Preparation Of Human APE1 Monoclonal Antibodies And Their Application In Serological Detection Of Cancer

The early detection and diagnosis is vitally important for the prevention and treatment of tumors. Detection of tumor markers,as one of the routine tools for the clinical diagnosis of tumors, plays an important role in the early diagnosis and therapeutic effect monitoring. With recent advances in biotechnology such as ELISA, RT-PCR, FISH, SELDI-TOF, proteomics, tissue microarray and gene microarray, many promising biomarkers have been identified and are currently under investigation and validation. However, it is unlikely that any one marker will predict perfectly the course of disease at present. Otherwise, the detection methods with high sensitivity and specificity in laboratory diagnosis of tumor are scarce and seriously needed. Masses of clinical practice suggest that the existing tumor markers are not quite effective in the early diagnosis of tumors because of their low sensitivity and specificity. Although combined detection of multiple tumor markers will be a realistic way to improve diagnosis of cancer sensitivity and specificity, total diagnosis accuracy can not be elevated efficiently. Hence, currently the main work of most laboratories is to search new tumor markers or combined detection methods with high sensitivity and specificity in the early diagnosis of tumors.Human apurinic/apyrimidinic endonuclease/redox effector factor (hAPE1) is a key enzyme in the base excision repair (BER) pathway which is responsible for the repair of DNA damage. As a protein with multifunction, hAPE1 is not only responsible for repair of AP sites, but also functions as a reduction-oxidation (redox) factor. It has been shown to be closely related to apoptosis, proliferation, differentiation and transformation of tumor cells, sensitivity of radiation therapy and chemotherapy as well as degenerative nervous system disease. Numbers of studies showed that hAPE1 expression is ubiquitous in tissue and organs. In some cancers, altered levels or cellular location of hAPE1 have been found, which are significantly different with the normal tissues and organs. Meanwhile, in our preliminary study, we found that the expression of hAPE1 is significantly increased in tumor patients'serum. Hence, alterations of hAPE1 expression levels and/or patterns may serve as a diagnostic factor in cancer screening. It indicates that hAPE1 is a good candidate for tumor early diagnose. Therefore, we speculated that detection of hAPE1 in serum may be helpful in early diagnosis of malignant tumors and evaluating radiosensitivity and chemosensitivityObjective1. To construct a prokaryotic expression vector of hAPE1 genes,induce its expression, purify hAPE1 fusion protein and analyze its biological function.2. To establish hybridoma cell lines with stable secretion of hAPE1 monoclonal antibody, prepare and purify hAPE1 monoclonal antibodies, evaluate their biological characteristics and present their preliminary application.3. To study the values of hAPE1 in diagnosis of malignant tumors with the serologic methodology, and evaluate its clinical values combined with multiple tumor markers.Materials and Methods1. Construction of prokaryotic expression vector for hAPE1 genes, purification and functional characterization of hAPE1 fusion proteinThe full-length of hAPE1 coding sequence amplified by PCR was subcloned into the prokaryotic expression vector pET28a(+). Then pET28a-hAPE1 was transformed into E.coli BL21 (DE3) and the expression of hAPE1 fusion protein was induced with IPTG. The expressed hAPE1 fusion protein was identified by SDS-PAGE and Western blot, and purified by affinity chromatographic with His column. The immunogenicity and activity of hAPE1 fusion protein was analyzed by Western blot and EMSA.2. Preparation, characterization and primary application of hAPE1 monoclonal antibodiesBalb/c mice were immunized with purified hAPE1 fusion protein by hypodermical and peritoneal injection. Hybridoma cells strains which could secrete monoclonal antibodies against human APE1 were prepared by cell conjugation, screened by indirect ELISA and amplified by cloning. Their chromosome nuclear type was identified with colchicine, specificity, genus cross-reaction titer and affinity were evaluate with Western blot and ELISA, and the antigenic epitope of monoclonal antibodies was determined by human APE1 15 peptide-array. The function of monoclonal antibodies was estimated with immuoblotting, immuocytochemistry, immunofluorescence and immunohistochemical test.3. Primary application of a sandwich ELISA for quantitative measurement of hAPE1 in serological detection of tumorsTo evaluate the diagnostic value of C-12 multiple tumor marker protein chip, the data of 12 tumor markers levels of sera in health examinations, benign disease, renal carcinoma, prostate cancer and testicular cancer patients were reviewed and analyzed. To detect the hAPE1 levels of serum and evaluate its diagnosis value of malignant tumors, the same serum specimens were evaluated using the sandwich ELISA method and compared with C-12 multiple tumor marker protein chip.Results1. Construction of prokaryotic expression vector for hAPE1 genes, purification and functional characterization of hAPE1 fusion proteinThe constrction of pET28a-hAPE1 was identified to be correct by DNA sequencing, and it could induce the high expression of hAPE1 fusion protein in E.coli BL21 (DE3) under the condition of 37℃and 0.4mmol/L IPTG. The protein was validated by SDS-PAGE and Western blot and its content accounted for nearly 50% of the total protein. The purity of hAPE1 fusion protein purified by His affinity chromatograph was higher than 90%. And it was verificated to have immunogenicity and AP endonuclease activity and redox function by SDS-PAGE,Western blot and EMSA.2. Preparation, characterization and primary application of hAPE1 monoclonal antibodiesTwo hybridoma cell strains named 2-G1 and 4-F6 were obtained successfully after cell conjugation. 2-G1 mAb and 4-F6 mAb with purity higher than 90% were obtained after concentration and purification of the bulk of abdominal dropsy. The potency was higher than 1:107, its Ig subtype contained IgG2b and IgG3, and affinity constants were 1.8×10-7 and 3.4×10-5 respectively. It could recognize both of nature and fusion hAPE1 protein, and showed no genus-cross reaction with rabbits, mice or rats. The antigenic epitope of 2-G1 mAb was verified to be the conformational epitope, which makes it to be a promising tool in immuoblotting, immuocytochemistry, immunofluorescence and immunohistochemistry regarding hAPE1.3. Primary application of a sandwich ELISA for quantitative measurement of hAPE1 in serological detection of tumorsThe serum hAPE1 concentration of health examination, benign disease, renal carcinoma, prostate cancer and testicular cancer patients were 8.97 (2.74,16.42) ng/ ml, 9.54 ( 2.36,14.22) ng/ml, 14.98 (7.14,45.33) ng/ml, 14.29 (10.83,33.68) ng/ml and 20.74 (8.76,58.81) ng/ml, respectively. These results demonstrated a significant increase of serum hAPE1 level in cancer patients compared to healthy control (P<0.05). The correct diagnosis rate of renal carcinoma, prostate cancer and testicular cancer using hAPE1 sandwich ELISA is 57.14%,53.33% and 66.67%,respectively. The correct diagnosis rate of renal carcinoma, prostate cancer and testicular cancer using C-12 multiple tumor marker protein chip is 48.65%,80.00% and 62.16%,respectively. The result indicated that there was no statistic difference in the above cancers. Except prostate cancer, hAPE1 has a great diagnostic value in the other two cancers combined with C-12 protein (P <0.05).Conclusion1. The prokaryotic expression vector of pET28a-hAPE1 is successfully constructed and highly expressed in E.coli BL21(DE3). The highly pure hAPE1 fusion protein which has immunogenicity and biological functions is successfully purified by His affinity chromatograph.2. Two hybridoma cell strains 2-G1 and 4-F6 which stably secrete hAPE1 monoclonal antibodies have successfully prepared. hAPE1 monoclonal antibodies with high titer, specificity and purity have been successfully produced. 2-G1 monoclonal antibody which has conformational antigenic epitope is an ideal agent for many immunologic experiments.3. Serum hAPE1 level in renal carcinoma, prostate cancer and testis cancer patients was significantly increased compared with healthy control. The hAPE1 Sandwich ELISA combined with C-12 multiple tumor markers protein chips could significantly increase the positive rate in diagnosis of renal carcinoma and testis cancer, which made it a promising biomarker for the diagnosis of malignant tumors.