Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization.
To study the expression of HLA-DQ beta chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 beta chain sequences. The splenocytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specific mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenocytes. Immunoblot analysis showed that several of the mAbs cross-reacted with DQ beta chain molecules. One mAb to a peptide representing DQw8 beta position [49-60] specifically recognised the DQw8 beta chain. Three mAbs to a peptide representing DQw8 beta position [39-52] specifically recognised an epitope consisting of Gly-Val-Tyr in position 45-47, i.e., all DQ beta alleles except DQw7 beta (position 45-47: Glu-Val-Tyr) and DQw2 beta (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound lymphocytes with the same specificity as found by immunoblotting analysis. Thus, by combining in vivo and in vitro immunization we have generated a number of epitope specific monoclonal IgG antibodies that distinguish closely related HLA-DQ beta chain alleles in predetermined positions.
Petersen
J S
JS
Hagedorn Research Laboratory, Gentofte, Denmark.
Dyrberg
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D000939:Epitopes; D005434:Flow Cytometry; D006681:HLA-D Antigens / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006681:HLA-D Antigens; D007074:Immunoglobulin G; D010455:Peptides