Comparison of three D-dimer assays for the diagnosis of DVT:ELISA, latex and an immunofiltration assay (NycoCard D-Dimer).

Ninety-two consecutive patients referred for suspicion of deep venous thrombosis (DVT) were analyzed for D-dimer using ELISA, latex test, and a new immunofiltration method (NycoCard D-Dimer). Contrast venography verified the diagnosis in 40, and excluded the diagnosis in 52 patients. The sensitivity, negative predictive values, specificity and positive predictive values were, for ELISA 98%, 95%, 38% and 54, for NycoCard D-Dimer 100%, 100%, 42% and 57% and for the latex test 73%, 78%, 75%, and 69%, respectively. Sensitivity and specificity were inversely related with increasing pathological cut-off value. Comparison of test results by concentration category revealed a good agreement between ELISA and NycoCard D-Dimer, but to less extent between latex and the two other tests. It is concluded that NycoCard D-Dimer and D-dimer ELISA are well-suited as exclusion tests for DVT. A plasma sample is tested with NycoCard D-Dimer in less than 2 min. Thus, this test combines advantageous analytical properties comparable to the ELISA-test, with rapidity and simplicity comparable to the latex test.

Assay of D-dimer based on immunofiltration and staining with gold colloids.

In this immunofiltration assay of D-dimer in plasma samples, the antigens are captured by a monoclonal antibody on a porous membrane, and labeled with the same antibody conjugated to gold colloids. The assay time is < 2 min, and a color of intensity proportional to the concentration of D-dimer is left on the membrane. The reference range (mean +/- 2 SD) was 0.336 +/- 0.133 mg/L (n = 69). Linearity was found up to 10 mg/L. Comparison with ELISA results (x) for 198 patients' samples demonstrated a linear regression equation of y = 0.99(+/- 0.05)x + 0.68(+/- 0.07) and a mean square error of 0.503. Comparison of visual reading of the color signal (y) vs reflectometric measurements (x) for 220 patients' samples demonstrated a linear regression equation of y = 2.5(+/- 0.06)x -0.22(+/- 0.04) and a mean square error of 0.095. Bilirubin, hemoglobin, fibrinogen, soluble fibrin, and fibrinogen degradation products and freezing/thawing of samples did not interfere. Some interference from rheumatoid factor, heparin, and the presence of cells or large lipid particles was seen. The variance (CV) was 8-12% within run, 10-18% between runs, and 13-20% between persons. The new assay constitutes a rapid and reliable analytical tool combining simplicity equivalent to that of latex tests with analytical information approaching that of ELISA.

A transition-state analysis of the enzyme catalysis in the affinity labelling of horse-liver alcohol dehydrogenase by bromoacids.

The effect of temperature on the inactivation of liver alcohol dehydrogenase and on the alkylation of a model thiol free in solution by bromoacetate, 2-bromopropionate, 3-bromopropionate and 2-bromo-3-(5-imidazolyl)-propionate has been studied and the thermodynamic activation parameters calculated. All the bromoacids had a favourable entrôpy of activation in reaction with the enzyme compared to the model reaction with the thiolate anion of cysteine. This results from the formation of a reversible bromoacid-enzyme complex prior to the irreversible inactivation. The enthalpy of activation is however unfavourable, due to the lower intrinsic reactivity of the metal-thiol in the enzyme, compared to the thiolate anion of cysteine. The difference in free energy of activation between the enzyme reaction and the model reaction was used to measure catalysis. The efficiency of the enzyme catalysis of alkylation increased in the order: bromoacetate less than 2-bromopropionate less than 3-bromopropionate less than 2-bromo-3-(5-imidazolyl)propionate. Promotion by imidazole of enzyme inactivation by bromoacetate is a pure enthalpy effect. This is due to imidazole when bound to the active-site metal improving the intrinsic reactivity of the metal-thiol of Cys-46.