Below are several commonly performed assays listed to measure the extent of complement activation. For additional assays please contact us.
Complement activation is an early marker of an inflammatory reaction. Activated complement components induce cell lysis, recruitment of granulocytes and adhesion of granulocytes to the activating agent (i.e. medical device, biomaterial). The subsequent response of granulocytes, being release of enzymes and oxygen radicals, is destructive for adjacent tissue. In vivo complement activation by medical devices and (bio)materials is quantified by generation of C3a. C3a, a split product of C3, is cleaved immediately into the more stable C3a-desArg. C3a-desArg can be determined by a sandwich-type enzyme immunoassay.
C5a is a split product of complement protein C5. Activation of complement proteins can be initiated soon after blood collection and also by the container in which incubations with biomaterials are performed. C5 activation is a step further downstream the complement cascade and therefore suffers less from non-biomaterial mediated activation than for instance C3 activation (note: because C3a generation suffers more from these ‘artifacts’ it is not recommended to use measurement of C3a for in vitro studies). Concentrations of C5a generated are determined by immunoassay of plasma which has been incubated with the biomaterial under study.
Terminal Complement Complex (TCC; C5b-9)
Terminal Complement Complex (TCC), or complement complex C5b-9, is part of the final complement pathway, which is activated through either the classical or alternative pathway. The complex is generated by the assembly of C5 through C9 as a consequence of activation of the complement system by either of these pathways. The Membrane Attack Complex (MAC) is a stable TCC which mediates irreversible target-cell membrane damage associated with complement activation. Complexes formed in the absence of a target membrane bind to a naturally occurring regulatory serum protein, the S protein. The sC5b-9 complex is the soluble, non-lytic form of the TCC, which is well-quantifiable via an enzyme immunoassay. From all complement products known, sC5b-9 is the least affected by blood sampling artifacts and in vitro handling.
Complement CH50 or AP50
Activation of complement can be assessed by measuring the extent of complement consumption as quantified by the hemolytic activity of complement. In this assay sheep erythrocytes, sensitized with rabbit antibodies are exposed to a sample. The sample complement components induce lysis of the erythrocytes. The amount of lysis is quantified spectrophotometrically by the presence of free hemoglobin. By testing several dilutions of the sample, the amount of plasma required to result in 50% lysis of the erythrocytes is determined. This amount represents one CH50 unit. If complement is consumed, the CH50 activity, as measured in units/mL, decreases. This assay is qualitative in nature (the CH50 scale is non-linear) and to obtain significant changes in CH50, extensive consumption of complement is required. Alternatively, consumption of the alternative complement pathway can be measured with washed rabbit erythrocytes without sensitization (AP50).
Complement Convertase Assay (CCA)
The complement system will be activated as result of the incubation of blood with (bio)materials and medical devices. Normally, to protect tissue and organs, tight controls are in effect that regulate the complement system. The cascade is naturally moderated by the instability of the enzymes (convertases) formed. Once a component is activated, failure to rapidly combine with its substrate causes it to decay. During the use of medical devices these regulatory mechanisms often appear inadequate due to the unnatural nature of the surface. Therefore, testing of complement convertase activity on the surface of (bio)materials and medical devices is an important variable to evaluate hemocompatibility.