Rabbit anti human iga hrp

BCA assay kit (Thermo Scientific) was used to measure total protein in all cell homogenates according to manufacturer’s instructions. This ensured equal loading of all cell samples for SDS-PAGE, western blotting and IgA enzyme-linked immunosorbent assay (ELISA).
IgA binding on all cell and saliva samples were measured using an ELISA, as previously described [22 (link)]. Rabbit anti-human IgA 1:1000 (Dako, Ely, UK) in carbonate buffer was used to coat the ELISA plates overnight. Three washes in phosphate buffered saline with 1% Tween (PBS-T) were completed. Samples were serially diluted down the plate in duplicate alongside the standard and incubated at 37°C for 1 hour, followed by 3 more PBS-T washes. Detecting antibody Rabbit anti-human IgA HRP 1:10000 was used for 1 hour (Dako) followed by 3 final PBS-T washes. Substrate was then added consisting of 20 ml sodium acetate, with 3 μl of H₂O₂ and 500 μl of 3,3’, 5, 5’ tetramethylbenzidine (3 mg/ml in dimethyl sulfoxide). The reaction was stopped with 2 M sulphuric acid and absorbance was read at 450 nm using a microplate reader (BioRad, Hemel Hempstead, UK).

Binding of soluble FcαRI or IgA Ab to plate‐bound peptides was tested with Pepscan‐based ELISA's, which were adapted according to the method previously described 19, 43, 44. The samples were washed to remove unbound fragments after each incubation step. The 455‐well credit‐card format polypropylene cards containing the covalently linked IgA‐peptides were incubated with blocking solution (4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween). Next, peptides were incubated with soluble FcαRI or 293T cells transfected with FcαRI and subsequently with mouse anti‐human FcαRI IgG mAb (1 μg/ml; BD, Franklin Lakes, NJ). Then, peptides were incubated with rabbit anti‐mouse IgG‐HRP (1/1000, Dako, P0212) for 1 h at RT. Alternatively, after blocking, the covalently linked FcαRI‐peptides were incubated with pooled human serum IgA (Cappel™, MP Biomedicals, Santa Ana, CA), and rabbit anti‐human IgA‐HRP (1 μg/mL, Dako, P0212) was added. The peroxidase substrate 2,2′‐azino‐di‐3‐ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% H₂O₂ was added (1 h, RT). Colour development was measured, which was quantified with a charge coupled device (CCD)‐camera and an image processing system. Values mostly ranged from 0 to 3000, a log scale similar to 1–3 of a standard 96‐well plate ELISA‐reader.