Preparation Of Monoclonal Antibody To Protein S100A6

The S100protein family is a class of calcium-binding protein withEF-hand, because of the solubility in neutral saturated ammonium sulfatesolution is100%. So far, it contains at least25members in the family,however, there are21members of the gene located on human chromosome1q21and the chromosome segment is less stable, prone to chromosomalamplification and rearrangement, closely associated with the tumor. S100A6is a member of the family, also known as calcium cycle protein (Calcyclin,Cacy) and is widely distributed in cells, including cytoplasmic, membraneetc(more abundant), initially detected by Kuznicki and Filipek in Ehrlichascites tumor.Monoclonal antibodies, are up to the individual cell proliferationand then monoclonal cells formed, the monoclonal cells can secretemonoclonal antibody to only one antigen of epitope. In1975, Kohler andMilstein created hybridization echnology, form which technology can getmonoclonal cells that has the ability of immune B lymphocytes secreteantibodies and the constantly proliferation split ability of myeloma cells.Because of the advantages of monoclonal antibodies (high specificity, purity and uniformity), it had been applicated in many medicalfields. First of all, monoclonal antibodies was a kind of important "tool" thatcould detect the function and distribution of the protein, or interactionsamong different protein. At the same time, it was an important tool fordetecting disease markers, so it played a very important role in thediagnosis of disease. On the other hand,especially in immune therapy,immune and drug combination of immune-chemotherapy, make full use ofmonoclonal antiboodies oriented functions, can block the cell signalingpathways, or will carry drugs to focal areas, which has benefit for thehuman disease diagnosis, prevention, treatment, especially for the diagnosisand the immunotherapy of cancer.It has been reported that S100A6was overexpressed in most of theepithelial tumours. If we make full use of the specificity of monoclonalantibody, we can set up the fast detection method of S100A6in tumor.After that, it can be expected to form the immune diagnosis technology andfurther realize the technology of targeted cancer therapy.Objective:This research is for the preparation and identification of monoclonalantibodies to the S100A6calcium binding protein, which will be afoundation for further research and development S100A6immunologyrelated diagnosis reagent.Methods: 1. Preparation and identification of human S100A6recombinantprotein: Transform pGST-moluc and pGST-moluc-S100A6into EColi.BL21by CaCl2method; induce the expression of GST or GST-S100A6protein by IPTG; prepare the bacteria lysate by ultrasound on ice; isolate andpurify GST and GST-S100A6from the lysate with the GlutathioneSepharose4B beads; after measuring the concentration through proteinconcentration cryoscope equipmentand identify by Western blot, store inaliquots at-80℃.2. Preparation and identification of monoclonal antibody to S100A6proteinBALB/c mices being immunized with the GST-S100A6fusion protein,the antibodies titer ofmice serum was detected by ELISA. Spleen cells inmice with higher titer was fusioned with SP2/0myeloma. TookGST-S100A6protein as the antigen for the original screening and then tookthe GST-tag protein as the antigen for the second screening. After culturedthe hybridoma, cloned S100A6monoclonal antibody cell lines through thelimited dilution and identified the antibody specificity in the supernatant ofmonoclonal cells medium by Western Blot.Results:1. The Western blot results showed that GST-S100A6protein could berecognized by S100A6commercialization antibody specifically, andappeared specific strip at36KD place. 2. Immuned the BALB/C mice by the GST-S100A6fusion proteinthree times, the serum antibody titer was detected by ELISA,which was upto106, and then immuned BALB/C mice for the last time through tail veinfollowed by cell fusioned, four weeks later,21strains hybridoma cellswere acquired. After Cloned S100A6monoclonal antibody cell linesthrough the limited dilution and identified, we got2strains, one of whichhas the antibody specificity in the supernatant of monoclonal cells mediumafter identified by Western Blot.Conclusion:In this experiment, we prepared one strains of S100A6monoclonalantibody cell lines successfully through the hybridoma technology.