Development and Optimization of an ELISA to Quantitate C3(H ₂ O) as a Marker of Human Disease

Discovery of a C3(H₂O) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(H₂O), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(H₂O). We initially utilized this assay to determine baseline C3(H₂O) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(H₂O) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(H₂O) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(H₂O) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(H₂O). To determine the potential use of C3(H₂O) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(H₂O) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(H₂O) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(H₂O) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(H₂O) as a diagnostic and prognostic biomarker in human diseases.