Establishment And Diagnostic Application Of Monoclonal Antibodies Against Tumor Markers HE4 And CA19-9

Malignant tumors are a serious threat to human health,and the prognosis of early tumors is significantly better than advanced tumors.Detection of serum tumor markers is a routine way of early screening of tumors.By detecting the presence and quantitative change of tumor markers in serum can reflect the occurrence and development of tumors in vivo.And play an important role in the diagnosis,classification,staging,monitoring,prognosis and guiding treatment of the tumor.At present,our tumor markers diagnostic reagents and diagnostic reagents required for the development of antigen and antibody are basically dependent on imports.Raw materials are controlled by foreign markets,and expensive price of the kit and other factors seriously restricted the pop?Larity of tumor marker early screening in China.Therefore,the development of home-reagents is very necessary.Because the key diagnostic epitopes of tumor markers are complex,it has become a diffic?Lt problem to develop diagnostic reagents for tumor markers with high specificity and high sensitivity.In this study,we choose two tumor markers with different structures,including human epididymis protein 4(HE4),which is located on the protein,and CA19-9(carbohydrate antigen 19-9,CA19-9).We try to develop antigens and antibodies and explore the feasibility of their use in diagnostic reagents.For tumor marker HE4,the recombinant antigen rHE4-Fe and eight recombinant antigen fragments were constructed by prokaryotic expression system.The recombinant epitopes of H03 and H05 were identified by using recombinant antigen.rHE4-Fe and rHE4-0164-32a recombinant protein were used to immunize Balb/C mice.The epitope of HE4 antibody was identified by ELISA platform and the binding ability with HE4-N was detected.Then,we obtained a pair of antibodies similar to the purchased reagent recognition epitope,coated antibody 60G1 and labeled antibody 6B9.The MB*60G1-6B9*AE was evaluated on the chemiluminescent microparticle immunoassay platform.The detection range was 5-3000 pmol/L,and have a good relevance with Roche reagent,the clinical serum detection correlation was 0.9931.For tumor marker CA19-9,glycoprotein antigen CA9-9 with nature activity was obtained by the use of biochemical extraction methods,which can meet the requirement of immunity.By trying different immunization programs with different adjuvants,the results showed that Freund's adjuvant immunized mice had the highest immunological titer of 104.42 monoclonal antibodies were obtained by natural antigens and competitive screening.The antibody subtypes were mainly IgM.Assessed by ELISA platform,we obtained 5 groups,9H7-17A12,8D11-5E7,2G4-14G2,17H11-14G2 and 13A12-15E8.The antibody pairs' P/N ratio and the linear correlation(R2>0.9)can reach the standard of control reagent.Using the chemiluminescent microparticle immunoassay(CMIA)technique,we We obtained the antibody pair MB*2G4-14G2*AE,which had a certain serum correlation with the Roche reagent(R2=0.7919).In summary,HE4 monoclonal antibody was successfp.Lly prepared in this study.And we established a HE4 CMIA diagnostic reagent.CA19-9 antibodies were developed,and we got some progress on the development of CA19-9 CMIA diagnostic reagents.It may be useful to the screening and early diagnosis of ovarian cancer and pancreatic cancer.