Production of a monoclonal antibody against the hRRM2 subunit of ribonucleotide reductase and immunohistochemistry study of human cancer tissues.
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.
Zhou
Bingsen
B
Department of Molecular and Clinical Pharmacology, City of Hope National Medical Center, Duarte, California.
Phan
Vu
V
Liu
Xiyong
X
Juhasz
Agnes
A
Chu
Peiguo G
PG
Yen
Yun
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000951:Antigens, Neoplasm / Q000276:immunology; D001943:Breast Neoplasms / Q000378:metabolism; D002277:Carcinoma / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000276:immunology*; D009959:Oropharyngeal Neoplasms / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012262:Ribonucleoside Diphosphate Reductase / Q000276:immunology*; D012264:Ribonucleotide Reductases / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D012264:Ribonucleotide Reductases; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase