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

Diana Pauly,Boris Ehrenstein,Jörg Reinders,Bernhard H F Weber,Peter F Zipfel,Benedikt M Nagel,Matthias Wulf,Susanne Ackermann,Christine Skerka,Tobias Killian,Cordula M Stover

doi:10.1371/journal.pone.0096371

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.

Highlights

The complement system serves as a bridge between the innate and the adaptive immune system
We demonstrated that our monoclonal antibody inhibits complement activation and allows defining properdin serum levels in patients with complement-mediated diseases
With the use of monoclonal antibody (mAb) 1340, we identified novel interaction partners like immunoglobulin G (IgG) and clusterin that seemed to include in a multimeric properdin protein complex in soluble human serum

Summary

Introduction

The complement system serves as a bridge between the innate and the adaptive immune system. Activated complement is a double-edged sword, capable of protecting from pathogens as well as causing self-tissue damage. Spontaneous or surface-dependent hydrolysis of complement protein 3 (C3) to C3(H2O) activates this pathway in human blood. C3(H2O) binds complement factor B and interacts with complement factor D to form a fluid phase C3convertase which cleaves C3 in C3a and C3b. C3b opsonizes target surfaces and binds factor B, which is cleaved by factor D, yielding Bb. Properdin stabilizes five to ten-fold both the fluidphase (C3(H2O)Bb) and surface-bound (C3bBb) C3-convertase of the alternative pathway [7]. Complement factor H (CFH) dissociates the C3-convertase and in combination with factor I inactivates the C3b protein [7,8]. In a positive feedback loop, C3convertase cleaves C3 and an additional C3b molecule complements C3bBb to form the C5-convertase. C5 cleavage initiates the terminal complement pathway and mediates inflammation as well as the formation of a cell membrane attack [9]

Methods

Results

Conclusion