Practical requirements for immunochemical analysis of antithrombin III in agarose gels

This report emphasizes the importance charged groups affixed to the agarose gel matrix have on immunoprecipitation of antithrombin III (AT) and of its protease complexes. Plasma and serum samples were analyzed for antithrombin III by electroimmunoassay and single radial immunodiffusion in different types of agarose. The results were compared with the amount of functionally active AT contained in the samples. A high correlation between the results was obtained provided the following criteria were fulfilled: (1) agarose with a sulphate content of 0.2% or more was used; (2) the antiserum should contain antibodies directed to AT plus antigenic determinant(s) that emerge(s) during the AT-protease interaction; (3) a gel buffer of about 75 mmol/l was used. Heparin added to the test system competed with sulphate groups affixed to the agarose gel matrix and inhibited immunoprecipitation of AT.