Improved assay of antithrombin-III. Effects of certain additives on thrombin and on chromogenic peptide substrates

When manual assays of antithrombin-III with use of different synthetic chromogenic peptide substrates are modified for a centrifugal analyzer, the downward deflection of the reference curve at the zero point raises a serious problem, because the position of the zero value is included in the calculation of the reference curve. Complete linearity was obtained by addition of polyethylene glycol 6000 to the thrombin solution and Tween 80 to the substrate solution in final concentrations of 1 g/L and 100 mg/L, respectively. The additives increased the concentration of active thrombin in the solutions by preventing its adsorption and aggregation, and the solubility of the substrates in aqueous media is increased. The precision of the optimized method, tested in routine assays during three months, yielded an estimated day-to-day CV of 3.4%.