Journal
ANALYTICAL CHEMISTRY
Volume 94, Issue 36, Pages 12427-12434Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.2c02241
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Funding
- Japan Society for the Promotion of Science (JSPS) [20J20465, 21H01957]
- JST COI from the Japan Science and Technology Agency (JST) [JPMJCE1303]
- MOONSHOT research & development program from Japan Agency for Medical Research (AMED) [JP21zf0127001]
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A highly sensitive immunoassay for human immunoglobulin G (IgG) using signal amplification of the coagulation cascade is reported. The reaction product, pNA, was quantified using square wave voltammetry with a glassy carbon electrode. Thrombin efficiently reacted with the substrate FPK-pNA and factor XIa was not inactivated during labeling. The coagulation cascade-based immunoassay showed high selectivity and sensitivity for human IgG.
Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.
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