Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.
An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.
Salata
R A
RA
Department of Medicine, Case Western Reserve University, School of Medicine, Cleveland, Ohio.
Malhotra
I J
IJ
Hampson
R K
RK
Ayers
D F
DF
Tomich
C S
CS
Rottman
F M
FM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D002417:Cattle; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D013006:Growth Hormone / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000882:Haplorhini; D007108:Immune Tolerance; D007114:Immunization; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012322:RNA Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D012322:RNA Precursors; D012333:RNA, Messenger; D013006:Growth Hormone