| Objective:To prepare MogV-HS-BSA and MogV-HS-HSA artificial antigen, establish the competitive-inhibitive ELISA method to detect MogV content and obtain hybridoma cell lines which can secrete MogV monoclonal antibody, these work will provide a technical basis for the application of the mogrosides V monoclonal antibodies and the cultivation of new varieties of the the high MogV content.Methods:(1) The preparation MogV-HS hapten were got through the reaction of SA and MogV; Two artificial antigen MogV-HS-BSA and MogV-HS-HSA were got through hapten respectively coupling with BSA or HSA, then the binding ratio of the artificial antigen were determined by Infrared spectroscopy (IR) and ultraviolet spectroscopy (UV). (2) The BALB/c mice were immuned with MogV-HS-BSA to get the antiserum, then serum antibody titer and specificity were measured by using MogV-HS-HSA as coating antigen. (3)The competitive inhibition enzyme-linked immunosorbent assay for MogV was established with the serum of highest titer, which was got through the MogV-HS-BSA immunized BALB/c mouse. (4) BALB/c mouse spleen cells were fused with mouse myeloma cell (SP2/0), then hybridoma cells that could secrete monoclonal antibodies were selected.Results:(1) In the artificial antigen MogV-HS-BSA, the conjugation ratio of MogV-HS and the carrier protein BSA was 44:1; In the artificial antigen MogV-HS-HSA, the conjugation ratio of MogV-HS and the carrier protein HSA was 64:1. (2)Their antiserum titers of the 3 mice were 1:128000,1:64000, 1:8000 respectively. The antiserum had anti-MogV specificity.(3) The coating antigen working concentration was 1μg/ml. The best antiserum dilution concentration in this work was 1:8000, under this contition MogV concentration within the the range of 1×10-4-1μg/ml the regression equation y=-0.158x+0.298, r2=0.982. (4) Three-hole positive hybridoma cells were screened out. After subcloning, a stable secretion anti-MogV monoclonal antibody hybridoma cell lines were obtained.Conclusion:(1) MogV-HS, MogV-HS-BSA and MogV-HS-HAS have been successfully prepared. (2)The artificial antigen MogV-HS-BSA has good immunogenicity, and the antiserums obtained by immunized mouse have high titer and good specificity.(3)The competitive inhibition enzyme-linked immunosorbent assay for MogV has been successfully established. (4)The hybridoma cell lines that can secrete anti-MogV monoclonal antibodies have been obtained. |
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