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Isolation and characterisation of antibodies which specifically recognise the peptide encoded by exon 7 (v2) of the human CD44 gene

Aims—Exon 7 of the human CD44 gene is overexpressed in many commonly occurring carcinomas. The aim of the study was to explore the diagnostic and therapeutic potential of this frequent abnormality. Methods—A new monoclonal antibody (mAb, M-23.6.1) and a polyclonal antibody (pAb,S-6127) to the corresponding antigen were raised by immunising mice and sheep, respectively, with a specially constructed fusion protein HIV2 (gp32)-CD44 exon 7. Results—Characterisation of mAb, M-23.6.1 by ELISA, western blotting, immunocytochemistry, and FACS analysis confirmed that it specifically recognises an epitope in the region between amino acids 19 and 33 of the peptide encoded by this exon. Western blotting experiments with two cell lines, RT112 and ZR75-1, known from RT-PCR data to be overtranscribing the exon, yielded a monospecific band of approximately 220 kDa, and immunocytochemistry showed discrete membrane staining on the same cell lines. Fluorescent antibody cell sorting (FACS) revealed binding to greater than 90% of the cells of each of these lines. Specificity of recognition of the antigen was shown by inhibition of the precise immunoreactivity typically seen in ELISA and Western blots, by pre-incubation with synthetic exon 7 peptide or fragments of it. Conclusions—The new antibodies will be useful tools for the further analysis of abnormal CD44 isoforms and their clinical implications.