Complement haemolytic activity is a functional test of the classical and alternative pathway of complement in plasma or serum. The classical pathway method (CH50) is based on lysis of sensibilized sheep erythrocytes in the presence of Ca++ and Mg++. It is helpful as screening test when complement depletion or deficiency is suspected. The test is sensitive to the reduction, absence and/or inactivity of any component of the pathway.
This method is also suited to assess the effects of pharmaceuticals on inhibition or consumption of complement components. Additionally, haemocompatibility of biomaterials and medical devices according to the international standard ISO 10993-4:2017 after blood, plasma, or serum contact with biomaterials can be evaluated. Activation of the complement system by biomaterials or pharmaceuticals can result in consumption of complement proteins, which reduces the CH50 level.
Application
Measuring classical pathway complement activity. Plasma or serum samples from the following species can be analyzed: human, guinea pig, hamster, rat, rabbit, bovine, swine, M. rhesus and beagle. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.
Principle
An erythrocyte suspension is incubated for 30 minutes with serial diluted serum or plasma at 37 ºC. The activation of the complement system will result in haemolysis. After incubation the samples are centrifuged to obtain supernatant. The free haemoglobin concentration in the supernatant, which is directly proportional to complement activity, is measured by means of a spectrophotometer at a wavelength of 415 nm. Plotting the dilution factor of plasma against the degree of haemolysis allows the calculation of the CH50 (i.e. the dilution of serum to obtain 50% lysis of erythrocytes).
The positive reference is total lysis induced by lysis fluid and the negative reference is obtained after incubation with dilution buffer.
The kit is designed to determine complement activity via the classical pathway of small samples (25 µL) and can be performed in a 96 well (round bottom) microplate. The assay can also be performed in microcentrifuge tubes.
