Chapter 14 : Analysis of Activity of Mannan-Binding Lectin, an Initiator of the Lectin Pathway of the Complement System

Complement activation pathways. The figure shows the events leading to the formation of enzyme complexes that cleave C3. The three pathways thus converge on C3. Thereafter, formation of the terminal membrane attack complex, opsonic factors (C3b), and chemotactic factors (e.g., C5a) may proceed. The classical pathway is activated by C1q in complex with C1s and C1r upon binding to antibody-antigen complexes. The lectin pathway is activated when MBL in complex with MASPs recognizes foreign carbohydrate patterns. As indicated in the figure, ficolins may activate complement via this pathway, but the assay described in this chapter is not influenced by these molecules, as they bind to a different set of ligands than those presented on the surface and used in the assay. The alternative pathway is constitutively activated by the formation of C3(H2O)-factor B complexes and may be propagated on foreign surfaces by the other factors indicated. The assay described in this chapter measures the cleavage of C4 and the following deposition of C4b by MASP-2 bound to MBL.

Assay of MBL pathway activity. Step 1, coating of the microtiter plate with mannan; step 2, MBL–MASP-2 complexes bind to immobilized mannan; step 3, incubation of C4, which is then cleaved, and deposition of C4b in the wells; step 4, anti-C4 antibody is bound to the deposited C4 fragment; step 5, binding of europium-labeled streptavidin to the biotinylated anti-C4 antibody; step 6, addition of enhancement solution to the wells, which is followed by reading of the plate by time-resolved fluorometry.

Appearance of a typical standard curve for the assay. The serum concentration is given on the x axis (log scale), and the signal is given on the y axis (log scale). In this figure, the highest serum concentration used is 1/20, followed by 3.5-fold dilutions. Other dilution schemes can also be used (e.g., 1/10 dilution followed by 3-fold dilutions).