A novel ELISA for the assessment of classical pathway of complement activation in vivo by measurement of C4-C3 complexes

Abstract

Measurements of complement split products by enzyme-linked immunosorbent assays (ELISA) are well established for the assessment of in vivo complement activation. We have combined two monoclonal antibodies (mAb) with specificities for C3b/iC3b/C3dg (mAb I3 15 and C4/C4b/C4d (mAb M4d2), respectively, in a sandwich ELISA to quantitate C4-C3 complexes as an indicator of complement activation. Serum incubated with heat aggregated IgG (HAG) was used as a standard and the C4-C3 levels expressed as μg equivalent HAG/ml (μg HAG-equ/ml). Normal values of C4-C3 complexes in plasma (EDTA) of healthy probands ( n = 11) were 6.3 μg HAG-equ/ml ± 1.5 (mean ± 1 standard deviation (SD), with a range from 3.6 to 9.1). In patients with systemic lupus erythematosus (SLE, n = 23) C4-C3 values were clearly elevated (48.8 μg HAG-equ/ml ± 52.9, range 7.5–184.7) as compared to samples from patients with idiopathic hypertension (IDH, n = 10) (6.5 μg HAG-equ/ml ± 1.7, range 4.1–9.4). For SLE patients C4-C3 levels significantly correlated with values for C3b/iC3b/C3d ( r = 0.69, p < 0.001) and C3 containing immune complexes ( r = 0.68, p < 0.001), but not with the C4d fragment ( r = 0.26). C4-C3 levels of 96% of the studied SLE patients were increased more than 2 SD above the normal mean as compared to 74% of C4d and activated C3 values, respectively. Serum treated with zymosan as an activator of the alternative pathway of complement did not exhibit higher C4-C3 values. These results demonstrate that the quantitation of in vivo generated C4-C3 complexes by ELISA provide a novel, sensitive parameter for classical pathway of complement activation.