Easy titer igg assay kit

The purification of serum total IgG (tIgG) was performed on the Protein G HP Spin Trap column as described by the manufacturer (GE Healthcare, USA). The tIgG samples were eluted at pH 2.5, immediately neutralized, dialyzed against phosphate buffered solution (PBS, 0.1% NaN₃), and stored at +4°C until being tested. About 8.5 mg of IgG was obtained from 1 mL of serum applied onto the Protein G Sepharose column. To obtain the IgG-depleted serum we used the same method on the Protein G HP Spin Trap column, except the serum volume applied to the Protein G column was three times lower, and the complete depletion of IgG was controlled using the Easy-Titer IgG Assay Kit (Thermo Scientific, USA).

To assess mAb ability to inhibit MP activity, a cell-based CD23 cleavage assay was performed, as described previously [6 (link)], in the presence of ADPs either concentrated from hybridoma supernatants or purified. Supernatants were concentrated (10×) with Amicon Ultra Centrifugal Filters (MilliporeSigma), dialyzed against PBS using Micro Float-A-Lyzer dialysis units (Spectrum Laboratories, Rancho Dominguez, CA, USA), and quantified with an Easy Titer IgG Assay kit (Thermo Fisher Scientific). Isotype-matched controls were IgG1 (clone G3G4), IgG2b (clone 10F1), and IgG2c (clone 6.3, ASB-1220, Nordic Biosite, Wayne, PA, USA). Anti-ADAM8 MAB1031 Ab (R&D Systems, RRID: AB_2305036) was used as positive control for MP inhibition. To assess the ability of ADPs to inhibit DI activity, we measured their effects on the adhesion of CHO cells expressing α9β1-Integrin to rHuADAM8, as reported previously [16 (link)], using either dialyzed or purified ADPs. Control IgGs and MAB1031 were used as negative and positive controls for DI inhibition, respectively.