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

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.

• Authors: Atsushi Kuno, Takashi Sato, Hiroko Shimazaki, Sachiko Unno, Kozue Saitou, Katsue Kiyohara, Maki Sogabe, Chikayuki Tsuruno, Youichi Takahama, Yuzuru Ikehara, Hisashi Narimatsu
• Journal: Proteomics. Clinical applications (Proteomics Clin Appl) Vol. 7 Issue 9-10 Pg. 642-7 (Oct 2013) ISSN: 1862-8354 [Electronic] Germany
• PMID: 23640794 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

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

Topics

• 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)