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Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans

We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct...

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Autores principales: Halkjær, Lene, Troldborg, Anne, Pedersen, Henrik, Jensen, Lisbeth, Hansen, Annette Gudmann, Hansen, Troels Krarup, Bjerre, Mette, Østergaard, Jakob Appel, Thiel, Steffen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214740/
https://www.ncbi.nlm.nih.gov/pubmed/32431705
http://dx.doi.org/10.3389/fimmu.2020.00774
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author Halkjær, Lene
Troldborg, Anne
Pedersen, Henrik
Jensen, Lisbeth
Hansen, Annette Gudmann
Hansen, Troels Krarup
Bjerre, Mette
Østergaard, Jakob Appel
Thiel, Steffen
author_facet Halkjær, Lene
Troldborg, Anne
Pedersen, Henrik
Jensen, Lisbeth
Hansen, Annette Gudmann
Hansen, Troels Krarup
Bjerre, Mette
Østergaard, Jakob Appel
Thiel, Steffen
author_sort Halkjær, Lene
collection PubMed
description We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested in vivo generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) (n = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals (n = 144) (p < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system.
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spelling pubmed-72147402020-05-19 Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans Halkjær, Lene Troldborg, Anne Pedersen, Henrik Jensen, Lisbeth Hansen, Annette Gudmann Hansen, Troels Krarup Bjerre, Mette Østergaard, Jakob Appel Thiel, Steffen Front Immunol Immunology We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested in vivo generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) (n = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals (n = 144) (p < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system. Frontiers Media S.A. 2020-05-05 /pmc/articles/PMC7214740/ /pubmed/32431705 http://dx.doi.org/10.3389/fimmu.2020.00774 Text en Copyright © 2020 Halkjær, Troldborg, Pedersen, Jensen, Hansen, Hansen, Bjerre, Østergaard and Thiel. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Halkjær, Lene
Troldborg, Anne
Pedersen, Henrik
Jensen, Lisbeth
Hansen, Annette Gudmann
Hansen, Troels Krarup
Bjerre, Mette
Østergaard, Jakob Appel
Thiel, Steffen
Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title_full Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title_fullStr Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title_full_unstemmed Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title_short Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
title_sort complement receptor 2 based immunoassay measuring activation of the complement system at c3-level in plasma samples from mice and humans
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214740/
https://www.ncbi.nlm.nih.gov/pubmed/32431705
http://dx.doi.org/10.3389/fimmu.2020.00774
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