Tumor Makers (Carbohydrate Antigen19-9) That Detection Of Pancreatic Cancer Prepar For Monoclonal Antibody And Initial Establishment Of Elisa (Antigen-sandwich)

CA19-9is a class of constitutive expression glycoprotein. It was expressed in both normal humanserum and patients’ serum who has certain cancer such as pancreatic cancer and gastric cancer. But，thelevel of CA19-9in normal human serum is generally low. Currently, CA19-9is a class of tumor markersapplied by clinical already, which is in high correlation with kinds of cancer, expecially, pancreatic cancer.The correlation rate appears to70%. So CA19-9can be used as a correlation detection index of cancer.Because the immunogenicity of carbohydrate anti is weak, routine immunization requires higher dosageand longer period, increasing the monoclonal antibody preparation cycle. This experiment aims to study theuse of cloning monoclonal antibodies with Quick Antibody’s new immune adjuvant and semi solid medium(methyl cellulose, MC), reducing the cycle of the monoclonal antibody preparation. Then on the basis ofthe above method, a double antibody sandwich ELISA detection method is builded by CA19-9monoclonalantibodies,and compare it with similar foreign kit for patients serum detection performance. Through theresults to value the preparation process that weather it can applicate to large-scale production and researchin future.Respectively, adopting different immune method, immune cycle,immune dose, we immunizedBALB/c mice by using Quick Antibody new immune adjuvant and freund’ s complete adjuvant and freund’s incomplete adjuvant, and evaluate its immune effects and mice to fusion according the serum titer values.We Cloned it by methyl cellulose and solid medium cloning method, and used octylic acid ammonium toprecipitate and pure the positive hybridoma cell lines after obtaining the abdominal ascites of mice. Aftermonoclonal antibodies were identified, the qualified were paired screening. Finally, we match abovemonoclonal antibodies, and ascertain the package concentration, the enzyme mark antibody dilution rate,optical package rate, enzyme label, color appearance system, and establish CA19-9double antibodies interlning ELISA detection method. After the experiment of Immune titers, we found the ways of QucikAntibody Immunization could achieve the immune effect in three weeks, which is the routine Freund’scomplete adjuvant might achieved as well. There are a total of four lab rats were in line with therequirements of cell fusion after rigorous screening. Then we conducted cell fusion operation, and threetimes cell fusion rates respectively were88.3%,83.9%and91.2%. Three monoclonal antibody strainswhich eventually selected by the way of semi-solid cloning (MC) were ZJY1-2C8, ZJY2-7F10, andZJY3-1G9. The matching results revealed that ZJY3-1G9could be used as coating antibody, ZJY2-7F10asHRP. And then ZJY3-1G9and ZJY2-7F10were utilized to established double antibody sandwich ELISAdetection method.Indoor reference products were determined in accordance with the foreign kit, and linearequation ultimately were obtained: y=0.0063x+0.0697. The linear range was about20U/mL~300U/mL.CV values of intra-assay and interassay was less than10%, so the precision meeting requirements.Thestability results well. Through detecting33patients serum to compare the double-antibody sandwichELISA which experimental constructed and foreign assay kit, the results showed that they had goodcorrelation. The correlation coefficient r=0.9504, and the regression equation y=0.9111x+2.076. Finally,the experiment gets three positive hybridoma and a matched pair of monoclonal antibodies, establishesCA19-9double-antibody sandwich ELISA detection method initially.