We report production of a
monoclonal antibody against the hRRM2 subunit of
ribonucleotide reductase and immunohistochemistry (IHC) staining of human
cancer tissues available in
paraffin block. BALB/c mice were immunized with purified hRRM2
protein, and splenocytes from these mice were fused with mice myeloma cell lines by using standard hybridoma production techniques. Resulting hybridomas producing anti-hRRM2
antibodies were screened by
enzyme-linked
immunosorbent assay (ELISA). The specificity was determined by limiting serial dilutions. Clones were chosen for antibody production based on their activities on
paraffin-embedded human tissues. They were then isotyped and shown to produce
immunoglobulin M (
IgM)
antibodies against hRRM2. Using these
antibodies, we performed Western blot on oropharyngeal KB
cancer cell lines and immunohistochemistry staining of available
paraffin-embedded
cancer tissues. Interestingly,
cancer tissues stained positive with the anti-hRRM2 antibody but not normal tissues. Colon, stomach, liver, lung, pancreatic, and
breast cancer had the strongest staining. No staining was identified on
astrocytoma,
mesothelioma, or myeloma. Our findings were validated with data from
reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrating overexpression of hRRM2 in
breast cancer tissues compared to matched noncancer tissues. We propose that IHC with this monoclonal anti-hRRM2 antibody may be useful for
ribonucleotide reductase research and as a
biomarker for tumorgenesis.