Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation

Abstract

Events in the mannan-binding lectin (MBL) pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented on the surface of microorganisms and the following activation of the proenzymes of the complement system. The protocols for MBL antigen assay are described in this chapter and are useful for routine quantification of MBL and evaluation of the MBL pathway activity in clinical samples. The assays are based on the robust, highly sensitive and reproducible time-resolved immunofluorometric assay (TRIFMA). TRIFMAs are similar to enzyme-linked immunosorbent assays (ELISAs), with the only difference being the type of labeling of detecting molecules. Different approaches have been used to measure the MBL concentration in plasma or serum. The first is based on a modification of the conventional sandwich ELISA, in which microtiter wells are coated with anti-MBL antibody and then incubated with dilutions of plasma or serum and the amount of bound MBL is measured by using europium-labeled anti-MBL antibody (MBL antigen assay). The second assay quantifies MBL on the basis of its lectin-binding activity (lectin assay), in which microtiter wells are coated with mannan instead of anti-MBL antibody. The MBL antigen assay is more sensitive (down to 2 ng of MBL/ml of plasma) than the lectin assay (10 ng/ml).