Purification and characterization of monoclonal antibodies against the free alpha-subunit of human chorionic gonadotrophin.
Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.
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Serviço de Bioquímica II do Departamento de Biotecnologia e Química Fina do INETI, Estrada do Paço do Lumiar, 1699 Lisboa.
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification*; D000918:Antibody Specificity; D003429:Cross Reactions; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D021122:Protein Subunits
D000911:Antibodies, Monoclonal; D015292:Glycoprotein Hormones, alpha Subunit; D021122:Protein Subunits