Cnbr sepharose

Anti-hMD-2 antibody (ab24182, Abcam) was coupled to CNBr-Sepharose (600 μg/ml gel; Sigma-Aldrich). Human SS plasma was diluted 5-fold in PBS and incubated at 4°C for 18 hours with the antibody-conjugated Sepharose. MD-2-depleted plasma was collected after pelleting the antibody-coated Sepharose by centrifugation and the MD-2 depletion was confirmed by western blot.

I(Glc) (3 mg) and the non-glucosylated adhesin I (3 mg) were dissolved in NaHCO₃ (0.1 M, 3 mL) containing 0.5 M NaCl, pH 8.3, and were coupled to CNBr-Sepharose (200 mg) matrix (Sigma). The NBD1 and the MS1 sera (3 mL) positive to both CSF114(N-Glc) and I(Glc) (diluted 1:10 in PBS) were passed three times through the non-glucosylated adhesin I column pre-equilibrated with PBS, pH 7.2. In the case of MS1, the un-retained flow-through fraction was directly loaded onto the column and recirculated three times. Adsorbed antibodies were eluted using glycine (0.1 M, 10 mL, pH 2.6) and recovered in D-PBS buffer pH 7.2. Protein content of specific anti-I(Glc) purified IgG antibodies was determined to be 46.5 μg/ml by UV spectroscopy measuring the absorbance of the solution at 280 nm (Shimadzu UV-1601PC) considering that the extinction coefficient is 1.4 for a solution of 1 mg/mL. The efficiency of this sequential immunoadsorbtion was confirmed by SP-ELISA.
The total IgG fraction from NBD1 serum was obtained by Protein G affinity chromatography using a prepacked column (Amersham Biosciences, NJ) according to manufacturer’s instructions. Protein content of the total IgG fraction from NBD1 serum was determined to be 1.7 mg/mL using the method above described.