Cell–cell adherence as a selection method for the generation of anti-melanoma monoclonal antibodies

• Published in: Journal of immunological methods Vol. 203; no. 1; pp. 103 - 109
• Main Authors: Barral, Ana Marı́a, Fernández, Alicia, Faxas, Marı́a Elena, Pérez, Xiomara, Garcı́a, Carlos A, Rosén, Anders
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 11-04-1997
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Neoplasm - biosynthesis; Antigens, Neoplasm - immunology; Antigens, Neoplasm - isolation & purification; Cell Adhesion - immunology; Chromatography, Affinity; Humans; Hybridoma selection; Hybridomas - chemistry; Immunohistochemistry; Malignant melanoma; MEDICIN; MEDICINE; Melanoma - immunology; Melanoma-Specific Antigens; Mice; Mice, Inbred BALB C; Monoclonal antibody; Neoplasm Proteins - immunology; Plasmacytoma; Tumor Cells, Cultured; 2-ME, 2-mercaptoethanol; PEG, polyethylene glycol; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Malignant melanoma; Monoclonal antibody; Hybridoma selection; FCS, fetal calf serum
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(97)00025-2

The aim of the present study was to obtain monoclonal antibodies (mAbs) recognising human melanoma-associated antigens after immunisation of BALB/c mice with a 70–150 kDa membrane fraction from melanoma tumour tissues. Screening of specific antibody- producing hybridomas was performed using a novel cell–cell adherence method with the melanoma cell line M-14. Three mAbs of IgG1 isotype were selected: Mel-1, Mel-2 and Mel-3 which recognised the immunogen by ELISA and stained several melanoma cell lines positive in immunofluorescence. The molecular weight of the antigen was studied by different methods; a 170-kDa band was identified following immunoblotting of tumour lysate and a 72-kDa band was observed following immunoaffinity purification. Cell–cell adherence appears to be a reliable procedure for the generation of mAbs against native cellular antigens.