[Preparation, characterization and application of monoclonal antibody against RecQ helicase]

Objective To prepare monoclonal antibodies (mAb) against RecQ helicase, characterize their biological properties, and then investigate their activity against RecQ helicase in tumor cells. Methods BALB/c mice were immunized with purified recombinant RecQ helicase from E.coli. Hybridoma technique was used to generate the mAb against RecQ helicase. The chromosome karyotype analysis of the hybridoma cells was performed by colchicine blocking. The Ig subtype and titer of mAb in ascitic fluid were determined by ELISA. The biological properties of mAb were detected via Western blotting and fluorescence polarization. Immunofluorescence was used to detect the interactions of mAb with BLM, RecQ4 and RecQ5 helicases in human breast cancer MDA-MB-231 cells. Immunohistochemistry was applied to detect the binding of mAb and RecQ helicase in K562 tumor cells and the corresponding stem cells. Results One stable hybridoma cell line expressing anti-RecQ mAb was obtained and named 6H5. The number of chromosomes was from 94 to 104, and Ig subtype was IgG1. The titer of 6H5 in ascitic fluid was 1×10^-7. The mAb could specifically recognize E. coli RecQ helicase and thus inhibit its DNA binding activity. Besides, the mAb could also recognize BLM, RecQ4 and RecQ5 in MDA-MB-231 cells, and sensitively detect the expression of the RecQ helicase in K562 tumor cells and the corresponding stem cells. Conclusion The mAb against RecQ helicase was successfully prepared with a high titer and specificity.