COMPLEMENT SYSTEM ASSAYS
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).
I) Quantitative Assay
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.
II) Functional Assays
1) Inhibition assay for CP50 (Effect of compstatin on classical complement activity)
Classical Pathway Standard Curve
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.