Development and evaluation of a modified colorimetric solid-phase microassay for measuring the activity of cellular and plasma (Factor XIII) transglutaminases.
Plasma TG (transglutaminase) [FXIII (Factor XIII)] stabilizes fibrin and plays an essential role in haemostasis. In the present paper, we report a simple colorimetric assay for measuring FXIII activity. The advantage of this approach, compared with all the other solid-phase assays described so far for measuring TG activity, is that the first substrate, namely the synthetic dipeptide, N-benzyloxycarbonyl-L-Gln-L-Gly, is coupled covalently by its C-terminus with amine-substituted polystyrene plates. Covalent coupling eliminates the problem of desorption of proteins such as casein and N,N'-dimethylcasein (first substrates) when 'blocking' buffers containing proteins, e.g. BSA, are employed, or when samples of plasma or cell homogenates are used. The principle of the assay itself is based on the incorporation of the well-known second substrate, 5-(biotinamido)pentylamine, into the gamma-carboxamide of glutamine in the immobilized dipeptide. The amount of biotinylated amine bound to the plate, as measured by the phosphatase activity of Extravidin phosphatase attached to the biotin moiety, is directly proportional to the TG activity. The method shows strong correlation (r = 0.96) with the radiometric assay for FXIII activity. For plasma samples, a linear response was obtained in the range 0-1.33 i.u. (international unit)/ml, versus the Behring standard. In a preliminary clinical investigation, the method was applied to normal and pathological plasma samples from patients with liver failure and disseminated intravascular coagulation. In the isolation of TG from guinea-pig liver, it was used to measure enzyme activity after each purification step. This method is rapid, sensitive and can easily be applied to routine clinical analyses and to specific research problems.
Thomas
Vincent
V
Covalab, 3, allée Pierre de Coubertin, 69007 Lyon, France. vthomas@covalab.com
El Alaoui
Saïd
S
Massignon
Denis
D
Clément
Sophie
S
Simonet
Frantz
F
Quash
Gerard
G
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D005176:Factor XIII / Q000032:analysis*; D006168:Guinea Pigs; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D017093:Liver Failure / Q000097:blood*; D008904:Miniaturization; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005176:Factor XIII