Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker.

Autor: Sofuku R; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: rina-sofuku@denka.co.jp., Miyazaki S; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: sayaka-miyazaki@denka.co.jp., Hosojima M; Department of Clinical Nutrition Science, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: hoso9582@med.niigata-u.ac.jp., Goto S; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan., Takemoto K; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan., Kabasawa H; Department of Clinical Nutrition Science, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: hkabasawa@med.niigata-u.ac.jp., Endo T; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: endotaeko@med.niigata-u.ac.jp., Komochi K; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: komochi@med.niigata-u.ac.jp., Sugita N; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: sugitana@med.niigata-u.ac.jp., Aoki H; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: hiroaoki@med.niigata-u.ac.jp., Kobayashi R; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: swymt701@med.niigata-u.ac.jp., Narita I; Division of Clinical Nephrology and Rheumatology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: naritai@med.niigata-u.ac.jp., Saito A; Department of Applied Molecular Medicine, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan. Electronic address: akisaito@med.niigata-u.ac.jp.
Jazyk: angličtina
Zdroj: Journal of immunological methods [J Immunol Methods] 2024 Nov; Vol. 534, pp. 113763. Date of Electronic Publication: 2024 Oct 05.
DOI: 10.1016/j.jim.2024.113763
Abstrakt: Megalin, a type I transmembrane protein, serves as a multi-ligand endocytic receptor in the apical membrane of proximal tubules. Its ectodomain and full-length forms are excreted into human urine, with the former being more abundant. We previously developed two types of sandwich enzyme-linked immunosorbent assays (ELISAs) utilizing monoclonal antibodies that target the amino-terminal ligand-binding domain-I and the carboxyl-terminal cytoplasmic region of human megalin, respectively. The former, termed "A-megalin" ELISA, primarily identifies ectodomains of megalin, whereas the latter, "C-megalin" ELISA, specifically recognizes full-length megalin originating from urinary extracellular vesicles. This study developed novel sandwich ELISAs to assess mouse urinary A-megalin and C-megalin, thereby facilitating studies involving these biomarkers in mouse disease models. Immunoblotting and immunohistochemistry of monoclonal antibodies against human megalin were performed to assess their compatibility with mouse megalin in novel sandwich ELISAs, which were constructed and validated using human assay protocols. Immunoblot analysis of megalin in urinary extracellular vesicles and supernatant was performed to investigate the ratio of ectodomain to full-length forms in mouse urine. Stable measurements having a precision and accuracy within 15 % were achieved in the measurement of quality control samples. A-megalin and C-megalin were detectable in the urine of C57BL/6 mice, whereas most urine samples from kidney-specific conditional megalin-knockout mice were below detection limits. Ectodomain forms of megalin were at least approximately 70 times more abundant than the full-length form, even in mouse urine. In conclusion, we successfully developed sandwich ELISAs for assessing mouse urinary A-megalin and C-megalin to evaluate primarily ectodomain and full-length forms of megalin, respectively.
Competing Interests: Declaration of competing interest A.S. received research grants from Denka Co., Ltd.
(Copyright © 2024. Published by Elsevier B.V.)
Databáze: MEDLINE
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