Molecular characterization of the GM 4672 human lymphoblastoid cell line and analysis of its use as a fusion partner in the generation of human-human hybridoma autoantibodies.
The GM 4672 lymphoblastoid cell line has been used in cell hybridization experiments with peripheral blood lymphocytes (PBLs) in order to generate human-human hybridomas that secrete immunoglobulins directed against a number of different autoantigens. The GM 4672 cells were fused with PBLs isolated from patients with rheumatoid arthritis or systemic lupus erythematosus, or from normal individuals, and the resulting hybridomas were screened for reactivity to platelets, erythrocytes, DNA, cardiolipin, human IgG-Fc, phosphatidylethanolamine, and for lupus anticoagulant activity. This report analyzes the results from 149 fusion experiments completed over a period of nine years. Fifty to sixty-six percent of the fusion experiments resulted in immunoglobulin-secreting clones, with an average of 15 clones/fusion. The hybridoma antibodies were predominantly of the IgM heavy chain isotype, and 67% expressed kappa light chains. Although most hybridoma antibodies (78%) recognized a single autoantigen, 22% recognized more than one autoantigen and were considered polyreactive. In addition, the light and heavy chain variable regions of the antibody secreted by the GM 4672 cell line were amplified by the polymerase chain reaction technique and sequenced. The GM 4672 light chain was encoded by a VkI gene and used a Jk4 minigene. The GM 4672 heavy chain was derived for the rearrangement of a gene from the VH4 subgroup and used a JH4 minigene. The 8 amino acid long diversity region was generated by the fusion of the DK1 and DLR2 genes. The hybridomas generated in fusion experiments, when examined, did not appear to secrete antibodies using the immunoglobulin variable regions derived from the GM 4672 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Rioux
J D
JD
Department of Medicine, McGill University, Montreal General Hospital Research Institute, Quebec, Canada.
Rauch
J
J
Zdarsky
E
E
Newkirk
M M
MM
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007132:Immunoglobulin Isotypes / Q000032:analysis; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data
D001323:Autoantibodies; D000939:Epitopes; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region