Biotinylated rat anti mouse iga clone c10 1

Murine blood samples were obtained from cheeks. Blood samples were allowed to clot for 2 h at RT before centrifugation for 10 min at 2000 g. Sera were collected and stored at −20 °C. The M315 myeloma protein, which is 2,4-dinitrophenol specific, was detected by enzyme-linked immunosorbent assay. Ninety-six-well plates were coated with 1 μg/ml dinitrophenol-conjugated bovine serum albumin and incubated at 4 °C overnight. Unspecific binding sites were blocked for 30 min at RT using PBS/5% bovine serum albumin followed by three washing steps with PBS/0.1% Tween. Serum samples were diluted 1:500–1:5000 in PBS containing 0.1 or 5% bovine serum albumin. M315 standard protein was serially diluted within a range of 10–2560 ng/ml. Plates were incubated for 2 h at 37 °C with serum samples and standard protein. After plates were washed three times with PBS/0.1% Tween, 1 μg/ml biotinylated rat anti-mouse IgA (clone C10-1, BD Pharmingen, Heidelberg, Germany) was added for 1 h at RT. Plates were again washed three times and incubated with streptavidin-conjugated alkaline phosphatase (1:3000; Roche, Mannheim, Germany) for 1 h at RT. After three washes, phosphatase substrate (Roche) was added at 1 mg/ml in substrate buffer and absorbance was measured at 415 nm. Sera were collected from vehicle-treated and GSI XII-treated mice at time points indicated (Figure 2a) or every 2–8 days (Figure 3a).

Paraprotein production by MOPC315.BM cells was evaluated by ELISA [23 (link)]. Briefly, 96 well Nunclon ELISA plates were coated with 2 μg/ml of anti-MOPC315.BM paraprotein idiotype (Ab2.1–4) (kindly provided by Prof Bjarne Bogen, University of Oslo, Norway) at 4 °C overnight. Wells were blocked with PBS/0.02% sodium azide/1% BSA, washed and incubated for 2 h at 37 °C with serum samples or standard paraprotein (ranging from 400 to 0.39 ng/ml) diluted in PBS/ 0.02% sodium azide/0.1% BSA/0.1% Tween 20. Then, the plates were incubated with 1 μg/ml biotinylated rat anti-mouse IgA (clone C10–1, BD Pharmingen, Germany) for 1 h at RT, washed, incubated with streptavidin- HRP (1:2000; Sigma-Aldrich) for 1 h at RT and washed again. TMB substrate (Merck Millipore, Billerica, MA, USA) was added for 10-min, the reaction was terminated with H₂O₂ and absorbance measured at 450 nm with a TECAN Infinite M200 ELISA reader.