Immunochemical Analysis of Active and Inactive Antithrombin III

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Abstract
Antithrombin III (AT) levels from normal and AT deficient persons were measured by electroimmunoassay (EIA) and the results compared with a chromogenic assay (S2238; D-phenylalanylpipecolylarginine p-nitroanilide). Discrepant results were obtained when plasma and serum were compared using 1 antiserum, and therefore did not always relate to functional activity. Another antiserum, however, when used was capable of differentiating active AT from inactive AT complexed with its proteases and demonstrated close correlation with all samples tested (r = 0.97). The specificity of the antisera and consequent anomalous results were elucidated when purified human thrombin was added to plasma samples and subsequently reanalyzed. Quantitative differences observed when serum samples were compared by single radial immunodiffusion and electroimmunoassay with 1 antiserum, illustrates the fundamental principle differences between the 2 methods. These results give some insight as to why previous anomalies with AT immunoassays have occurred. They also indicate that plasma and not serum should be used as clinical test material. AT antisera should be capable of recognizing and distinguishing free AT from AT/protease complexes if the results obtained by electroimmunoassay are to correlate with functional activity. [Individuals who have reduced plasma levels of AT have a tendency to develop thrombotic disease].