Production and characterisation of a monoclonal antibody specific for apoptotic bodies derived from several tumour cell lines

• Published in: Journal of immunological methods 1999-08, Vol.228 (1), p.49-58
• Main Authors: Gautier, Fabien, Lassort, Danielle, Le Cabellec, Marie-Thérèse, Videau, France, Minet, Lorette, Meflah, Khaled, Pradal, Gilbert, Harb, Jean
• Format: Article
• Language: English
• Subjects: Ageing, cell death; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - isolation & purification; Antibody Specificity; Apoptosis - immunology; Apoptotic bodies; Autoantibodies; Autoantigens; Biological and medical sciences; Cell physiology; Colonic Neoplasms - immunology; Colonic Neoplasms - pathology; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Humans; Immunization; Microscopy, Fluorescence; Microscopy, Immunoelectron; Molecular and cellular biology; Monoclonal antibody; Rats; Tumor Cells, Cultured
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00093-9

We have recently shown that apoptotic bodies (apobodies) derived from rat colon carcinoma cell lines (PROb) after sodium butyrate (NaB) treatment were able to cure rats with induced peritoneal carcinomatosis ( Boisteau et al., 1997). A specific immune response was assumed to be involved since the serum of cured rats contained antibodies specific for apobodies. In the present study, a mAb (clone 6E8) produced by immunisation of rats with apobodies strongly recognized apobodies but had little reactivity with parental tumour cell lines, as demonstrated by enzyme-linked immunosorbent assay (ELISA), immunostaining and flow cytometry. Immunoelectron microscopy showed that 6E8 mAb mainly stained the hyaloplasm or cytosol of apobodies. A protein was detected at 67 kDa by immunoprecipitation of apobodies with mAb, followed by immunoblotting, using serum of rats immunised with apobodies. The 6E8 mAb recognized apobodies derived from several rat or human colon cancer cell lines and a rat glioma cell line, regardless of the apoptosis stimulus used (NaB, staurosporine or UV). Our results clearly show that 6E8 mAb defines an epitope specifically generated during apoptosis, which suggests that the protein recognized may be involved in the molecular cascade of apoptotic cell death.