title: Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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
  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.
authors: Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
authors: Atsushi
authors: A
authors: Kuno
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Takashi
authors: T
authors: Sato
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Hiroko
authors: H
authors: Shimazaki
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Sachiko
authors: S
authors: Unno
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Kozue
authors: K
authors: Saitou
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Katsue
authors: K
authors: Kiyohara
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Maki
authors: M
authors: Sogabe
authors: Immunology & Chemistry Product Engineering, ICH Business Unit, Sysmex Corporation, Kobe, Hyogo, Japan.
authors: Chikayuki
authors: C
authors: Tsuruno
authors: Immunology & Chemistry Product Engineering, ICH Business Unit, Sysmex Corporation, Kobe, Hyogo, Japan.
authors: Youichi
authors: Y
authors: Takahama
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Yuzuru
authors: Y
authors: Ikehara
authors: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors: Hisashi
authors: H
authors: Narimatsu
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003933:Diagnosis; D054794:Glycomics / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D034781:Receptors, N-Acetylglucosamine / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D008562:Membrane Glycoproteins; D037121:Plant Lectins; D011134:Polysaccharides; D034781:Receptors, N-Acetylglucosamine; C114782:TAA90K protein, human; C040129:wisteria lectin
keywords: Antibody; Glycoproteomics; Lectin; Liver fibrosis; Sandwich immunoassay