An assay for the mannan-binding lectin pathway of complement activation

Abstract

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.