Hitrap n hydroxy succinimide activated high performance affinity column

This experiment was performed according to the method that we established in a previous study. Auto-IgG was purified from 15 ml of mixed serum from patients with bullous pemphigoid using HiTrap Protein G and a HiTrap N-hydroxy-succinimide-activated high-performance affinity column (Amersham Biosciences, Little Chalfont, UK) coated with the BP180 NC16A domain. HaCaT cells seeded on coverslips in 6-well plates were then incubated overnight with 1 μg/ml purified pathogenic IgG at 37°C, before being incubated for 2 h with 10 μg/ml recombinant CD55 protein and 1 ml of fresh serum from healthy controls containing complement components to initiate complement activation as a simulation of the bullous pemphigoid phenotype. C3b deposition at the DEJ and cell membrane was used as a measure of the degree of complement activation.

Auto immunoglobulin IgG was purified from 15 ml of mixed serum from BP patients using HiTrap Protein G and a HiTrap N-hydroxy-succinimide-activated high-performance affinity column (Amersham Biosciences, Little Chalfont, UK) coated with the BP180 non-collagenous 16A domain (NC16A). HaCaT cells seeded on coverslips in 6-well plates were incubated overnight with 1 μg/ml purified pathogenic IgG from BP patients, followed by addition of 10 μg/ml recombinant CD46 protein and 1 ml of fresh serum and incubation for 2 h to initiate complement activation to simulate the BP phenotype. C3 deposition at the DEJ and cell membrane was used as a measure of the degree of complement activation.