Preparation And Preliminary Application Of The Anti-human HE4 Monoclonal Antibody

Background:With the aggravation of the pollution of people's living environment,the aging of the population and the emergence of bad living habits,the health threat caused by cancer is becoming more and more serious.According to the data from Globocan2018,the number of cancer cases in China accounts for about 23.7%of the world,and the number of cancer deaths in China accounts for about 30.2%of the world,with both the number of deaths and the number of cases ranking first in the world.In the past two decades,with the rapid development of medicine,although great progress has been made in the diagnosis and treatment of tumors,the incidence and mortality of tumors are still remains high.Recently,one of the most effective ways to reduce cancer death is to improve the rate of early diagnosis and achieve early treatment.Tumor markers are substances secreted by tumor cells that are mainly distributed in the tissues,serum,urine,ascites and other body fluids of cancer patients.Depending on tumor markers,simple,non-invasive and highly sensitive detection tool can be developed,which is suitable for early screening of tumors and can improve the rate of early diagnosis of tumors.They are also helpful for to the differential diagnosis of tumors,observation of curative effects and judgment of prognosis.Human epididymis protein 4(HE4)is a new tumor marker that has appeared in recent years.Its role in the early diagnosis of ovarian cancer is widely recognized and used in clinical detection.Recently,a large number of scientific studies have found the expression of HE4 is increased in other malignant tumors such as lung cancer,gastric cancer,breast cancer and pancreatic cancer.These results suggest that it has important significance and good application prospect in tumor screening and diagnosis.At present,most of the in vitro diagnostic reagents and the required high-quality materials such as antigens and antibodies account for a high proportion of imports,and their prices are expensive.These factors hinder the progress of using tumor markers to screen tumors in our country.Therefore,the development of domestic HE4 recombinant protein,specific anti-human HE4 monoclonal antibody and HE4 detection kit is of great significance to promote the early screening of tumor and improve the rate of early diagnosis.Purpose:The recombinant HE4 protein and anti-human HE4 monoclonal antibody were prepared and applied to the preliminary exploration of the development of HE4 detection kit.Methods:1:The recombinant human HE4 protein was expressed in E.coli expression system and apurified by nickel column affinity chromatography with gradient elution.The purity and activity of the protein were verified by SDS-PAGE and Western-blot2:The recombinant human HE4 protein was used as immunogen to immunize animals.The HE4 hybridoma cell lines were screened by Hybridoma technique and anti-human HE4 monoclonal antibody were prepared.The subtypes,affinity and specificity of antibodies were evaluated.3:The anti-human HE4 monoclonal antibodies were applied to the development of the ELISA kit and colloidal gold immunochromatographic kit by double antibody sandwich method for detecting HE4.Their clinical application value was evaluated by testing their sensitivity,specificity and other properties.Results:1:The purity of the prepared human HE4 recombinant protein can reach 92.3%and its biological activity was good.2:Three HE4 hybridoma cell lines(3F7,3E5,5F10)were screened and anti-human HE4 monoclonal antibodies were successfully prepared.The affinity of antibodies to HE4 were 252 pM,89 pM and 104 nM,respectively.The antibody did not cross-react with CA125,AFP,CEA,CYFRA21-1,CA72-4,BSA and HCG.They have good specificity.The subtype of 3F7 and 3E5 antibodies were IgG2a,and 5F10 antibody was IgG1.3:The coating concentration of antibody in ELISA kit of HE4 was 1 ?g/mL,the working concentration of the enzyme labelled antibody was 1:5000,and the sensitivity was 1 nmol/L.The sensitivity of the HE4 colloidal gold immunochromatography kit was 100 pmol/L.When these two kinds of HE4 detection kits were used to detect the serum of clinical samples,the detection results were consistent with the clinical results.Conclusion:In this study,we prepared anti-human HE4 monoclonal antibodies with high affinity and specificity,and then successfully established the development method of HE4 ELISA kit and colloidal gold immunochromatographic kit.