Elsevier

Molecular Immunology

Volume 56, Issue 3, 15 December 2013, Pages 232-239
Molecular Immunology

Review
An international serum standard for application in assays to detect human complement activation products

https://doi.org/10.1016/j.molimm.2013.05.221Get rights and content

Abstract

The importance of the complement system in clinical medicine has become evident during the last decades and complement therapeutics has now reached the clinic. Thus, there is an increased interest in and need for assays to evaluate complement activity and dysfunction. Pathologically increased complement activation can indirectly be evaluated by quantification of complement components, but in order to exactly measure such activation, assays for quantification of products formed during activation are required. Progress in this field is hampered by lack of standardization. Therefore, members of the International Complement Standardization Committee, a joint initiative of the International Complement Society and the International Union of Immunological Societies (IUIS), prepared a defined standard for application in assays for complement activation products. We here report on the production and properties of this International Complement Standard #2 (ICS#2). ICS#2 was made from a pool of sera from healthy blood donors (ICS#1) that was activated with a combination of heat-aggregated IgG and zymosan, and subsequently stabilized by adding EDTA and nafamostat mesylate. The protocol was optimized to make the standard applicable in the following activation product assays: C1rs-C1-inhibitor complexes, C4a, C4bc, C4d, Bb, C3bBbP, C3a, C3bc, C3dg, C5a and the soluble terminal C5b-9 complement complex (SC5b-9, TCC). ICS#2 was defined as containing 1000 complement activation units (CAU)/mL for all activation products measured. All activation products were stable after 10 times thawing and freezing and most of the activation products were stable during storage at 4 °C for up to 21 days. ICS#2 was produced large-scale and is considered a valuable tool for standardization, calibration and reference control for complement activation assays, providing the necessary prerequisite for quality assessments between complement laboratories.

Introduction

Along with the increasing understanding of complement as an important part of etiology and pathophysiology of a number of disease conditions, the interest for complement has increased and the first specific complement inhibitor (eculizumab) is already in clinical use, FDA approved to treat paroxysmal nocturnal hemoglobinuria and atypical hemolytic-uremic syndrome. To further explore the role of complement in human diseases, complement laboratories must expand their assay repertoire for evaluating complement pathophysiology. Several recent reviews on complement analysis in research and clinical practice have been published and are here referred to for background information (Mollnes and Kirschfink, 2006, Mollnes et al., 2007, Oppermann and Wurzner, 2010, Harboe et al., 2011, Nilsson and Ekdahl, 2012).

Among assays to measure complement activity and function, only a few are standardized and can be compared between laboratories worldwide. A wide range of assays, both commercial and in-house made, are used to measure different complement activation products. Many of these assays are not standardized, making it difficult to compare results between different laboratories. In order to meet these challenges, the International Complement Society as well as the International Union of Immunological Societies (IUIS) established an International Complement Standardization Committee. Members of this committee have previously prepared and evaluated a serum standard for quantitative and functional complement analysis in normal (non-activated) human serum. This standard was named “Standard 1” (ICS#1). An activated standard was prepared for assays to quantify complement activation products, “Standard 2” (ICS#2), which we present in this paper.

We aimed to obtain a uniform activated human serum standard containing defined and stable amounts of all relevant complement activation products. For this purpose, optimal conditions to prepare a single preparation suitable for classical, lectin and alternative pathway activation were defined and characterized with respect to stability in a number of assays reflecting complement activation generated in all complement pathways. The final International Complement Standard 2 (ICS#2) was then prepared on a larger scale.

Section snippets

Reagents

Zymosan A (from Saccharomyces cerevisiae), 2,2′-azino-di(3-ethylbenzthiosoline sulfonate) (ABTS), ethylenediaminetetraacetic acid (EDTA), polyethylene sorbitan monolaurate (Tween 20) and phosphate-buffered saline (PBS) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Na2CO3·H2O, NaHCO3, KH2PO4, Na2HPO4, NaCl and NaC2H3O2 were obtained from Merck (Darmstadt, Germany). Gammanorm® (human IgG, 165 mg/mL) was obtained from Octapharma AB, Stockholm, Sweeden. Nafamostat mesylate (Futhan, FUT-175)

Re-establishing five previously described in-house ELISAs

The following in-house assays were re-designed using the ICS#2 as standard-curve: C1rs-C1-inhibitor complexes (C1rs-C1-INH), C4bc, C3bBbP, C3bc and TCC. The assays have been described previously (see Section 2 for references). The detailed assay procedures used when re-designing the assays for ICS#2 are presented in Table 1. Inter-and intra-assay CV, lower detection limits and reference range based on samples from human healthy donors are described in Table 2.

Optimization of the conditions for preparation of ICS#2

Pilot data indicated different

First International Complement Activation Standard

We here describe production, stability and application of the first international standard for use in assays for detecting and quantifying complement activation products (ICS#2). The standard contained high amounts of activation products generated from the whole complement cascade, and turned out to be remarkably stable, resisting changes from thawing and freezing, and for many products, also for storage at 4 °C.

Complement activation products

Complement analysis has become increasingly important in research and the clinic, as

Conclusion

We here present a complement activation standard with the broadest possible panel of activation products in high concentration that can be prepared by incubating a human serum pool at 37 °C with HAIgG (1 mg/mL) and zymosan (10 mg/mL) for 4 h, followed by adding 20 mM EDTA and 0.2 mg/mL nafomastat mesylate for stabilization. Repeated careful thawing and freezing up to 10 times did not affect the concentration of any complement activation product tested. Furthermore, the standard was stable at 4 °C for

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