Elsevier

Molecular Immunology

Volume 66, Issue 2, August 2015, Pages 164-170
Molecular Immunology

Antibodies reactive to cleaved sites in complement proteins enable highly specific measurement of soluble markers of complement activation

https://doi.org/10.1016/j.molimm.2015.02.029Get rights and content

Highlights

Abstract

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.

Introduction

The complement system consists of more than 30 proteins organized in a cascade of proteolytic events and conformational changes, which support sequential interactions of activated complement components eventually leading to lysis of target cells (Walport, 2001). Proteolysis takes place during the initiation of the alternative complement pathway (cleavage of C3 and factor B) and classical/lectin pathway (C2 and C4), during generation of C3a and C3b by C3 convertases, during inactivation of C3b and C4b opsonins by factor I (FI) (Nilsson et al., 2011), and finally at the transition from opsonic to lytic stage, i.e. splitting of C5 molecule to C5a and C5b (Walport, 2001). Proteolysis triggers conformational changes resulting in exposure of binding sites for complement components placed downstream in the cascade, which in turn also adopt new conformations allowing interaction with the next component. The best-known soluble complement activation products are anaphylatoxins C3a and C5a (and their desArg forms lacking the C-terminal arginine). However, free anaphylatoxins are rapidly cleared from circulation (Webster et al., 1982) thus making the interpretation of their systemic measurement problematic. Notably, not all of the cell-bondable, activated complement components bind target cells but a certain amount remains in their soluble, stable forms, e.g. Bb, C4d, sC5b-9 (Bergseth et al., 2013). Although this gives an opportunity to monitor complement activation in clinical material like plasma and body fluids, measurement of such soluble markers is challenging, as it demands highly specific antibodies not reactive to abundant, non-activated complement components.

Most of the existing assays make use of antibodies against conformational neoepitopes but these sites may be mimicked or decayed in the course of sample handling. Manufacturers seem to be aware of the problem as they recommend very strict sampling guidelines and discourage prolonged storage or heat-inactivation of the sample before measurement as well as repetitive freezing and thawing of the samples to be measured; a precaution not always easy to follow, especially with archival clinical material. Therefore, we tested a novel strategy of raising antibodies specific to very short linear neoepitopes exposed in activated complement components.

Section snippets

Reagents, sera, antibodies and purified complement proteins

Normal human serum (NHS) was prepared as described (Blom et al., 2014) and heat inactivation was performed by heating at 56 °C for 30 min. C6 and C9-depleted sera were purchased from Complement Technology (Tyler, TX). Function-blocking antibody against FI was bought from Quidel (San Diego, CA). The C5-blocking antibody eculizumab was purchased from Alexion Pharmaceuticals (Lausanne, Switzerland). Zymosan was purchased from Sigma (St Louis, MO). Mouse monoclonal antibody mk54 against protein S was

Specificity of C4d detection by ELISA based on antibodies reactive to a C-terminal, linear C4d neoepitope

Given that complement proteases are characterized by very narrow specificity (Sim and Tsiftsoglou, 2004), there is likely a chance that antibodies targeted to short aa sequences exposed at the cleavage sites might specifically recognize only activated complement components. A challenge of raising such antibodies is that such short peptides may be of low immunogenicity due to impaired presentation in MHC class II molecules (Nelson et al., 1997) or that raised antibodies would recognize a region

Funding

This work was supported by The Swedish Cancer Society (to Anna Blom), the Swedish Research Council grants (K2012-66X-14928-09-5 to Anna Blom and K2010-80X-21514-01-4 to Marcin Okroj), grant for Clinical Research (ALF, to Anna Blom) and the Foundations of Österlund (to Anna Blom), Greta and Johan Kock (to Anna Blom), Malmö Cancer (to Marcin Okroj and Anna Blom), Knut and Alice Wallenberg (to Anna Blom) and Inga-Britt and Arne Lundberg (to Anna Blom), as well as grants from the Skåne University

Conflict of interest statement

Anna M. Blom and Marcin Okroj are named as inventors in a patent application including claims to inventions described in the present article. Anders Österborg has received grants for research from Glaxo-SmithKline and has been principal investigator of clinical trials on ofatumumab. Tom E. Mollnes declares no conflict of interest.

Acknowledgements

The help from Ms Sofia Metzner to collect patient samples and Ms Ingrid Eriksson to freeze and store samples is highly appreciated. We also thank Dr Niels Foged for his comments and remarks on the manuscript.

Glossary

sC5b-9 and “sC5b-9 intermediates”
the soluble forms of terminal complement complex (TCC). In contrast, TCC being present on the surface of target cell is termed as membrane attack complex (MAC).

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