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Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

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Abstract

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 α-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-αhCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free α-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 α-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: αhCG 100%; intact hCG 1.8%; βhCG 0.14%; αFSH 24.5%; intact FSH 0.8%; βFSH 0.09%; αLH 20.5%; intact LH 0.9%; βLH 0.08%; αTSH 50.5%; intact TSH 3.7%; βTSH 0.07%;

The affinity constant (K) of this Mab with respect to free α-subunit of hCG was found to be 1.5 × 107 1/mol as determined by the simple antibody dilution analysis method.

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  1. Laboratório de Engenharia Bioquímica do Departamento de Engenharia Química do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1900, Lisboa, Portugal

    Amin Karmali

  2. Serviço de Bioquímica II do Departamento de Biotecnologia e Química Fina do INETI, Estrada do Paço do Lumiar, 1699, Lisboa Codex

    Carlos Novo & Ana Domingos

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Novo, C., Domingos, A. & Karmali, A. Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin. Mol Biotechnol 17, 119–128 (2001). https://doi.org/10.1385/MB:17:2:119

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