Hinge region of human IgG2 protein: conformational studies with monoclonal antibodies

We previously produced three anti-human IgG2 mAbs with high specificity and found that they recognize distinct epitopes in the hinge region and neighboring residues in human IgG2: HG2-6A was reactive with the hinge region (Glu216-Pro230); HG2-56F with the Pro234 residue and HG2-30F with the Val235 residue. In this study, we evaluated the reactivities of those three mAbs with human IgG2 protein under various conditions. The results obtained using HG2-6A mAb indicated that the hinge region was concealed in the native form, but exposed after heat treatment at 63 degrees C, or chemical treatment with 3 M KSCN, 3 M guanidine, 30% CH3CN, 8 M urea or acid at pH 2.0 as well as by adsorption onto polystyrene beads. The IgG2 hinge region was also exposed after binding to specific antigens. The Pro234 residue recognized by HG2-56F mAb was exposed under all conditions studied. The neighboring Val235 residue recognized by HG2-30F, however, was completely concealed in the native and antigen-bound states. Only treatment with 3 M guanidine and acid at pH 2.0, or physical adsorption induced conformational changes to partially expose the Val235 residue.