|Identification of epitopes recognized by a panel of six anti-human IgG2 monoclonal antibodies.|
Harada, S, et al.
J. Immunol. Methods, 141: 89-96 (1991)
Human IgG2 contains several subclass specific amino acid residues or deletions in the CH1 and CH2 domains and also in the hinge region. These substituted residues are the structural correlates for IgG2 specific epitopes. Since human IgG2 has different biological properties from other subclasses, some IgG2 epitopes may be located in regions correlating with sites determining the biological functions. Previously, we produced three anti-IgG2 monoclonal antibodies (mAbs) with highly specific and interesting reactivities using improved immunization protocols. However, it has been almost impossible to identify epitopes conventionally, because human IgG2 is so resistant to proteolysis that various proteolytic fragments could not be isolated. In this study, we identified the epitopes recognized by anti-IgG2 mAbs by SDS-PAGE, Western blotting, amino acid sequence analysis and peptide/mAb binding ELISA, thus overcoming the need for fragment isolation. A panel of six anti-human IgG2 mAbs, including the current WHO/IUIS specificity standards (HP6002, HP6008, HP6014) and our own (HG2-6A, HG2-30F, HG2-56F), reacted with distinct epitopes. The residues essential to expression of the epitopes recognized by the mAbs were: Pro234, Val235 and Val309 for HG2-56F, HG2-30F and HP6008, respectively. HP6014 reacted with the epitope expressed by Thr214 and its neighboring residues. HG2-6A was reactive with the hinge region, and HP6002 was assumed to be directed against discontinuous epitopes requiring intact Fc for expression. Through these studies, the pepsin and papain cleavage sites of human IgG2 were also clarified.
|Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes.|
Reimer, C B, et al.
Hybridoma, 3: 263-75 (1984)
Stable clones of 31 mouse hybridomas that produce monoclonal antibodies (MAbs) against human IgG antigenic determinants were obtained. The number of hybridomas of different specificity described are: 2 anti-IgG1 Fc, 1 anti-IgG2 Fc, 1 anti-IgG2 Fd, 2 anti-IgG3 Fc, 2 anti-IgG3 hinge, 3 anti-IgG4 Fc, 3 anti-IgG4 Fd, 2 anti-nG4m(b), 4 anti-IgGFc, 2 anti-IgGFd, 1 anti-kappa, 1 anti-lambda, 1 anti-non IgG1, 2 anti-non IgG2, 2 anti-non-IgG3, 2 anti-non-IgG4. Evidence is presented validating their specificity. Some MAbs demonstrated to be avid, potent, and specific for well defined IgG-subclass epitopes may be partially or completely inactive in other assay systems, presumably because of different presentations of antigen epitopes. In general, this problem requires careful writing of protocols describing the use of MAbs.