Characterization Of Monolonal Antibody Of Human NY-ESO-1

Objective: To develop and character the monolonal antibodies of human NY-ESO-1, a member of cancer-testis antigen family.Methods: Human NY-ESO-1 gene was prepared by RT-PCR and sub-cloned into the prokaryotic expression vector pGEX-4T-2; The recombinant plasmid was transformed into E.coli and fusion protein was purified through GSTrapFF column for immunizing Balb/c mouse. The traditional protool for preparing monolonal antibody was followed with modification for some proedures. The monolonal antibody of anti-NY-ESO-1 screened in this study was identified for its Ig subclass, subtype and specificity. The distribution of NY-ESO-1 in breast cancer tissues was detected by IHC with mAb 6A4 and mAb 7D6 as well, in which mAb 6A4 revealed a better result than mAb 7D6.Results: A high level expression of GST-NY-ESO-1 was obtained and followed by purification with more than 90 percent. The fusion protein was cleaved by bovine thrombin and subjected to immunize Balb/c mice. Murine monolonal antibody were prepared using traditional hybridoma technique.the titer of monolonal antibody were detected by indirect ELISA and two monolonal antibody were obtained with titer 10^-5 and 10^-6 respectively. Type were identified as mouse IgG_2b /K with a specificity for NY-ESO-1. The monolonal antibody indicate specificity and only reacted with NY-ESO-1. About 25% of the samples expressed NY-ESO-1 antigen, eight out of the 23 samples (34.8%) were found to be NY-ESO-1 mRNA positive.