Activation Of Terminal Complement Pathway Research Articles

Cfhr1 gene deficiency exacerbates Staphylococcus aureus-induced sepsis and acute lung injury through complement alternative pathway hyperactivation

ObjectiveTo explore the role of excessive activation of the complement alternative pathway (AP) in acute lung injury (ALI) and sepsis induced by Staphylococcus aureus. Subsequently, we aimed to define the effects of Cfhr gene deletion on Factor H expression, AP activation, and the development of sepsis-induced ALI. MethodsA sepsis-induced ALI model was established in Cfhr1-knockout mice by tail vein injection of S. aureus. Sepsis scores, bacterial load in lungs, and cytokine and complement factor levels in blood and lung tissues were evaluated at 6, 12, and 24 h after model establishment. Real-time quantitative PCR and RNA sequencing (RNA-seq) were employed to assess the expression of complement pathway-associated molecules and identify differentially expressed genes (DEGs) related to immune responses. ResultsCompared to wild-type mice, Cfhr1-knockout mice exhibited significantly increased C3a formation in lung tissues following S. aureus infection, indicating enhanced terminal complement pathway activation. Notably, these mice also had higher bacterial colony counts in the lungs, suggesting impaired S. aureus clearance. Transcriptome analysis provided further insights into the impact of Cfhr1 deletion on biological processes and signalling pathways involved in immune response regulation. ConclusionCfhr1 deletion leads to excessive AP activation, exacerbating S. aureus-induced sepsis and ALI.

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Anti-SARS-Cov-2 S-RBD IgG Formed after BNT162b2 Vaccination Can Bind C1q and Activate Complement.

The ability of vaccine-induced antibodies to bind C1q could affect pathogen neutralization. In this study, we investigated C1q binding and subsequent complement activation by anti-spike (S) protein receptor-binding domain (RBD) specific antibodies produced following vaccination with either the mRNA vaccine BNT162b2 or the inactivated vaccine BBIBP-CorV. Serum samples were collected in the period of July 2021-March 2022. Participants' demographic data, type of vaccine, date of vaccination, as well as adverse effects of the vaccine were recorded. The serum samples were incubated with S protein RBD-coated plates. Levels of human IgG, IgA, IgM, C1q, and mannose-binding lectin (MBL) that were bound to the plate, as well as formed C3d, and C5b-9 were compared between different groups of participants. A total of 151 samples were collected from vaccinated (n = 116) and nonvaccinated (n = 35) participants. Participants who received either one or two doses of BNT162b2 formed higher levels of anti-RBD IgG and IgA than participants who received BBIBP-CorV. The anti-RBD IgG formed following either vaccine bound C1q, but significantly more C1q binding was observed in participants who received BNT162b2. Subsequently, C5b-9 formation was significantly higher in participants who received BNT162b2, while no significant difference in C5b-9 formation was found between the nonvaccinated and BBIBP-CorV groups. The formation of C5b-9 was strongly correlated to C1q binding and not to MBL binding, additionally, the ratio of formed C5b-9/bound C1q was significantly higher in the BNT162b2 group. Anti-RBD IgG formed following vaccination can bind C1q with subsequent complement activation, and the degree of terminal complement pathway activation differed between vaccines, which could play a role in the protection offered by COVID-19 vaccines. Further investigation into the correlation between vaccine protection and vaccine-induced antibodies' ability to activate complement is required.

Terminal complement pathway activation drives synaptic loss in Alzheimer’s disease models

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis.

Demonstration of eculizumab deposition by direct immunofluorescence on a renal biopsy in a case of atypical haemolytic uraemic syndrome

Eculizumab is an IgG2/IgG4 kappa monoclonal antibody that blocks C5 and subsequent terminal complement pathway activation. It can be useful in complement-mediated atypical haemolytic uraemic syndrome occurring as a consequence of complement dysregulation. We report a case demonstrating direct immunofluorescence (DIF) findings suggestive of eculizumab binding to renal tissue.

Modulation of complement activation by pentraxin-3 in prostate cancer

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12–36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell invasion and migration.

Soluble terminal complement activation fragment sC5b-9: a new serum biomarker for traumatic brain injury?

Terminal complement pathway activation after traumatic brain injury (TBI) leads to formation of the membrane attack complex (MAC/C5b-9) which induces neuronal cell death and host-mediated secondary brain injury. Serum levels of soluble MAC (sC5b-9) have not been previously determined in patients with isolated TBI. A prospective observational cohort study was performed during a 5-year time-period on adult patients with isolated TBI admitted to an academic level I trauma center in the United States. Controls consisted of patients with femur shaft fractures with or without TBI to mitigate the effect of systemic complement activation by peripheral trauma. Healthy volunteers served as internal controls. The sC5b-9 serum concentrations were measured on the day of admission by enzyme-linked immunosorbent assay (ELISA) and compared between the study cohorts. Univariate analysis was performed to determine independent predictive variables of major complications during hospital admission. Serum sC5b-9 levels were significantly elevated in patients with isolated TBI (n = 42), compared to patients with isolated femoral shaft fractures (n = 36) or combined TBI and femoral shaft fractures (n = 30; p < 0.05). There was no significant difference in serum sC5b-9 levels between the femur group and the combined injury group, compared to the healthy volunteers (n = 21). Univariate analysis revealed serum sC5b-9 levels as an independent predictor of major postinjury complications after isolated TBI (p < 0.01). The soluble terminal complement complex sC5b-9 represents a potential novel serum biomarker specific for isolated head injuries, since peripheral trauma did not appear to affect the serum sC5b-9 levels.

Complement-mediated thrombotic microangiopathy as a link between endothelial damage and steroid-refractory GVHD

AbstractTransplant-associated thrombotic microangiopathy (TA-TMA), a complication of hematopoietic cell transplant (HCT), is associated with significant morbidity and mortality. The pathophysiology and overlap of TA-TMA with other posttransplant complications such as graft-versus-host disease (GVHD) is poorly understood. We retrospectively identified cases of TA-TMA among patients with grade 3/4 gastrointestinal (GI) GVHD, reviewed intestinal biopsy specimens, and performed correlative testing of biomarkers associated with TA-TMA. TA-TMA was more common in patients with steroid-refractory GVHD compared with steroid-responsive GVHD (79.3% vs 42.1%; P = .001). Among patients surviving 100 days post-HCT, 1-year survival from day 100 was significantly better for patients who had not developed TA-TMA in the first 100 days (69.5% vs 36.7%; P < .001). Only 1 of 7 proposed TA-TMA histology criteria (mucosal hemorrhage) differed significantly based on GVHD steroid response. In multivariable modeling, steroid-refractory GVHD was a risk factor for development of TA-TMA (hazard ratio, 3.09; 95% confidence interval, 1.68-5.67; P < .001). There were no differences in complement activation at GVHD onset; however, 2 to 6 weeks later, patients with TA-TMA had higher levels of BBPlus and C5b-9, markers of alternative and terminal pathway activation (BBPlus: median, 600 vs 209.3 ng/mL; P = .0045) (C5b-9: median, 425.9 vs 258.4 ng/mL; P = .029). TA-TMA is associated with poor overall survival (OS) following HCT and may be detected early by histologic findings and may be differentiated from GVHD by measurement of alternative and terminal complement pathway activation. It is unknown whether treatment of TA-TMA will improve survival in steroid-refractory GVHD.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Changes in diagnostic criteria of thrombotic microangiopathy after stem cell transplantation

Hematopoietic stem cell transplantation associated thrombotic microangiopathy is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. The diversity of activity parameters, like elevated laktát-dehidrogenáz, hematological parameters and kidney function are not specific variables after stem cell transplantation. Dysregulation of the classical and alternative pathway can play an important role in the pathomechanism of thrombotic microangiopathy, but the understanding of the role of complement activation under transplantation conditions requires further investigation. Monitoring of complement parameters, including terminal complement pathway activation complex during transplantation may help physicians to improve diagnostic strategy, to evaluate therapeutical options and to predict and follow up efficacy of complement blockade methods and supportive therapy. This review focuses on the development of diagnostic criteria and therapeutical options in thrombotic microangiopathy, and presents some preliminary findings while using different diagnostic criteria in pediatric patients. Orv Hetil. 2017; 158(27): 1043-1050.

Preclinical Evaluation of Orally Bioavailable Small-Molecule Inhibitors of Complement Factor D As a Potential Treatment for Paroxysmal Nocturnal Hemoglobinuria

BACKGROUNDComplement factor D, a serine protease, plays an essential role in the activation of the alternative complement pathway and provides important amplification of the classical and lectin complement pathways. Cleavage of factor B by factor D generates C3 convertase that leads to opsonization of targeted surfaces with complement activation fragments and to the formation of the terminal complement complex (TCC); both events lead to cell lysis. Complement dysregulation underlies multiple hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycophosphatidylinositol-anchored complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antibody eculizumab. Although eculizumab lessens intravascular hemolysis, it does not prevent opsonization of erythrocytes and subsequent extravascular hemolysis by immune cells. In contrast, factor D inhibitors are expected to inhibit both terminal complement pathway activation as well as opsonization and should, therefore, be well-positioned to potentially serve this unmet medical need. Herein, we present the preclinical evaluation of our small-molecule inhibitors of factor D including potency, off-target activities, metabolism, and pharmacokinetic properties.METHODS & RESULTS: Initial inhibitors were discovered through laboratory and virtual screening efforts. A high-resolution (1.5 Å) X-ray structure of an early proprietary inhibitor co-crystallized with factor D aided our optimization campaign that culminated in compounds with IC50 values below 100 nM in biochemical protease assays using natural and non-specific substrates. The potent inhibitory effect of the compounds on factor D protease activity translated to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-digit nM. These small molecules are highly selective for factor D and displayed no significant inhibitory effect on a panel of human serine proteases. In accordance with the high selectivity, the inhibitors showed minimal cellular toxicity, no effect on multiple human receptor-ligand interactions, and no inhibition of hERG potassium channel current. Finally, oral and intravenous administration of selected lead compounds to preclinical animal species showed pharmacokinetic properties that suggest their suitability for oral dosing in humans. CONCLUSIONSWe have discovered highly active small-molecule inhibitors of factor D that demonstrate oral bioavailability and low off-target activities. These key attributes, and the potential to inhibit both intravascular and extravascular hemolysis through inhibition of both terminal complement pathway and opsonization, position these inhibitors as promising development candidates for the oral treatment of PNH. DisclosuresWiles:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Terminal complement pathway activation and neuroaxonal damage in a mouse model of traumatic brain injury

Terminal complement pathway activation and neuroaxonal damage in a mouse model of traumatic brain injury

Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts.

Compstatin, a newly described C3-binding peptide, inhibits complement activation by blocking C3 convertase-mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Pig kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid-phase and in the tissue were examined. The survival of the Compstatin-perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs-C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immune electron microscopy showed less terminal SC5b-9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62L (L-selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P-selectin or beta-thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.

Terminal complement complexes in acute poststreptococcal glomerulonephritis

Activation of the complement cascade occurs in most cases of acute poststreptococcal glomerulonephritis (APSGN) and results in the formation of the terminal complement complexes (TCC). To examine the possible role of TCC in the pathogenesis of glomerular injury in APSGN, we studied 30 patients with the clinical diagnosis of APSGN. All patients had an elevated plasma SC5b-9 concentration at the onset of clinical nephritis. Serial plasma concentrations showed an inverse linear relationship with time after onset of clinical disease (r = -0.59, P = 0.0008), while plasma C3 concentrations showed a positive linear relationship (r = 0.78, P = 0.0001). Renal biopsies of 5 patients demonstrated co-localization of C5b-9, S-protein, and C3 deposition in a glomerular capillary loop and mesangial distribution. Urinary excretion of TCC in the acute phase of APSGN was not elevated and was not a useful marker of disease activity. These data suggest that in APSGN with terminal complement pathway activation the local generation of TCC may contribute to the pathogenesis of the disease.

Anaphylatoxins and Terminal Complement Complexes in Pancreatitis

Complement activation has been proposed as a mediator of remote complications of acute pancreatitis. Thirty-seven patients with acute pancreatitis were studied with respect to the formation of anaphylatoxins (C3a/C3adesArg, C5a/C5adesArg) and terminal complement complexes (TCC) in plasma and ascites fluid. The patients were classified according to Ranson's criteria. Eighteen patients with moderate or severe pancreatitis had higher maximum plasma C3a/C3adesArg and TCC concentrations than 19 patients with mild pancreatitis. During convalescence, the concentrations had returned to normal. High concentrations of C5a/C5adesArg and TCC were also found in ascites and pancreatic cyst fluid, drawn from patients with moderate or severe pancreatitis. As the terminal complement pathway activation is involved in reactive lysis and anaphylatoxins increase vascular permeability, anemia and impaired respiration in these patients may be influenced by complement activation.

Serum complement activation in central nervous system disease in sjögren's syndrome

Central nervous system disease and vasculitis are extraglandular manifestations of Sjögren's syndrome. In our experience, central nervous system disease develops in approximately 70 percent of patients with Sjögren's syndrome and biopsy documented peripheral vasculitis. In order to further investigate the pathogenesis of central nervous system disease and its relationship to peripheral vasculitis in Sjögren's syndrome, we examined sera of patients with Sjögren's syndrome with and without focal central nervous system involvement for evidence of terminal complement pathway activation. Patients were classified as having active focal central nervous system involvement only when they had focal neurologic deficits on physical examination, plus at least one abnormal neurodiagnostic test result. Two thirds of these patients also had cognitive or psychiatric dysfunction. Patients were classified as having peripheral vasculitis if they had clinical and histopathologic documentation of vascular inflammation. Serum SC5b-9 was measured by a sensitive enzyme-linked immunoabsorbent assay. Total hemolytic complement assay, measurement of serum C3 and C4 by radial immunodiffusion, and determination of immune complexes were performed. Fluid-phase terminal complement complexes (SC5b-9) were detected in the sera of 25 of 30 (83 percent) patients with focal central nervous system involvement, but in only seven of 21 (33 percent) patients with Sjögren's syndrome without focal central nervous system disease (p = 0.00084 by Yates' chi-square analysis). Four of these seven patients without focal central nervous system disease, but who had serum SC5b-9, had psychiatric or cognitive dysfunction. SC5b-9 was also detected in sera from 14 of 15 (93 percent) patients with active biopsy-documented peripheral vasculitis in contrast to 18 of 36 (50 percent) patients without clinical evidence of peripheral vasculitis (p = 0.0094). Serum SC5b-9 was a more sensitive indicator of complement activation than circulating immune complex or complement assays. These findings suggest that terminal complement activation may participate in the pathophysiology of both central nervous system and peripheral vasculitis in Sjögren's syndrome. Serum SC5b-9 appears to be a useful diagnostic indicator of vascular inflammation in Sjögren's syndrome and appears to identify those patients at risk for central nervous system complications.

Quantitation of activation of the human terminal complement pathway by ELISA

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigen c determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.