Development and Optimization of an ELISA to Quantitate C3(H 2 O) as a Marker of Human Disease.

Michelle Elvington,Alexis R Liszewski,Thalachallour Mohanakumar,Hrishikesh S Kulkarni,John P Atkinson,Alfred H J Kim,Ramsey R Hachem,M Kathryn Liszewski

doi:10.3389/fimmu.2019.00703

Abstract

Discovery of a C3(H2O) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(H2O), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(H2O). We initially utilized this assay to determine baseline C3(H2O) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(H2O) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(H2O) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(H2O) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(H2O). To determine the potential use of C3(H2O) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(H2O) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(H2O) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(H2O) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(H2O) as a diagnostic and prognostic biomarker in human diseases.

Highlights

The complement system is a well-established component of innate immunity and an instructor of adaptive immunity
To determine if C3(H2O) might serve as a disease biomarker for conditions associated with complement activation as well as to further evaluate the sample handling issues, we set out to develop an ELISA specific for C3(H2O) that lacks crossreactivity with C3, C3b or C3b fragments (Figure 1A)
The hydrolysis of the internal thioester bond is accompanied by a substantial conformational change in C3(H2O) such that it closely mimics the three-dimensional structure of C3b [28,29,30,31,32,33,34,35] (Figure 1B)

Summary

Introduction

The complement system is a well-established component of innate immunity and an instructor of adaptive immunity. While the CP and LP have specific triggers (antibodies and lectins, respectively), the AP is thought to be independently engaged by the spontaneous activation of C3 to a form with a cleaved thioester bond [1, 2]. The prevailing hypothesis is that this occurs by the spontaneous, low-level hydrolysis of the internal thioester bond in native C3 in the fluid phase [3,4,5]. Within microseconds this form of C3 becomes covalently bound to a target (such as a microbe or cell) or interacts with water to form C3(H2O) [4]. Since C3(H2O) may be a trigger for complement activation at sites of cellular injury, degeneration or autoimmune attack, a simple, quantitative and specific assay for this form of C3 could provide insights into how the complement system functions in human disease

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Conclusion