A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood
Elizabeth C. Schramm, Nick R. Staten, Zhouning Zhang, Samuel S. Bruce,
Christopher Kellner, John P. Atkinson, Vasileios C. Kyttaris, George C. Tsokos,
Michelle Petri, E. Sander Connolly, Paul K. Olson
Reference: YABIO 11968
To appear in: Analytical Biochemistry
Received Date: 20 September 2014
Revised Date: 27 January 2015
Accepted Date: 27 January 2015
Please cite this article as: E.C. Schramm, N.R. Staten, Z. Zhang, S.S. Bruce, C. Kellner, J.P. Atkinson, V.C. Kyttaris,
G.C. Tsokos, M. Petri, E. Sander Connolly, P.K. Olson, A Quantitative Lateral Flow Assay to Detect Complement
Activation in Blood, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.01.024
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A Quantitative Lateral Flow Assay to Detect Complement Activation in
Elizabeth C. Schramm1, 2, Nick R. Staten2, Zhouning Zhang2, Samuel S. Bruce3,
Christopher Kellner3, John P. Atkinson1, Vasileios C. Kyttaris4, George C. Tsokos4,
Michelle Petri5, E. Sander Connolly3, Paul K. Olson2, 6 1 Division of Rheumatology, Washington University School of Medicine, Saint Louis,
MO 63110 2 Kypha, Inc., Saint Louis, MO 63108 3 Department of Neurological Surgery, Columbia University Medical Center New York,
NY 10032 4 Beth Israel Deaconess Medical Center, Boston, MA 5 Johns Hopkins University School of Medicine, Baltimore, MD 6. Corresponding author: email@example.com
Short title: Measuring complement activation in blood
Subject category: Immunological procedures
Corresponding authors: firstname.lastname@example.org, email@example.com 2
Complement is a major effector arm of the innate immune system that responds rapidly to pathogens or altered self. The central protein of the system, C3, participates in an amplification loop that can lead to rapid complement deposition on a target and, if excessive, can result in host tissue damage. Currently, complement activation is routinely monitored by assessing total C3 levels, which is an indirect and relatively insensitive method. An alternative approach would be to measure downstream C3 activation products such as C3a or iC3b. However, in vitro activation can produce falsely elevated levels of these biomarkers.
To circumvent this issue, a lateral flow immunoassay system was developed that measures iC3b in whole blood, plasma and serum and avoids in vitro activation by minimizing sample handling. This assay system returns results in 15 minutes and specifically measures iC3b while having minimal cross-reactivity to other C3 split products. While evaluating the potential of this assay, it was observed that circulating iC3b levels can distinguish healthy individuals from those with complement activation-associated diseases. This tool is engineered to provide an improved method to assess complement activation at point-of-care and could facilitate studies to monitor disease progression in a variety of inflammatory conditions.
Complement activation, iC3b biomarker, lateral flow assay, lupus, intracerebral hemorrhage (ICH) 3
The complement system is a phylogenetically ancient branch of the innate immune system that primarily serves to eliminate foreign pathogens from the host [1,2]. A second function of the complement system is to recognize and mark altered self, such as apoptotic or necrotic cells, for clearance . The complement system is activated via three distinct pathways, the classical, lectin, and alternative. While the classical and lectin pathways are initiated by antibodies recognizing antigens and lectins binding sugars, respectively, the alternative pathway (AP) is spontaneously triggered at a continuous, low rate in the blood through a process known as tick-over (reviewed in ). The three activation cascades converge at the central step of activation of component 3 (C3) (Figure 1). Additionally, recent studies have identified an extrinsic pathway, which allows activation of C3 (and the downstream C5) via enzymes of the coagulation pathway and other proteases [5,6].
C3 is the most abundant protein of the complement system and its key opsonic protein .
Proteolytic activation of C3 leads to two split products, C3a and C3b. Deposition of C3b marks pathogens and waste material for clearance by phagocytic cells through immune adherence and ingestion . Release of the anaphylatoxins C3a and the downstream C5a lead to recruitment of inflammatory cells such as neutrophils to a site of infection. Furthermore, initiation of the terminal pathway leads to membrane perturbation and subsequent target cell lysis by the membrane attack complex (MAC). 4
C3 is activated by the C3 convertases, enzymatic complexes formed via all three pathways which convert C3 to C3a and C3b (Figure 1A). During this conversion, the protein undergoes a dramatic conformational change that results in exposure of the thioester bond [8,9]. This is a highly reactive species that enables the transfer of the protein from the fluid phase to nearby targets through a covalent interaction. C3b is itself a component of the AP
C3 convertase and thus participates in its own activation. This results in a powerful positive feedback loop (the AP amplification loop) that can result in the rapid deposition of many copies of C3b on a target (reviewed in ).
To prevent damage to the host, complement activation is stringently regulated (Figure 1A).
Once produced, C3b can be rapidly converted to iC3b by the serine protease Factor I (FI) and a cofactor protein, which releases a small fragment of 18 amino acids, C3f, into the fluid phase (reviewed in [11,12]). iC3b cannot participate in the amplification loop of the AP and is normally cleared from circulation.