Affi gel protein a maps 2 kit

The isolation was according to previously described methods [6 (link)]. The β₂GPI previously purified was coupled to Cyanogen bromide-activated Agarose (Sigma-Aldrich, MO, USA). Briefly, 1 g of the CNBr activated agarose was mixed with 1 mM HCl, then 15 mg of pure unnicked β₂GPI dissolved in 0.1 M NaHCO3/0.5 M NaCl, pH 8.3 was binding to 4.0 ml of the activated agarose. The solution was stirred at 4 °C over night. The unreacted sites were blocked with 0.2 M glycine pH 8.3. Finally it was washed 10 times alternating with 0.1 M NaHCO3/0.5 M NaCl, pH 8.3 and 0.1 M acetate buffer/0.5 M NaCl, pH 4, loaded to the column, equilibrated by PBS pH 7.4 and stored at 4 °C. The IgG fraction from mice pooled plasma that was isolated with Affi-Gel Protein A MAPS II Kit (Bio-Rad, CA, USA) was applied to the CNBr-β₂GPI column. After washing with the same buffer, bound anti-β₂GPI antibodies were eluted with 0.1 M glycine-HCl pH 2.5. Eluates were collected from 1 ml and neutralized immediately with saturated solution of Na₂CO₃. The absorbance was reader to 280 nm, the peaks (containing anti-β₂GPI antibodies) were collected, concentrated and dialyzed against PBS buffer pH 7.4. Finally, we analyzed the capacity to react of this antibody with two different β₂GPI (reference and purified) by ELISA and Western Blot, the fraction was maintained at −80 °C.

OVA-IgG1-IC was formed as previously described (Baker et al., 2011 (link)). In brief, mouse monoclonal anti-OVA-IgG1 antibodies purified using Affi-Gel Protein A MAPS II kit (Biorad) from hybridoma (clone 01–4), a kind gift of H. Karasuyama (Tokyo Medical and Dental University, Tokyo, Japan), was incubated at 1:2 molar ratio of anti-OVA-IgG1 to OVA at 37°C for 30 min. OVA-IgG1-IC (100 µg) was given i.p. to BALB/c WT female mice once weekly for 6 wk during pregnancy and breastfeeding, or for 3 wk during breastfeeding.