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