Preparation Of Anti-HE4 Monaclonal Antibodies And Development Of HE4/CA125 Joint Detection

Objective: Preparation of anti-HE4 monaclonal antibodies and development of based on fluorescence and quantum dot dual-signal system for HE4/CA125 joint detection Methods: Preparation and identification of anti-human HE4 monoclonal antibodies:(1)Identification of recombinant human WFDC2(HE4)full-length protein and immunized mice;(2)Preparation and screening of hybridoma cells;(3)Preparation and identification of anti-HE4 monoclonal antibodies;(4)Condition optimization and establishment of HE4 double antibody sandwich ELISA detection method.Establishment of a combined detection method for HE4 and CA125:(1)Establishment of HE4 and CA125 double antibody sandwich ELISA methods to provide conditions for the establishment of joint detection methods;(2)Optimize the coupling ratio between the detection antibody and fluorescent microspheres and quantum dots,and determine the best working concentration at the same time;(3)Establish joint detection methods and test clinical samples to verify the application value of the method.Results: 1.Five anti-human HE4 monoclonal antibodies(7D4,7A4,8G4,4C10,1G8)were successfully screened,and a double-antibody sandwich ELISA assay with detection range of 0.5-8 ng/m L(20-350 pmol/L)was established.The correspondence between ELISA and clinical results was 92%,indicating that the antibodies have clinical application value.2.A double-antibody sandwich ELISA for the detection of HE4 and CA125 was established,and the detection range of HE4 and CA125 was 0.5-10 ng/m L and 8-200 U/m L,respectively.A dual-signal system based on fluorescent microspheres and quantum dots was developed for the detection of HE4 and CA125 by optimizing the conditions.In this method,the detection ranges of CA125 and HE4 were 1-20 ng/m L(40-840 pmol/L)and 12-200 U/m L,respectively,and it has be successfully applied in clinical samples.Conclusion: This study has successfully prepared five anti-human HE4 monoclonal antibodies and established a double-antibody sandwich ELISA.At the same time,a parallel analysis method for combined detection of HE4 and CA125 was established and it could improve the detection of conventional ELISA method efficiently.