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

Abstract

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.

Hydrogen deuterium exchange and other mass spectrometry- based approaches for epitope mapping

“…Subsequently, others described polyclonal antibodies for study of immune response against tumor necrosis factor α (TNF-α) (Abbott et al, 2013) and factor H-binding protein (fHbp) (Ständer et al, 2021). Further, other investigators compared the immune response for a given antigen in different serum samples (Yang et al, 2016) to evaluate different diagnostic polyclonal antibodies against C-reactive protein, an important biomarker for inflammation (Sun et al, 2022) and recently against Cardiac troponin I (cTnI), a biomarker for acute myocardial infarction (Song et al, 2023). Such studies showcase the exquisite tolerance of HDX-MS for handling molecular heterogeneity, which is difficult with other structural methods.…”

“…Further, monoclonal antibodies are one of the fastest growing drug classes (Ecker et al, 2015;Martin, 2016;Reichert, 2023) (Figure 1) (as of this writing, 104 approved monoclonal antibodies are IgG, three Fabs, two ScFvs, one nanobody, and one DsFv (https://www.antibodysociety.org/resources/approvedantibodies/). Additionally, antibodies are widely used as analytical reagents for diagnostic tests and in research and development (Leinikki et al, 1993;Sun et al, 2022;Song et al, 2023). The introduction of monoclonal antibodies for therapeutic use was a paradigm shift in the pharma industry.…”

Ultrasensitive Electrochemical Immunosensor for Multiplex Sandwich Bioassaying Based on the Functional Antibodies

“…A wide array of detection methods support IVDs, including enzyme-linked immunosorbent assay (ELISA), − immunofluorescence analysis (IFA), , radioimmunoassay (RIA), immuno-PCR, , photoelectrochemical (PEC), , nucleic acid hybridization, high-performance liquid chromatography (HPLC), mass spectrometry, electrochemiluminescence, − fluorescence immunoassay, electrochemical immunoassay , and so on. Recent research efforts have increasingly focused on the development of advanced IVD devices.…”

A carbon quantum layer modified BiVO4 photoelectrochemical aptamer biosensor for ultra-sensitive cTnI biomarker detection based on the interface nephelauxetic effect and heterojunction assistance

“…1,2 Cardiac troponin I(cTnI) is a specific structural protein found in cardiac tissue that inhibits the binding of myosin to actin, and plays a crucial role in the process of cardiac contraction. 3,4 Numerous studies have demonstrated that cTnI exhibits significant changes in its concentration levels during the onset of acute myocardial infarction (AMI). 5 Additionally, cTnI offers advantages such as clear diagnostic thresholds, a wide window of detection, and rapid testing, making it a core biomarker for AMI diagnosis.…”