Reconstruction of a Robust Glycodiagnostic Agent Supported by Multiple Lectin-Assisted Glycan Profiling

Proteomics Clin Appl. 2013 Oct;7(9-10):642-7. doi: 10.1002/prca.201300010. Epub 2013 Aug 7.

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.

Keywords: Antibody; Glycoproteomics; Lectin; Liver fibrosis; Sandwich immunoassay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Neoplasm / metabolism*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Diagnosis*
  • Glycomics / methods*
  • HEK293 Cells
  • Humans
  • Membrane Glycoproteins / metabolism*
  • Plant Lectins / metabolism*
  • Polysaccharides / metabolism*
  • Receptors, N-Acetylglucosamine / metabolism*

Substances

  • Antigens, Neoplasm
  • Membrane Glycoproteins
  • Plant Lectins
  • Polysaccharides
  • Receptors, N-Acetylglucosamine
  • TAA90K protein, human
  • wisteria lectin