A human monoclonal antibody, produced following in vitro immunization, recognizing an epitope shared by HLA‐A2 subtypes and HLA‐A28

Abstract

In vitro immunization and subsequent immortalization of peripheral blood cells of a multiparous woman has resulted in the production of a stable human mouse heterohybridoma, 5C2A2, secreting an HLA-A2/A28-specific human monoclonal antibody. Although possibly exposed to HLA-A2 by transfusions, the cell donor showed no HLA-A2-specific serum antibodies. The present protocol for in vitro immunization includes the elimination of suppressor cells from the responder cell population, the presence of irradiated allogeneic lymphocytes as a source of antigen, as well as stimuli--recombinant interleukin-2 and a B-cell specific nucleoside analogue--causing the proliferation of B lymphocytes, prior to immortalization. The ability of the antibody 5C2A2 to detect all known HLA-A2 subtypes, except A2.3, and A28, allows identification of the serological epitope on the HLA-A2 molecule. Application of this in vitro immunization method allows the production of a set of HLA monoclonal antibody-secreting human hybridomas, independent of the existence of serum HLA antibodies in the lymphocyte donors.

Monocyte Recruitment by HLA IgG-Activated Endothelium: The Relationship Between IgG Subclass and FcγRIIa Polymorphisms

“…Human monoclonal allele specific antibodies against HLA-A2 (clone SN230G6) or HLA-A3 (clone MUL2C6), were produced by human hybridomas generated as previously described (77–79), and were purified by protein A affinity chromatography. The UCLA Immunogenetics Center maintains a repository of polyclonal reference sera from multiparous women consented for research purposes (80) that are well defined for HLA antibodies.…”

Structural aspects of human leukocyte antigen class I epitopes detected by human monoclonal antibodies

“…For the 5 eplets in this study, we have calculated from the models in Fig. 1 that eplets and their corresponding 15-Å 2 surface areas have an average of 33 residue locations (range [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. These numbers are higher than the general 15 to 25 range of contact residues in structural epitopes of experimentally tested protein antigens.…”

“…This study was performed using IgG-and IgM-type mAbs derived from Caucasoid women (Dutch) who became sensitized during pregnancy by paternal antigens [32][33][34]. All were supernatants of cloned hybridomas generated from Epstein-Barr virus-transformed B cells.…”

Down-regulation of HLA-A and HLA-Bw6, but not HLA-Bw4, allospecificities in leukemic cells: an escape mechanism from CTL and NK attack?

“…HLA antibody-producing EBV cell lines were stabilized by electrofusion and rigorous cloning. [22][23][24][25] The HLA specificity of mAbs was determined by testing with a large (n Ͼ 240) panel of HLA-typed peripheral blood mononuclear cell suspensions in the conventional complement-dependent microcytotoxicity (CDC) assay. For flow cytometry application, HLA hu-mAbs were used as undiluted hybridoma culture supernatants.…”