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

Area of Science:

• Immunology
• Complement System Biology

Background:

• The mannan-binding lectin (MBL) pathway is the third complement activation route.
• MBL, a serum protein, complexes with MASPs to activate complement components C4 and C2 upon microbial binding.
• This activation leads to opsonization via C4b and C3b deposition.

Purpose of the Study:

• To develop a specific assay for MBL pathway complement activation.
• To inhibit the classical complement pathway for accurate MBL pathway measurement.
• To assess MBL complex activity in serum and plasma samples.

Main Methods:

• Developed a complement activation assay utilizing high ionic strength buffers (1 M NaCl) to specifically target the MBL pathway.
• Inhibited classical pathway activation by disrupting the C1 complex under hypertonic conditions.
• Quantified MBL pathway activity by measuring C4b deposition on mannan-coated microtiter wells using ELISA.

Main Results:

• The developed ELISA assay demonstrated an interassay coefficient of variation of 7.3%.
• MBL-deficient serum showed no MBL pathway activity.
• Serum MBL levels correlated with C4b deposition, but a threefold variation in functional capacity was observed among individuals with similar MBL concentrations.

Conclusions:

• The assay accurately measures MBL pathway-specific C4b deposition.
• The findings highlight a significant inter-individual variability in MBL pathway functional activity.
• This assay can evaluate the clinical relevance of MBL-mediated complement activation.