Epitope mapping of antibodies in C-reactive protein assay kits by hydrogen-deuterium exchange mass spectrometry explains differential results across kits

Sun, Haofeng; Xiao, Peng; Zhou, Yi; Li, Hongmei; Shen, Min; Sun, Keqi; Wang, Xiaojian; Zhou, Min; Song, Da

doi:10.1007/s00216-022-04029-z

Cited by 4 publications

(7 citation statements)

References 27 publications

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“…In general, manufacturers of immunoturbidimetry test systems do not pay sufficient attention to the epitopes of antibodies. However, our previously published work showed that the epitope information of the antibody in the immunoturbidimetry kit is also worthy of attention …”

Section: Resultsmentioning

confidence: 99%

“…However, our previously published work showed that the epitope information of the antibody in the immunoturbidimetry kit is also worthy of attention. 22 Because assay kits A and C use the same principle of quantification (i.e., the chemiluminescence method), they were compared in detail. Antibodies S13 and D2 originated from different test systems but identified the same region of residues 91−96 in cTnI.…”

Section: Comparison Of Antibodiesmentioning

confidence: 99%

“…We previously studied the relationship between quantitative bias and the epitope differences in C-reactive protein immunoturbidimetry kits using a conventional HDX-MS workflow . Herein, we analyzed antibodies against cTnI using a similar workflow for different cTnI diagnostic test systems from three manufacturers labeled as A, B, and C. Moreover, we investigated the epitope changes in two antibody cocktails(D1, D2, pAb2) and (R195, F12, S13).…”

mentioning

confidence: 99%

“…20,21 We previously studied the relationship between quantitative bias and the epitope differences in C-reactive protein immunoturbidimetry kits using a conventional HDX-MS workflow. 22 Herein, we analyzed antibodies against cTnI using a similar workflow for different cTnI diagnostic test ■ EXPERIMENTAL SECTION Materials and Reagents. Native cardiac troponin I (>98%) lyophilized powder was purchased from Hytest (Turku, Finland).…”

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confidence: 99%

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In-Vitro Diagnostic Reagent Evaluation of Commercially Available Cardiac Troponin I Assay Kits Using H/D Exchange Mass Spectrometry for Antibody-Epitope Mapping

Song

Sun

et al. 2023

Anal. Chem.

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Cardiac troponin I (cTnI) is the biomarker of choice and considered a gold standard for the diagnosis of acute myocardial infarction. However, the quantitative results of cTnI assay kits from different manufacturers are not comparable. Based on the H/D exchange mass spectrometry (HDX-MS) workflow, we developed an in-vitro diagnostic reagent antibody evaluation strategy to analyze the interactions of epitopes and antibody cocktails(R195, F12, S13) and (D1, D2, pAb2). The HDX results indicate that the quantitative result bias of the different reagents originates from the ability of antibodies to recognize various cTnI complex forms, such as free cTnI, hydrolyzed cTnI, and cTnI combined with cTnT or TnC as binary or ternary complexes (cTnIC, cTnTIC), in blood based on different epitopes. The data obtained from the peptide HDX of interest after treatment with various antibody cocktails clearly indicated epitope specificity. The consistency of quantitative results can be improved by a thorough investigation into the epitopes recognized by the antibodies of various diagnostic kits, which will lead to the standardization of cTnI diagnosis.

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Section: Resultsmentioning

confidence: 99%

Section: Comparison Of Antibodiesmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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In-Vitro Diagnostic Reagent Evaluation of Commercially Available Cardiac Troponin I Assay Kits Using H/D Exchange Mass Spectrometry for Antibody-Epitope Mapping

Song

Sun

et al. 2023

Anal. Chem.

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“…To maintained 25% concentration, cooled 95% ethanol was added and maintained the temperature between -5°C and -6°C. Centrifugation removed the precipitated IgG and suspended in PBS [3]. Another method to obtain fractions from blood plasma is shown in Fig 2 . Electrophoretic methods: In electrophoresis, various particles bearing different charges are being separated.…”

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confidence: 99%

A comprehensive review on preparation of pure immunoglobulins Authors

Shehzad¹,

Tayyab²,

Bibi³

et al. 2022

IJNMS

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Immunoglobulins are also known as antibodies. Plasma cells are responsible for the production of immunoglobulins. Beta cells are activated against a pathogenic attack and facilitated the formation of different types of immunoglobulins naturally. These immunoglobulins are also artificially synthesized by non-specific laboratorial techniques include fractionation precipitation, electrophoretic methods, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and by the specific immuno- adsorbent methods. Third generation immunoglobulins are effectively used for therapeutic purpose against viral infections intravenously. Second generation immunoglobulins synthesis involved removal of anti-complement contaminants and IgG aggregates, through enzymatic degradation and chemical modification. The harmful effects of intravenous immunoglobulins can be reduced by using ultraviolet light, P-propiolactone, and pepsin at pH 4. Intravenous immunoglobulin drugs lead to anti-inflammatory and immunomodulatory effects in various infections. The mechanism of action of intravenous immunoglobulins is dependent on the binding between the Fc portion of injected IgG and target cell receptors. Radiolabeling is performed by two methods as in vivo, in which radiolabeled antibodies are incorporated into the body to bind with the antibodies and in vitro method, radioactive material is bound with already formed antibodies. This chapter highlighted the artificial methods adopted for production of radiolabeled immunoglobulins holding significant therapeutic and diagnostic applications.

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Investigating how intrinsically disordered regions contribute to protein function using HDX-MS

Parson

Jenkins

Burke

2022

Biochemical Society Transactions

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A large amount of the human proteome is composed of highly dynamic regions that do not adopt a single static conformation. These regions are defined as intrinsically disordered, and they are found in a third of all eukaryotic proteins. They play instrumental roles in many aspects of protein signaling, but can be challenging to characterize by biophysical methods. Intriguingly, many of these regions can adopt stable secondary structure upon interaction with a variety of binding partners, including proteins, lipids, and ligands. This review will discuss the application of Hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a powerful biophysical tool that is particularly well suited for structural and functional characterization of intrinsically disordered regions in proteins. A focus will be on the theory of hydrogen exchange, and its practical application to identify disordered regions, as well as characterize how they participate in protein–protein and protein–membrane interfaces. A particular emphasis will be on how HDX-MS data can be presented specifically tailored for analysis of intrinsically disordered regions, as well as the technical aspects that are critical to consider when designing HDX-MS experiments for proteins containing intrinsically disordered regions.

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Epitope mapping of antibodies in C-reactive protein assay kits by hydrogen-deuterium exchange mass spectrometry explains differential results across kits

Cited by 4 publications

References 27 publications

In-Vitro Diagnostic Reagent Evaluation of Commercially Available Cardiac Troponin I Assay Kits Using H/D Exchange Mass Spectrometry for Antibody-Epitope Mapping

In-Vitro Diagnostic Reagent Evaluation of Commercially Available Cardiac Troponin I Assay Kits Using H/D Exchange Mass Spectrometry for Antibody-Epitope Mapping

A comprehensive review on preparation of pure immunoglobulins Authors

Investigating how intrinsically disordered regions contribute to protein function using HDX-MS

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