AP50 Test

HComplement hemolytic activity is a functional test of the classical and alternative pathway of complement in plasma or serum.
AP50 test kit (100 determinations)

Description

Complement haemolytic activity is a functional test of the classical or alternative pathway of the complement system in plasma or serum. The alternative pathway method (AP50) is based on lysis of rabbit erythrocytes in the presence of 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 AP50 level.

Application

Measuring alternative pathway complement activity. Plasma or serum samples from the following species can be analyzed: human, guinea pig, hamster, rat, bovine, swine, sheep, 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 °C. 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 AP50 (i.e. the dilution of plasma 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 alternative pathway of small samples (75 µL) and can be performed in a 96 well (round bottom) microplate. The assay can also be performed in microcentrifuge tubes.

References that used the AP50 assay:

1. Sülzen H, Began J, Dhillon A, Kereïche S, Pompach P, Votrubova J, Zahedifard F, Šubrtova A, Šafner M, Hubalek M, Thompson M, Zoltner M, Zoll S. Cryo-EM structures of Trypanosoma brucei gambiense ISG65 with human complement C3 and C3b and their roles in alternative pathway restriction. Nat Commun. 2023 Apr 27;14(1):2403.

2. Pinnapireddy SR , Giselbrecht J , Strehlow B , Janich C , Husteden C , Meister A , Loppnow H , Sedding D , Erdmann F , Hause G , Brezesinski G , Groth T , Langner A , Bakowsky U , Wölk C . A triple chain polycationic peptide-mimicking amphiphile - efficient DNA-transfer without co-lipids. Biomater Sci. 2019 Dec 17;8(1):232-249.

3. Giselbrecht J, Wiedemann S, Reddy Pinnapireddy S, Goergen N, Loppnow H, Sedding D, Erdmann F, Bakowsky U, Hause G, Lúcio M, Langner A, Wölk C. Nucleic acid carrier composed of a branched fatty acid lysine conjugate-Interaction studies with blood components. Colloids Surf B Biointerfaces. 2019 Dec 1;184:110547.