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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).

 I) Quantitative Assay 

 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 

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 
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