Assay of complement activity in human serum using large unilamellar liposomes

Abstract

Liposomes incorporating 2,4,6-trinitrophenyl-aminocaproyl-dipalmitoylphosphatidylethanolamine (TNP-cap-DPPE) as a membrane hapten can be lysed by human complement in the presence of anti-TNP antibody. Liposomes were composed of L-α-dimyristoylphosphatidylcholine, cholesterol, TNP-cap-DPPE and dicetylphosphate at a molar ratio of 1:1:0.005:0.02. Large unilamellar liposomes were prepared by reverse-phase evaporation with an entrapped marker, carboxyfluorescein, which is self-quenching in liposomes at high concentrations such as 0.2 M. When the marker is released by complement action, a strong fluorescence is produced. The amount of marker released from liposomes is able to quantify complement activity of both the classical and alternative pathways. CL50 is the complement activity obtained by lysis of 50% of the liposomes. The number of CL50 units in sera obtained with liposomes correlates well with those obtained using the hemolytic complement test CH50 ( r = 0.98). The advantages of this method include stability of reagents, accuracy, simplicity and speed. In addition, it is well suited for developing an automated system. This method is a useful substitute for the hemolytic assay for determination of human complement activity.