Protein a hp spintrap

IgG antibodies were isolated from whole serum from two individual rabbits using a Protein A HP SpinTrap (GE Healthcare, Pittsburg PA) according to the manufacturer’s antibody capture and cross-linking protocol. The resultant rabbit anti-native BP affinity columns were then utilized to isolate antigenic proteins from native BP protein extracts. Two different types of columns were made, one with D0 serum and one with D84 anti-BP serum. Briefly, serum was diluted 1:10 with binding buffer (50 mM Tris, 150 mM NaCl, pH 7.5), and 200 μL was incubated in the column with end-over-end rotation for 30 min. The column was then washed with 400 μL binding buffer, followed by 400 μL of 200 mM triethanolamine and cross-linked with 400 μL of 50 mM dimethyl pimelimidate dihydrochloride (DMP) in 200 mM triethanolamine for 1 h with end-over-end rotation. Columns were blocked with 400 μL of 100 mM ethanolamine, and unbound antibodies removed with 200 μL of pH 2.9 elution buffer (0.1 M glycine with 2 M urea). Columns were incubated with 200 μL of native BP protein extract for 1 h with end-over-end rotation. Bound proteins were then eluted using sequential washes with elution buffer at a pH of 5 and 4 for non-specific binding and final specific-binding elution at pH of 2.9. All washes and run-throughs were collected for later analysis and stored at −80 °C.

An affinity batch spin column (3.2 mL) was prepared for the purified anti-male-attracting peptide rabbit IgG or purified normal rabbit IgG by mixing the IgG (32 mg) with Protein A-coupled Sepharose (Protein A HP SpinTrap; GE Healthcare), followed by covalent cross-linking of the bound antibody using dimethoxypropan. The equivalent amount to water conditioned by 2.5 females was dissolved in binding buffer (50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl; 6.4 mL), introduced into the spin column, mixed by manual inversion and then incubated with slow end-over-end mixing for 1 hour. The non-adsorbed fraction was collected by centrifugation. The column was then washed five times with 50 mM Tris-HCl and 150 mM NaCl with 2 M urea at pH 7.5 (12.8 mL), and the adsorbed substances were eluted with 0.1 M glycine with 2 M urea at pH 2.9 (22.4 mL).

Polyclonal antibodies were commercially raised in rabbits against recombinant [Syb (1-75)] antigen (Covalab, UK). Immunoglobulins were purified against Protein A from 1 ml of rabbit serum using a Protein A HP SpinTrap (28-9031-32, GE Healthcare) following the manufacturer's instructions.