidase A and biotin-labeled helix aspersa agglutinin after incubating. After another incubation, the plates were further incubated with horseradish peroxidase-ExtrAvidin (Sigma). The plates were then developed with the peroxidase chromogenic substrate o-phenylenediamine-hydrogen peroxide (Sigma). The color reaction was stopped with 1 M sulfuric acid, and the absorbance was measured at 490 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments Inc., Winooski, VT).