Preparing The M Abs Against Cucurmosin And Applying In Cucurmosin's Purification

Objective:To construct the hybridomas against cucurmsoin and screen the ones that could secrete positive antibodies;To prepare and purify the monoclonal antibodies against cucurmosin;To construct the immunoaffinity chromatography column against cucurmosin and explore the further applications.Methods: I.Preparing and Purifying the Monoclonal Antibodies against Cucurmosin 1.Balb/C mice were immunized with cucurmosin extracted from pumpkins;Construct the hybridomas against cucurmsin,which could secrete aimed antibodies constantly and steadily,by fusing the mice's SP2/0 myeloma cells and the immunized Balb/C mice's spleen cells;2.The hybridomas,which could secrete positive antibodies against cucurmosin,with HAT medium and subclone culture;3.After sensitizing the Balb/C mice with fluid-wax,obtain the monoclonal antibodies by injecting screened positive hybridoma cells into Balb/C mice's abdominal cavity to induce ascites;4.Purify the monoclonal antibodies against cucurmosin with saturated ammonium sulfate(SAS)salting-out method and protein G chromatography assay;5.Compare the fore-and-aft purification effect of the monoclonal antibodies against cucurmosin with SDS-PAGE;II.Identifying and Detecting the Monoclonal Antibodies against Cucurmosin and Cucurmosin-effecting Immunotoxins Molecules 1.Identify the monoclonal antibodies against cucurmosin and cucurmosin-effecting immunotoxins molecules with western blotting(WB)assay;2.Measure the valence of antibodies against cucurmosin with indirect enzyme linked immunosorbant assay(i ELISA);3.Detect the affinity value of the antibodies against cucurmosin with Octect assay;4.Measure the subclass of the antibodies against cucurmosin with anitibody isotyoping kit.III.Constructing and Applying the Immunoaffinity Chrotomagraphy Columns with CNBr-activated Sepharose 4B;1.Compare the fore-and-aft purification effect of cucurmosin and cucurmosineffecting immunotoxins with SDS-PAGE;2.Detect the purified cucurmosin and purified cucurmosin-effecting immunotoxins' anti-tumor activity in vitro with SRB assay;Results: 1.Obtain three monoclonal antibodies-1B2,1G9,2E12 against cucurmosin with strong specificity and affinity successfully;2.The same subclass of all the antibodies is Ig G1,kappa;3.By detecting,the valence of the monoclonal antibodies is respectively 1:1000000;4.By measuring,the subclass of all the three monoclonal antibodies is the same-Ig G1,kappa;5.By detecting,the affinity value of the monoclonal antibodies,KDs is all low than 10-12;6.Successfully purify the two monoclonal antibodies-1B2,1G9;7.Successfully construct immunoaffinity chromatography column against cucurmosin;8.By detecting,the purified cucurmosin and the purified cucurmosin-centered immunotoxins still remain anti-tumor acitivity in vitro.Conclusion: 1.Successfully prepare the three monoclonal antibodies(1B2,1G9,2E12)against cucurmosin with high specificity and affinity,whose subclass is all Ig G1,kappa;2.Successfully construct the immunoaffinity chromatography column against cucurmosin;3.Purify natural cucurmosin,recombinant cucurmosin and cucurmosin-centered immunotoxins with the IAC column.