Demonstration and quantitation of activation of the first complement in human serum

Abstract

Activation of the first component of human complement (C1) in human sera can be readily detected in double immunodiffusion studies with anti-C1q, anti- C1r, and anti-C1s as it produces a characteristic pattern quite different from that of precursor C1. Native macromolecular C1 gives a continuous line of precipitation with antisera to C1q, C1r, and C1s in double diffusion studies. After activation of C1 by incubation of serum with complement activators, three major changes occurred in the Ouchterlony pattern. First, spurring of the C1s precipitin line over that of macromolecular C1, indicating release of C1s from C1, was observed with low doses of activator. Release of C1s was quantitated by single radial diffusion and shown to be complete with the highest activator dose examined. Second, C1q was released with larger activator doses as shown also by spurring of the precipitin line due to this component over the remaining macromolecular C1. Third, and most surprising, C1r antigenicity was progressively lost as the activator dose was increased and no C1r line remained with the highest dose of activator tested. This was not true with C1s as there was no change in the total C1s concentration in serum incubated with various activator doses. These observations provide two approaches to the quantitation of C1 activation in human serum. First, C1r and C1s can be quantitated by single radial diffusion. A decrease in the C1r:C1s ratio correlates with activation. Second, C1s released by the activation can be quantitated by single radial diffusion if the agarose contains high concentrations of anti-C1q to confine C1, also containing C1s, to the area near the application well, and lesser concentrations of anti-C1s to permit free C1s to produce a measurable ring. The extent of release of C1s also correlates with activation. These immunochemical techniques to quantitate C1 activation directly inserum do not require specialized reagents. It is hoped that they will be useful in screening pathological sera and in monitoring the status of the complement system in patients.

Activation of the classical pathway of complement by Hageman factor fragment.

“…That the activation of complement proceeded via the classical pathway is indicated by the ability of HFf to directly activate and dissociate the C1 macromolecule with concomitant release of Clq and Cls and disappearance of C lr antigenicity as assessed by single and double radial immunodiffusion studies using antisera to Clq, Clr, and Cls. Native, macromo'lecular C1 gives a continuous line of precipitation with antisera to C 1 q, C l r, and C l s in double diffusion as was previously shown by Ziccardi and Cooper (22). Upon incubation of serum with complement activators, however, spurring of the C ls precipitin line over that of macromolecular C 1 occurred, indicating release of C ls from activated C 1 (22).…”

“…Fig. 4 depicts the result of a representative experiment in which NHS human serum (0.1 ml) was incubated with 5 #g HFf or 2 mg/ml aggregated IgG, which under similar conditions is known to deplete complement hemolytic activity (22). After incubation the mixture was made into a 1:100 dilution with GVB ++, and samples ranging from 0.5-1 ml were mixed with 0.2 ml EA (5 X 108/ml) in a total volume of 1.5 ml GVB ++ and further incubated for 60 min at 37°C.…”

“…Radial double or single immunodiffusion analyses were carried out in 0.8% agarose in barbital-buffered saline, pH 7.2, containing 2.5 mM CaC12 and 0.09 M NaC! at 4°C for 48 h as described (22). Immunoelectrophoresis was performed in 1% agar or 0.8% agarose in barbital-buffered saline, pH 8.6, containing 0.09 M NaCI and 2.5 mM calcium, at 4.7 V/era at 4°C for 2 h. Sodium dodecyl sulfate (SDS) (9%) polyacrylamide gel electrophoresis was performed according to Laemmli (23).…”

Effect of Aspirin on the Complement System <i>in vitro</i>

“…This can be demonstrated by immunoelectrophoresis with anti-CIs. The C l complex remains at the application site, while the C ls released appears in the a region [18].…”

Mechanisms of activation of the classical pathway of complement by Hageman factor fragment.

“…The (31). The disappearance of CIr antigenicity in this assay is therefore a measure of Cir activity and the degree of activation could be quantitated as described (30).…”