Portal de Periódicos da CAPES

Evidence for the presence of a free N-terminal fibronectin 30-kDa domain in human plasma by quantitative determination with an indirect immunosorbent assay

1988; Elsevier BV; Volume: 173; Issue: 2 Linguagem: Inglês

10.1016/0003-2697(88)90183-2

1096-0309

J Škrha, Hartmut Richter, Helmut Hörmann,

Protease and Inhibitor Mechanisms

The free N-terminal 30-kDa domain of the fibronectin subunit chains had previously been shown to mediate binding of soluble fibrin to phagocytic cells. In order to demonstrate whether the fragment is available in plasma in a suitable concentration, an indirect immunoassay procedure for its quantitative evaluation was developed. The free form of the 30-kDa domain was separated from fibronectin and the bulk of the plasma proteins by two-step affinity chromatography on gelatin- and heparin-Sepharose. In the eluate of the heparin-Sepharose the 30-kDa fragment was determined by its capacity to inhibit the immune reaction between a specific antiserum and the 30-kDa fragment immobilized on microtiter wells. The procedure offered reproducibility comparable with other immunoassays (coefficient of variation 4.0 to 8.0%); the lowest amount of detectable 30-kDa fragment was 0.1 μg/ml. In human plasma this method detected for the first time ca. 5 μg/ml 30-kDa fragment. This concentration is in the range required for binding of fibrin to cells.