Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling

Authors

  • Atsushi Kuno,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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    • These authors contributed equally to this work.

  • Takashi Sato,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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    • These authors contributed equally to this work.

  • Hiroko Shimazaki,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Sachiko Unno,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Kozue Saitou,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Katsue Kiyohara,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Maki Sogabe,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Chikayuki Tsuruno,

    1. Immunology & Chemistry Product Engineering, ICH Business Unit, Sysmex Corporation, Kobe, Hyogo, Japan
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  • Youichi Takahama,

    1. Immunology & Chemistry Product Engineering, ICH Business Unit, Sysmex Corporation, Kobe, Hyogo, Japan
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  • Yuzuru Ikehara,

    1. Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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  • Hisashi Narimatsu

    Corresponding author
    • Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
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Correspondence: Dr. Hisashi Narimatsu, Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

E-mail: h.narimatsu@aist.go.jp

Fax: +81-29-861-3201

Abstract

Purpose

Wisteria floribunda agglutinin positive human Mac-2-binding protein (WFA+-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin–antibody sandwich immunoassay. In this study, we supplied recombinant WFA+-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA+-hM2BP quantification.

Experimental design

The optimum conditions for producing recombinant WFA+-hM2BP were selected by cell glycome analysis based on a lectin microarray. Interlot variability of recombinant WFA+-hM2BP was determined using an antibody-overlay lectin microarray. Screening of anti-M2BP mAb was completed by incorporating a WFA–antibody sandwich ELISA and an antibody-overlay lectin microarray.

Results

The lectin microarray analysis revealed that human embryonic kidney 293 cells efficiently and stably produced WFA+-hM2BP in DMEM containing 10% FCS without any variation in the M2BP glycosylation level. A spiking experiment with recombinant WFA+-hM2BP was mostly effective for antibody screening. The reconstituted sandwich immunoassay was useful for the continuous quantification and cutoff index expression of serum WFA+-hM2BP.

Conclusions and clinical relevance

The multiple use of lectin-assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.

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