| 1 Purpose and significance Transient receptor potential channel 3 belongs to non-selective and non-voltage-gated ion channels that controls transmembrane calcium flux. The purpose of this experiment is to prepare TRPC3 peptide monoclonal antibodies, and to establish the monoclonal antibodies-based sandwich ELISA for detecting TRPC3 protein. The significance lies in the determination of the established ELISA system will be used in determination of TRPC3 protein expression in various kinds of tissues and cells, which can be further help to atherosclerosis diagnosis; The preparation of monoclonal antibody can be also widely applied in the immune markers(such as gold marker, fluorescent tags, isotope labeling). Therefore, this study has important application value no matter where the field of medical research and clinical diagnosis and treatment technology.2 Main research, methods and results 2.1 Design and synthesis of TRPC3 peptides According to TRPC3 amino acid sequence, we analysed TRPC3 transmembrane by using Uni Prot database to predicte the extracellular region. Then we predicted the surface probability, antigenic index, hydrophilicity and the secondary structure of TRPC3 by Prot Param, IEDB and DNA Star to screen synthetic peptide sequences. The most likely B cell epitope of TRPC3 is located in the N-terminal protein 509YFTYARDKWLPSDPQ523 section. This epitope has 15 amino acid, and its antigenicity, hydrophilic, flexibility andnso on, all complied with the design requirements and structure analysis. The peptide after economic cooperation into purity is greater than 98%. 2.2 Preparation and identification of Mc Ab against TRPC3 peptides The TRPC3 peptide coupling with KLH was used as antigen to immune Balb/c mice. After three times immunization and strengthen immune, the splenocyte of Balb/c mice immunized with highest serum immune titer were fused with SP2/0 cells by PEG. 8 hybridomas secreting monoclonal antibodies against TRPC3, 100% secrete antibody, designated respectively as A5, B8, B10, C6, F6, G11, H7 and H9, were selected with inderect ELISA and cloned by the method of limiting dilution. On this basis, after repeated screening hybridoma cell line, T1, T2 and T3 were proved that the 3 strains of ascites fluid were the best monoclonal antibodies against TRPC3. After the concentration T1 is 1.884 mg/ml, T2 is 1.787 mg/ml, T3 is 1.648 mg/ml. The SDS-PAGE electrophoresis analysis result showed that 3 strains of ascites fluid at 55 KD and 25 KD all have clear bands, which are monoclonal antibody heavy chain and light chain. 2.3 Establishment and preliminary application of the Mc Ab-based sandwich ELISA By the relative affinity determination results we known that relative affinity of T1 was highest, 8.255×10-3μg/ml. In order to determine the optimal amount of antibody and enzyme mark antibody, c ELISA were introduced in the sensitivity of the monoclonal antibodies. IC50 of T1 is 10ug/ml, IC50 of T2 is 1.3μg/ml, IC50 of T3 is 2.6μg/ml. It is the best matching working antibody concentration when the package concentration of T3 antibody is 0.842μg/ml, matching the concentration of T1 antibody is 0.235μg/ml. Established sandwich ELISA method for detecting range is 0.681-8μg/ml, and the detection sensitivity is 0.681μg /ml.3 Conclusion 3.1 By comparing antigenicity, hydrophilic and flexibility, we ultimately selected 509YFTYARDKWLPSDPQ523 as a sequence of TRPC3 peptide synthesis. Synthetic peptides with KLH coupling can be used as immune animal immune to the original. 3.2 Through synthetic peptide antigen immune mice against TRPC3 peptide monoclonal antibody was prepared, and set up three hybridoma cell line(T1、T2、T3). 3.3 The double antibody sandwich ELISA could be based on clinical samples test. |
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