Factors influencing the in vitro interaction between immunoglobulins and isolated C1: a critical study

Abstract

The C1 fixation test is widely used for the study of the interaction between immunoglobulins, their fragments and the complement system. Some factors influencing the apparent extent of the C1 fixation ability of immunoglobulins and immunoglobulin fragments have been studied. C1 preparations purified by two different methods (euglobulin precipitation and affinity chromatography) were used in parallel in the majority of the experiments. It was demonstrated that the haemolytic activity of both types of C1 preparation was considerably decreased during the incubation at 30° for 10 min (first step of the C1 fixation assay) and the C1s-dependent C4 inactivating effect of C1 also was reduced approximately to the same extent. Gelatin, Trasylol (GORDOX®) and 1,4-diaminobutane in low concentration (0.01 M) were shown to cause an increase in the apparent haemolytic activity of C1. This effect was not due to the reduction of the rate of C1 inactivation, nor to the blocking of the adsorption of C1 to the glass vessel walls; but was based on the ability of these compounds to increase the strength of binding between C1 and EAC4 cells. Similarly, the apparent extent of the C1 fixation by immunoglobulin preparations and their fragments was significantly increased in the presence of gelatin. In the absence of gelatin, a dual effect of immunoglobulins on C1 was observed: (1) fixation to the immunoglobulin; (2) reduction of the inactivation of C1 in the presence of immunoglobulins. The significance of these findings to the mechanism of the C1 binding to immunoglobulins is discussed.