The Establishment Of APE1 Serological Assay And Its Application In Lung Cancer Diagnosis

Lung cancer is the most common malignant tumor with high morbidity and mortality in China, and the morbidity is increasing year by year. The World Health Organization pointed out, 90~95 percent cancer is curable with early diagnosis and timely treatment. Early diagnosis of lung cancer is still the most effective way to improve the survival rate. Tumor markers have been used in the tumor screen, diagnosis, prognosis, evaluation of efficacy and the follow-up of high-risk groups. With recent advances in biotechnology such as proteomics, FISH, ELISA, RT-PCR, and SELDI-TOF, many promising biomarkers have been identified and are currently under investigation and validation. However, it is unlikely that any tumor marker will perfectly diagnose lung cancer. At present, the recombination of multiple predictive factors will be a realistic way to improve lung cancer in screening and staging. At present, searching for more specific and sensitive tumor markers is the most promising way to improve cancer diagnosis.APE1 is a multifunctional protein that not only repairs the apurinic/apyrimidinic sites of DNA lesions, acting as a rate-limiting enzyme of BER pathway, but also functions as a reduction-oxidation factor protecting cells against the toxic effects. We believe the dysfunction of APE1 have the potential in increasing susceptibility of cancer. Most studies show that, lung cancer existed APE1 overexpression and altered localization, these alteration are likely to be the abnormal cell phenotype determinants. Since the tumor cell multiplication is excessively quickly, within unceasingly cell apoptosis and necrosis, a large number of cellular proteins released into the blood, so we proposed that APE1 protein in serum may be increased. APE1's altered expression, primarily at the protein or subcellular localization level may stimulate organism to produce antibodies. As the antibody amount is more than the antigen and antibody exist in serum for a long time, so the detection of specific antibody may allow for identification of an underlying tumor. APE1 is expected to become a new biomarker for lung cancer. To our knowledge, there is no report about APE1 proteins or antibodies level in serum. Studies on screening APE1 alterations were mostly by immunohistochemical staining or western blot. So far, this is the first examination of serum APE1 level in relation to cancer diagnosis. Our main objective of this prospective study is 1) to prepare rabbit anti-human APE1 polyclonal antibody 2) to evaluate clinical value of serum APE1 protein and antibody or in combination with CEA, CA125 and CA242 in distinguishing between lung cancer patients and healthy.Objective:1. To prepare rabbit anti-human APE1 polyclonal antibody and then characterize it.2. To establish a sandwich ELISA for quantitative measurement of APE1 protein, measure serum APE1 level in healthy and lung cancer patients, and to study its clinical application in combination with CEA, CA125 and CA242.3. To detect serum APE1 antibody in lung cancer patients and healthy by indirect ELISA, monitor patients before and after chemotherapy, combine with CEA, CA125 and CA242 to evaluate if serum APE1 antibody can be used as a new tumor marker and predictor of therapeutic efficacy.Materials and Methods:1. Preparation and characterization of rabbit anti-human APE1 polyclonal antibody Rabbit anti-human APE1 polyclonal antibody was prepared by a modified rapid immune procedure followed by purification of specific avidity column. The efficacy of this polyclonal antibody was certificated by ELISA, Western blot and immunohistochemistry.2. Establishment and primary application of a sandwich ELISA for quantitative measurement of APE1The assay was based on the sandwiching of the antigen between a monoclonal mouse anti-APE1 antibody, pre-coated on a 96 well polystyrene plate, and a polyclonal rabbit anti-APE1 antibody, which was then detected with a peroxidase-labeled goat anti-rabbit antibody. The optimal concentrations were defined by titration. Standard curve was performed using purified APE1 protein and was judged by sensitivity, reproducibility and recovery rate. The sandwich ELISA was tried to measure the APE1 levels in healthy and lung cancer patients, and then combined with CEA, CA125 and CA242 to study its clinical application. 3. Establishment and primary application of indirect ELISA in lung cancer and healthy Indirect ELISA was carried out to detect APE1-Abs in 345 lung cancer patients and 350 healthy. Ninety-one patients were monitored before and after chemotherapy. The data was analyzed by independent t-test, ROC curve and chi-square test. APE1 antibody was combined with CEA, CA125 and CA242 to study its clinical application.Results:1. The titer of the antiserum obtained in this experiment determined by ELISA was up to 1∶128 000.The kaff value of the antibody was 8.96×10-6mol/L. Western blot results showed the antibody has high specificity to APE1 protein. The final purified antibody was highly specific to native form of APE1 protein in human, mouse and rat.2. In the range of 8.0 ng/mL to 200 ng/mL, the APE1 antigen showed a good linearity in the standard curve. The sensitivity of this assay was 2.0 ng/mL. The intra-assay precision and inter-assay precision were 8.37%～8.97% and 10.47%～14.57% respectively. The average recovery rate was 90.83 %. APE1 protein levels in serum were skewed distribution, The 95% CI of serum APE1 concentration in healthy and lung cancer patients were 3.67ng/mL～16.58ng/mL and 7.06ng/mL～24.90ng/mL respectively. The results demonstrated a significant increase of serum APE1 level in cancer patients compared to healthy (P<0.05).3. Retrospective analysis found that, CEA, CA125 and CA242 were more specific for diagnosis of lung cancer and had higher positive rate. APE1 combined with these three makers can elevate the diagnostic sensitivity and correct rate. In this study, the diagnostic sensitivity, specificity and correct rate were 68.57%, 80.00% and 74.15%, respectively. The diagnostic sensitivity and accuracy were improved than a single indicator, but at the same time the specificity of diagnosis were reduced.4. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy volunteers (P=0.000). For each possible cut-point, the resulting sensitivity and specificity were indicated as a point on the ROC graph. The area under ROC was 0.735. One hundred and nineteen (34.49%) lung cancer patients and 12 (3.43%) healthy were positive with APE1-Abs. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs (P>0.05, respectively).5. We also found serum APE1-Abs were significantly associated with response to therapy. Serum APE1-Abs of responders were significantly higher after chemotherapy (P=0.000), while APE1-Abs level in non-responders, the difference was not significant (P=0.393).6. Diagnostic sensitivity, specificity and correct rate of APE1 antibody with combination of CEA, CA125 and CA242 were 71.17%, 95.57% and 84.03%, respectively.Conclusion:1. Rabbit anti-human APE1 polyclonal antibody, with high titer and specificity, was successfully produced by a modified rapid immune procedure. This polyclonal antibody can be further applied in ELISA, Western blot and immunohistochemistry to elucidate the roles of APE1 protein played in many important cellular procedures.2. Successfully established a sandwich ELISA assay for APE1 protein with good sensitivity and reproducibility for the first time.3. Serum APE1 level in cancer patients was significantly increased compared to healthy. Serum APE1 combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate, APE1 may become a new diagnostic biomarker for cancer.4. Successfully established an indirect ELISA assay with good sensitivity and reproducibility for the first time. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs levels.5. Serum APE1-Abs was also significantly correlated with response to therapy, and it may be a predictor of therapeutic efficacy.6. APE1 antibody combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate.