Premium
Reconstruction of a robust glycodiagnostic agent supported by multiple lectin‐assisted glycan profiling
Author(s) -
Kuno Atsushi,
Sato Takashi,
Shimazaki Hiroko,
Unno Sachiko,
Saitou Kozue,
Kiyohara Katsue,
Sogabe Maki,
Tsuruno Chikayuki,
Takahama Youichi,
Ikehara Yuzuru,
Narimatsu Hisashi
Publication year - 2013
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.201300010
Subject(s) - lectin , recombinant dna , antibody , biology , microbiology and biotechnology , immunoassay , antibody microarray , microarray , glycan , glycoprotein , immunology , gene expression , biochemistry , gene
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.