Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay.
The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg-p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.
D000818:Animals; D001777:Blood Coagulation; D001780:Blood Coagulation Tests / Q000379:methods*; D002863:Chromogenic Compounds; D005172:Factor XI / Q000032:analysis*; D005173:Factor XI Deficiency / Q000097:blood; D015945:Factor XIa; D006801:Humans; D007704:Kininogens / Q000502:physiology; D009842:Oligopeptides; D011288:Prekallikrein / Q000502:physiology; D011480:Protease Inhibitors / Q000502:physiology; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D002863:Chromogenic Compounds; D007704:Kininogens; D009842:Oligopeptides; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; C040190:S 2366; D005172:Factor XI; D011288:Prekallikrein; D015945:Factor XIa; D011761:Pyrrolidonecarboxylic Acid