Preparation of monoclonal antibodies against human telomerase

Telomerase, a ribonucleoprotein enzyme that extends telomeres of eukaryotic chromosomes, consists of the catalytic protein submit telomerase reverse t ranscriptase (TERT) and a telomerase RNA subunit. Nearly 85% of human tumor s have tested positive for high telomerase activity. Telomerase activity is very low or not present in normal cells, whereas it is up-regulated in imm ortalized cells. Telomerase, partially purified from the breast tumor cell line MCF-7, was used to immunize Balb/C mice. Monoclonal antibodies (MAbs) were prepared by conventional hybridoma technology and screened by enzyme-l inked immunoadsorbent assay (ELISA), followed by a polymerase chain reactio n (PCR) based telomeric amplification repeat protocol (TRAP) assay to detec t binding to or inhibition of telomerase activity. Reactive MAbs were found to be of IgM type by mu -specific ELISA. Two MAbs were characterized, one that neutralizes telomerase activity in TRAP assay and the other non-neutra lizing. In Western blotting, crude telomerase extract and HIV-1 virus lysat e (control) were blotted on nitrocellulose membranes and the strips were tr eated with both MAbs and a nonrelated HIV polymerase-specific MAb, also IgM type. A band of approx. 65-kDa was detected in extracts of 293 cells with both the MAbs, but no reaction occurred with the HIV polymerase-specific MA b used as control. Similarly, when HIV-1 virus lysate strips were treated w ith HIV polymerase-specific MAb, a 65-kDa band was detected and no band was observed with either of the hybridoma supernatants. These antibodies may b e useful for studying regulatory mechanism of telomerase and inhibition of its activity in vitro and in vivo.