Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies.

Monoclonal antibodies (mAbs) were raised against human C3a, C3b, C5a, and C5b after immunization of BALB/c mice with the native components C3 and C5. Using different combinations of these mAbs we have developed four sensitive sandwhich-enzyme-linked immunosorbent assays (ELISAs) for the detection of native C3 or C5 in samples with low concentrations of these proteins, e.g., in cell culture supernatants or synovial fluids and cerebrospinal fluids (CSF) and for the detection of the anaphylatoxic peptides (AT-peptides) C3a or C5a in human EDTA-plasma. The C3- and C5-ELISAs were found to be specific for the uncleaved complement proteins. Two different anti-C3a or anti-C5a mAbs were combined for the C3a- and C5a-ELISA. Before assaying a sample in the C3a- or C5a-ELISA a precipitation step to eliminate uncleaved C3 and C5 was necessary. The sensitivity and specificity of the four ELISAs were tested with purified antigens and EDTA-plasma or Cobra venom factor-activated EGTA-plasma samples as a source of C3a and C5a. The detection limits were 1 ng/ml for C3, 1 ng/ml for C3a, 2 ng/ml for C5, and 100 pg/ml for C5a. Plasma samples from patients undergoing cardiopulmonary bypass (CPB) surgery were used as a source of pathological material.