Preparation, Identification And Application Of Monoclonal Antibody Against Synthetical Peptides Derived From TRPC6

1Purpose and significanceThe study aims to the preparation of anti-TRPC6monoclonal antibody, and thenestabilishing MoAb-MoAb-sandwich ELISA. The significance of the research is todetermine the highly expressed TRPC6protein of all kinds of tissues and cells by theELISA determine system we found,and then to assist in diagnosis of focalsegmentalglomerulosclerosis (FSGS). The monoclonal antibody we found can be extensivelyused in immunolabelling technique,such as gold labeling,fluorescence labeling andisotope labeling,etc. Therefore,this study has a significant value both in medicalresearch and clinical diagnosis and treatment technology.2main content,methods and results of the research2.1Design and synthesis of TRPC6polypeptideTRPC6(Transient Receptor Potential cation Channel6) is a cation channelprotein with six transmembrane channels and931amino acids. Based onpeptide-design principles and protein software analysis results, an amino acidsequence of KDLTKVTLGDNVKYY was selected among5sequences comparison.This objective sequence contained15amino acids which originally located onsection565-579of TRPC6molecule, and satisfied well with all the designrequirements of antigenicity, hydrophilicity, flexibility and structural analysis, etc..The synthesized polypeptide was98%above in purity and could be an immunogenwhen coupled with Keyhole Limpet Hemocyanin(KLH). 2.2Preparation and identification of Monoclonal Antibody Against SyntheticalPeptides Derived from TRPC6Mithridatized Balb/c mice three times with the synthesized and KLH-coupledTRPC6polypeptide antigen and detected serum immunopotency by indirect ELISA,we boostered the chosen mice which were with the highest immunizing titer. Fusioncells of splenocytes from immunized mice with myeloma cells（P3-X63-Ag8.653）were made by polyethylene glycol method, and were elected by HAT selective media.Positive clones were selected from the supernate titer detection among growth holes.After3times of cloning by the limiting dilution method,7hybridoma cell linessecreting monoclonal antibody with the rate of100%were obtained, which werenamed A2, D8, E9, G3, G11, H8and F11accordingly. After repeated optimation,3hybridoma cell lines （A2, H8and F11) steadily secreting monoclonal antibody weresuccessfully established. They were all confirmed to be IgG1and their light chains beκ type.Titres of their purified ascitic fluid were1:1600，1:3200and1:1600respectively. Concentrations and purities of their monoclonal antibodies were4.1mg/ml,1.04mg/ml, and1.03mg/ml, respectively. Results of SDS-PAGE exhibitedthat molecular weight was55KD for heavy chains and25KD for light chains and theirpurity get to more than90%by Bandscan. Relative affinity was6μg/ml for both H8and F11,12.5μg/ml for A2. Results of additive ELISA assessed that H8and F11recognized the same epitope and A2recognized the different one. The match testdetermined that the optimal match was A2with F11, therefore, laid the foundation forour newly established double antibody sandwich ELISA assay.2.3Establishment and preliminary application of the double antibody sandwichELISAThe double antibody sandwich ELISA was established and preliminaryapplicated to the TRPC6protein detection in rat brain, based on the best matchbetween A2and F11. In order to obtain the optimal amounts of coated antibody andenzyme-labeled antibody, cELISA was done first to evaluate their sensibility. IC50was0.6μg/mL for F11, and2.8μg/mL for A2. F11exhibited higher sensibility than A2.The results from matrix titration showed that optimum working concentrationand dilution ratio of coated A2were5μg/mL,1:200, respectively. And that of HRP-F11were7μg/mL,1:150, respectively. The results from double antibodysandwich ELISA, with the standard curve for TRPC6protein detection,y=0.0519x+0.1869（R2=0.992, P＜0.05) in the linear range of0.625-10μg/mL,approved the credibility of this assay for TRPC6protein detection, although optimalreaction conditions and multi-sample assays remained further investigation. From thisassay, the content of TRPC6protein in rats brain could be detected, and finally, thedata of its over-expression in histiocyte obtained.3Conclusion3.1The novel designed and synthesized sequence of TRPC6polypeptide,KDLTKVTLGDNVKYY, was finally elected to be the synthesized sequence of TRPC6,from the comparison screening test of antigenicity, hydrophilicity and flexibility. Thissequence, coupled with KLH, was used as an immunogen for animal immunizing.3.2Monoclonal Antibody against synthesized TRPC6polypeptide was obtainedthrough the mice immunized by synthetic peptide antigen.3hybridoma cell lines, A2and F11and H8, were established. F11and H8shared the approximate values ofrelative affinity, which were greater than that of A2. Additive ELISA indicated that H8and F11recognized same epitopes, while A2recognized different ones. Therefore, thedouble antibody sandwich ELISA could be based on.3.3Double antibody sandwich ELISA, aiming at detecting TRPC6protein, wasinitially established and could be used in the TRPC6protein over-expression assayin histocyte.