Preliminary Investigation Of A New Tumor Marker ELISA Based On REGγ Protein

It is key to improve the detection rate of early tumor patients due to the low cure rate of advanced tumor patients.The development of detection kit based on tumor markers is one of main approach to early diagnosis of tumors.Currently,over 100 tumor markers has been reported and 20 tumor markers have been used in clinical diagnosis[1].The sensitivity and specificity of detection kits are generally low based on only one kind of tumor marker due to individual difference of patient with tumor and tumor heterogeneity.It is urged to develop the high sensitivity of detection kits for tumor diagnosis.REG gamma,belongs to the members of the family of 11 s proteasome activation factor [2],also known as PA28 gamma,PSME3,K i antigen [3],can be combined with 20 s proteasome mediates the degradation of protein [4].REG gamma mediated the degradation of proteins via non-ubiquitin,which different from ubiquitin protein degradation pathway.It has been reorted that REG gamma was involved in many important physiological process,including the development of cancer,autoimmunity,apoptosis,metabolic senescence and the development of pluripotent stem cells[3].Studies have shown that REG gamma is overexpressed in the serum of patients with breast cancer,thyroid cancer,colorectal cancer and other tumors [2].Here,the aim of this study is to preliminarily develop and evaluate a sensitive sandwich ELISA kit based on the level of REG in patient serum which was used for early diagnosis of colorectal cancer and other related cancers.Methods: the purified monoclonal antibody was used as the coated antibody.Purified REG,gst-reg and his-reg protein were used as protein antigens.The polyclonal antibodies,as detection antibodies,from commercial biological company were added to the 96-well enzyme marker plate with 100 ul per well followd by the large-capacity reaction system sandwich ELISA test.In order to improve the detection sensitivity of the ELISA kit,we further take advantage of the biotin-streptavidin system and optimized the relevant physical and chemical factors.Actually,we has build and confirmed the optimal test conditions,and established the appropriate standard curve.Finally,the performance of sandwich ELISA was evaluated by specificity test and repeatability test.Results: Firstly,we got the high concentration(about 1mg/ml)antigenic proteins with different labels by affinity chromatography and molecular sieve.The monoclonal antibody,the concentration was up to 1mg/ml,from the supernatant of hybrid tumor cells were purified and used for encapsulation.The polyclonal antibody(3.2mg/ml)from company was used for detection.The optimal dilution ratio of coated antibody(1:2000)and the optimal dilution ratio of detected antibody(1:4000)were confirmed by indirect ELISA assay.The combination method of monoclonal antibody coating,polyclonal antibody detection and four-parameter equation fitting regression curve were determined by the experimental analysis and comparison.The sensitivity of ELISA detection kit in biotin-streptin-avidin system(BAS)(the minimum concentratio n:375pg/ml)is significantly higher than that in the conventional volume-reaction system(the minimum concentration: 3ng/ml).In addation,3% skim milk was determined as the best sealing reagent by comparing different sealing conditions.The serum test results showed that this method was highly specific and no cross-react with other proteins in the serum.However,the cell lysate could not be detected by our ELISA kit.Repeated tests showed that the coefficient of variation between plates was 1.22% to 6.22%,and the coefficient of variation within plates was 0.88% to 5.04%,both of which were less than7%,indicating that the method was stable.The new techniques were used to improve the sensitivity of ELISA kit in our further study.Finally,we can confirm the sensitivity of ELISA kit with the patient’s serum.In this study,a new ELISA kit was initially developed,which is expected to provide more choices for the early diagnosis of tumor,thus improving the sensitivity and specificity of early diagnosis of tumor.