Double Antibody Sandwich ELISA Methods For The Detection Of Mouse IL-1β And IL-12p40

The concentration of interleukin-1βand interleukin-12p40 of human blood are lower than 12.50 pg m L^-1 in normal physiological state,and more than the level,induces the inflammatory disease.Therefore,developing sensitive and specific detection methods of IL-1βand IL-12p40 are crucial.Mouse IL-1βand mouse IL-12p40 protein were obtained by the E.coli expression system.The monoclonal antibodies were fabricated by hybridoma technology and mouse ascites induction.And then the paired antibodies were identifyed by checkerboard titration.Based on these work,double antibody sandwich ELISA methods which towards the mIL-1βand mIL-12p40 detection were established and showed good commercial application prospect.The works as follows:（1）Seven strains of hybridoma cell（1B7,2A12,2C1,3A6,3B1,3C7,5F6）which showed efficiency secreted anti-mIL-1βmonoclonal antibody were obtained in this study.A double antibody sandwich ELISA which towards mIL-1βdetermination was established by using 3B1 as capture antibody and 5F6 as detection antibody.The linear range was 11.72pg m L^-1-1500 pg m L^-1（y=0.0015x+0.0227,R²=0.9943）,and the detection of limit was1.55 pg m L^-1（2σ）.This method showed the superiority with commercial kit.（2）Six strains of hybridoma cell（1C3,1B4,1G5,1H3,3E7,5G3）which showed stability and efficiency secreted anti-mIL-12p40 monoclonal antibody were obtained in this study.A double antibody sandwich ELISA which towards mIL-12p40 determination was established by using 1C3 as capture antibody and 1G5 as detection antibody.The linear range was 62.5 pg m L^-1–2000 pg m L^-1（y=0.0003x+0.0297,R²=0.9829）,and detection of limit was 10 pg m L^-1（2σ）.This method showed the comparable with some commercial kit.