Production of monoclonal antibodies to thyroglobulin by in vitro immunization with a free synthetic peptide.
A synthetic peptide corresponding to amino acids 1-19 of thyroglobulin was used to test the possibility of generating protein-reactive monoclonal antibodies by immunization in vitro with a synthetic peptide as antigen. Splenocytes from non-immunized Balb/c mice were cultured in serum-free medium for 3 days in the presence of thymocyte-conditioned medium and the synthetic peptide prior to fusion with SP2/0 murine myeloma cells. The synthetic peptide was used in its free form, i.e. not coupled to a protein carrier. Hybridomas secreting monoclonal antibodies reactive with the synthetic peptide were obtained after immunization in vitro with as little as 10 ng/ml of the synthetic peptide. Between 50 and 70% of the primary clones obtained in different experiments produced monoclonal antibodies also reactive with the intact protein. Six stable hybridomas were isolated; all produced antibodies of the IgM class. We conclude that immunization in vitro with a free synthetic peptide is an efficient method for the generation of monoclonal antibodies reactive with the intact protein.
de Boer
M
M
Laboratory of Biochemistry, Biotechnology Centre, University of Amsterdam, The Netherlands.
Ossendorp
F A
FA
Al
B J
BJ
Hilgers
J
J
de Vijlder
J J
JJ
Tager
J M
JM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001324:Autoantigens / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology*; D013154:Spleen / Q000276:immunology; D013954:Thyroglobulin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D007075:Immunoglobulin M; D010446:Peptide Fragments; D013954:Thyroglobulin