Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement

Area of Science:

• Immunology
• Complement System Biology
• Biochemistry

Background:

• The complement system's terminal pathway generates C5b-9 complexes, either membrane-bound (C5b-9m) or fluid-phase (SC5b-9).
• Accurate quantification of these complexes is crucial for understanding complement activation and related pathologies.
• Existing methods for SC5b-9 determination may require re-evaluation.

Purpose of the Study:

• To develop and validate a sensitive and reliable sandwich ELISA for quantifying human C5b-9m and SC5b-9.
• To assess SC5b-9 levels in normal human serum and plasma under various conditions.
• To characterize the composition of circulating terminal complement complexes.

Main Methods:

• Development of a sandwich ELISA utilizing a monoclonal antibody against a C5b-9 neoantigen and polyclonal rabbit antibodies.
• Calibration of the ELISA with purified C5b-9m and SC5b-9.
• Analysis of SC5b-9 levels in pooled human serum, normal EDTA plasma, and serum incubated at 37°C; partial purification and characterization of plasma-derived complexes.

Main Results:

• The developed ELISA can detect 3 ng/ml C5b-9m and 20 ng/ml SC5b-9.
• Maximal complement activation in serum yielded 220 ± 40 µg/ml SC5b-9.
• 65% of normal plasma samples contained 100-600 ng/ml SC5b-9; serum incubation at 37°C led to spontaneous SC5b-9 generation (mean 1848 ± 1031 ng/ml after 60 min).
• Partially purified plasma complexes contained C8, C9, and S-protein, consistent with genuine SC5b-9.
• No significant age- or sex-dependent variations in SC5b-9 levels were observed.

Conclusions:

• A novel, sensitive ELISA for SC5b-9 and C5b-9m has been established.
• Spontaneous generation of SC5b-9 occurs readily in serum upon incubation.
• The findings necessitate a critical review of previous SC5b-9 quantification methods in human plasma and serum.