IMMUNOCHEMICAL STUDIES OF HUMAN FACTOR-XIII - RADIOIMMUNOASSAY FOR THE CARRIER SUBUNIT OF THE ZYMOGEN

Abstract

Plasma factor XIII circulates as a noncovalently associated, tetrameric zymogen (a2b2). The b-subunit may act as a carrier protein for the a-subunit, which possesses the potential catalytic function. In order to define interactions that occur between the 2 subunits, a sensitive and specific RIA [radioimmunoassay] for the b-subunit was developed. Purified plasma factor XIII was incubated with thrombin and chromatographed on organomercurial agarose to separate the subunits. Pure b-chain eluted in buffer containing CaCl2. This material was used as the standard b preparation, both for preparing a monospecific antiserum and for establishing the assay. The linear range of the assay is 7-700 ng/ml (.apprx. 0.1-10 nM b-subunit), with a minimum detectable dose of 1 ng/ml. Data were analyzed by use of the logit-log transformation of antigen-binding curves. The dose-response slope for standard b is -2.76 .+-. 0.20, with a potency (ED50) of 74.9 .+-. 6.5 ng/ml. This RIA is valid for determining b-subunit concentration in plasma and serum. The dose-response slope for the plasma system is -2.69 .+-. 0.20 with an ED50 identical to that of the purified system. By this method the mean b-subunit concentration in normal human plasma (corrected for anticoagulant) is 13.8 .mu.g/ml (.apprx. 0.15 .mu.M). The concentration in serum is 13.6 .mu.g/ml, which shows that b-subunit is quantitatively recovered after coagulation has occurred.