Human alfa-fetoprotein: isolation and production of monoclonal antibodies.
Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.
LNETI-DTIQ Bioquímica, Estrada das Palmeiras, Portugal.
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A
Karmali
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C
Novo
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005312:Fetal Blood / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D000509:alpha-Fetoproteins / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000509:alpha-Fetoproteins