Production Of Monoclonal Antibodies To Human Antithrombin Ⅲ And Its Primary Application

AT-â…¢ is very important for the inhibition of thrombin in plasma. AT-â…¢ is the recognized early diagnosis index of DIG and it also plays role in the prevention and treatment of DIG. The production of anti-AT-III monoclonal antibody with high specificity and affinity is required for the establishment of ELISA and affinity chromatography method. Therefore, we developed the anti-human AT-III monoclonal antibodies and do some advanced research about them.A method for preparation of antithrombin â…¢ (AT-â…¢) concentrated from human plasma's fraction has been described. After preliminary treatment of plasma with the cold ethanol, the isolation of AT-III from the precipitate was performed by affinity chromatography on heparin-sepharose CL-6B; desalted by dialysis and concentrated. On disc gel electrophoresis, the AT-III concentrates prepared showed single band or fraction. The reclaim rate of AT-III calculated by plasma is 8.0% and the relative specific activity is 7.7 lU/mg. The isolated AT-III will be used in the screening of positive clones of hybridoma cell lines.SP2/0 myeloma cells are fused with spleen cells of BALB/c mice (the ratio is 1:10) immunized with AT-â…¢ by routine method. Positive clones are detected by ELISA. Three monoclonal antibodies of AT-III have been produced. The rate of fusion is 92.2% and the positive rate of antibody is 5.8%. The Ig class is IgGi. McAb of AT-III provides very efficient tools for the detection and purification of AT-III.Sandwich ELISA is an immunoassay technique in common use, especially for the determination of macromolecular protein antigen. In order to increase sensitivity, we take advantage of polyclonal antibody in the assay to measure the AT-III content in blood serum. The specificity of the assay can be improved by using monoclonal antibody as enzyme-linked antibody. One of the three monoclonal antibodies we screened is suitable to this assay. This assay has been proved that it can be used in clinic.