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A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

ABSTRACT: The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1?50. Reproducibility assays showed a variation below 25% at dilutions less than 1?1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.

SUBMITTER: Pauly D

PROVIDER: S-EPMC4010523 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

Pauly Diana D Nagel Benedikt M BM Reinders Jörg J Killian Tobias T Wulf Matthias M Ackermann Susanne S Ehrenstein Boris B Zipfel Peter F PF Skerka Christine C Weber Bernhard H F BH

PloS one 20140505 5

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mo ...[more]

PMID: 24797388

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Project description:The complement system alternative pathway (AP) can be activated excessively in inflammatory diseases, particularly when there is defective complement regulation. For instance, deficiency in complement regulators CD55 and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), whereas Factor H mutations predispose to atypical hemolytic uremic syndrome (aHUS), both causing severe thrombohemolysis. Despite eculizumab being the treatment for these diseases, benefits vary considerably among patients. Understanding the molecular mechanisms involved in complement regulation is essential for developing new treatments. Properdin, the positive AP regulator, is essential for complement amplification by stabilizing enzymatic convertases. In this study, the role of properdin in red blood cell (RBC) lysis and endothelial cell opsonization in these AP-mediated diseases was addressed by developing in vitro assays using PNH patient RBCs and human primary endothelial cells, where the effects of inhibiting properdin, using novel monoclonal antibodies (MoAbs) that we generated and characterized, were compared to other complement inhibitors. In in vitro models of PNH, properdin inhibition prevented hemolysis of patient PNH type II and III RBCs more than inhibition of Factor B, C3, and C5 (>17-fold, or >81-fold, or >12-fold lower molar IC90 values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC90 values than inhibition of Factor B, or C3, respectively (P < 0.0001). When comparing target/inhibitor ratios in all hemolysis assays, inhibiting properdin was at least as efficient as the other complement inhibitors in most cases. In addition, using in vitro endothelial cell assays, the data indicate a critical novel role for properdin in promoting complement activation on human endothelial cells exposed to heme (a hemolysis by-product) and rH19-20 (to inhibit Factor H cell-surface protection), as occurs in aHUS. Inhibition of properdin or C3 in this system significantly reduced C3 fragment deposition by 75%. Altogether, the data indicate properdin is key in promoting RBC lysis and complement activation on human endothelial cells, contributing to the understanding of PNH and aHUS pathogenesis. Further studies to determine therapeutic values of inhibiting properdin in complement-mediated diseases, in particular those that are characterized by AP dysregulation, are warranted.

Dataset Information

A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

Publications

A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

Similar Datasets

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