Quantitative and Functional Assays of Complement Complex

The complement (C) system consists of more than 30 serum and cellular proteins, including positive and negative regulators, linked in 3 biochemical cascades, the classical, alternative and lectin pathways.

IMMUNI T offers a variety of assays to analyze activation (Immunogenicity Assays) or inhibition of complement system using quantitative assays (levels of complement components) and/or functional assays (CH50 and AP50) and we can design a custom program tailored to your specific needs 

Assay of In Vitro Complement Activation

Compstatin as complement inhibitor in this assays is a 13-residue disulfide-bridged peptide (ICVVQDWGHHRCT-NH2) which binds to human and non-human primate (NHP) native C3, and to its bioactive fragment C3b, preventing the cleavage of C3 into C3b, and disabling the incorporation of C3b to form C3/C5 convertases. IC50 values are 63 μM for CP (Classical Pathway) and 12 μM for AP (Alternative Pathway).

Complement activation by the 3 concentrations of TI product

sC5b-9 (soluble, non-lytic form of the MAC) is the end product of activation of the whole C cascade and can therefore be taken as direct indicators of C activation and anaphylatoxin production. sC5b-9 levels was measured by ELISA in sera of healthy subjects 30 min after TI treatment  at 37°C.  Zymosan used as positive control showed high and comparable complement functionality for both frozen and fresh sera from normal human serum (NHS). Negative control incubations contained endotoxin free D-PBS for assessing background levels.

 I) Quantitative Assay 

 Quantitative Assay 

II) Functional Assays

1) Inhibition assay for CP50 (Effect of compstatin on  classical complement activity)

Classical Pathway Standard Curve 

OD Value

Positive Control (%)

III) Inhibition Assay for CH50 

 sC5b9 was quantified in serum obtained from healthy donors.  In vitro inhibition of sC5b9 production by compstatin. Increasing amounts of compstatin (final concentrations from 0 to 96 μM) were added to NHS.

Effect of compstatin on classical complement activity in hemolytic assay

Inhibition of classical pathway-mediated hemolysis of sensitized sheep erythrocytes (SRBCs) in 1/4 diluted NHS in the presence of increasing amounts of compstatin.

Inhibition Assay for CH50