The terminal complement complex (TCC), consisting of C5b, C6, C7, C8, and C9, contains neoantigens that are absent from the individual native components. Neoantigens are present both in the membrane-bound (MAC) and the fluid-phase (SC5b-9) complex. The present study describes production of monoclonal antibodies against neoantigens of both forms of the TCC. A convenient screening and detection system, based mainly on enzyme-linked immunosorbent assays, crossed immunoelectrophoresis with autoradiography, and affinity chromatography with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis including immunoblotting, is described in detail. Two monoclonal antibodies were specific for a neoantigen located in the poly(C9) moiety of the TCC. One of these antibodies, MCaE11, was used for immunohistochemical detection of MAC in tissue and for quantification of the fluid-phase TCC in ethylenediaminetetraacetic acid plasma.

A sensitive double antibody enzyme-linked immunosorbent assay (ELISA) for quantification of C3 activation products in human plasma, synovial fluid, and cerebrospinal fluid is described. The monoclonal antibody MoAb bH6, which is specific for a C3 neoepitope expressed on C3b, iC3b, and C3c, was used as capture antibody. Detection antibody was a polyclonal rabbit anti-human C3c followed by development with a peroxidase-conjugated anti-rabbit Ig antiserum. The activity in normal human EDTA plasma was 1.5% of that in zymosan-activated serum (ZAS). The interassay and intra-assay coefficients of variation were 15% and 3%, respectively. The lower detection limit was 0.0005% of the ZAS standard. Reference range (1.1-2.1% of ZAS) was obtained by measuring EDTA plasma from 40 healthy blood donors. A positive correlation rs = +0.92; P less than 0.0002) was found between the present assay and an already established C3'g' activation ELISA, when samples from 16 patients were examined in both assays simultaneously. The present assay and an assay detecting the terminal complement complex showed virtually identical activation patterns in consecutively drawn samples from a patient undergoing extracorporal circulation.

Background-Complement activation contributes to ischemia-reperfusion injury. Patients undergoing thoracoabdominal aortic aneurysm (TAAA) repair suffer extensive ischemia-reperfusion and considerable systemic inflammation. Methods and Results-The degree and mechanism of complement activation and its role in inflammation were investigated in 19 patients undergoing TAAA repair. Patients undergoing open infrarenal aortic surgery (nϭ5) or endovascular descending aortic aneurysm repair (nϭ6) served as control subjects. Substantial complement activation was seen in TAAA patients but not in controls. C1rs-C1-inhibitor complexes increased moderately, whereas C4bc, C3bBbP, C3bc, and the terminal SC5b-9 complex (TCC) increased markedly after reperfusion, reaching a maximum 8 hours after reperfusion. Interleukin (IL)-1␤, tumor necrosis factor ␣ (TNF-␣), and IL-8 increased significantly in TAAA patients but not in controls, peaking at 24 hours postoperatively and correlating closely with the degree of complement activation. IL-6 and IL-10 increased to a maximum 8 hours after reperfusion in the TAAA patients, were not correlated with complement activation, and increased moderately in the control subjects. Myeloperoxidase and lactoferrin increased markedly before reperfusion in all groups, whereas sICAM-1, sP-selectin, and sE-selectin were unchanged.No increase was observed in complement activation products, IL-1␤, TNF-␣, or IL-8 in a mannose-binding lectin (MBL)-deficient TAAA patient, whereas IL-6, IL-10, myeloperoxidase, and lactoferrin increased as in the controls. Two other MBL-deficient TAAA patients receiving plasma attained significant MBL levels and showed complement and cytokine patterns identical to the MBL-sufficient TAAA patients. Conclusions-The data suggest that complement activation during TAAA repair is MBL mediated, amplified through the alternative pathway, and responsible in part for the inflammatory response.

Background-Acquired deficiencies of