Improved lectin ELISA for glycosylation analysis of biomarkers using PS-tag-fused single-chain Fv.

In this study, we successfully developed a novel lectin enzyme-linked immunosorbent assay (lectin ELISA) for the detection of glycosides linked to carcinoembryonic antigen (CEA) as a model antigen using a scFv-immobilized hydrophilic polystyrene (phi-PS) plate and 12 different HRP-labeled lectins. Anti-CEA scFv genetically fused with a PS-tag at its C-terminus (scFv-PS) was successfully over-expressed in an insoluble fraction by cultivation of recombinant E. coli. A solid-phase refolding method was adopted to immobilize and refold scFv-PS on the surface of the phi-PS plate. Consequently, in sandwich ELISA using phi-PS plates immobilized with scFv and scFv-PS as well as Maxisorp™ plate with whole antibody (whole Ab), the highest sensitivity and S/N ratio were obtained from the scFv-PS-immobilized phi-PS plate as it was the antigen-binding domain with the highest surface immobilization density and remaining activity displayed on the phi-PS surface. During the lectin ELISA, high background signals were detected from the whole-Ab-immobilized Maxisorp™ plate, indicating that HRP-labeled lectins, particularly PHA-E, Con A, LCA, PSA, DSA, and MAA, directly recognized the glyco-chains of a whole Ab. However, a considerable amount of low background signals were detected from the scFv-PS-immobilized plate since the ligand antibody, namely scFv-PS, was produced by E. coli cells that had no potential for glycosylation during the process of post-transcriptional modification. Signals for glyco-chains conjugated with CEA were detectable using 6 kinds of HRP-labeled lectins, namely PHA-E, PHA-L, SBA, Con A, LCA, and DSA, with much higher sensitivities and S/N ratios. Thus, the lectin ELISA using scFv-PS as a ligand was considerably useful for detecting the glyco-chains of glycoproteins, including glyco-biomarkers, with much higher sensitivities and S/N ratios compared with a conventional whole Ab. By preparation of a phi-PS plate immobilized with different species of scFvs, this method is capable of the glyco-chain analysis of a number of glyco-biomarkers in the fields of clinical diagnosis and biochemical research. Copyright © 2012 Elsevier B.V. All rights reserved.

Citation

Yoichi Kumada, Yuji Ohigashi, Yusuke Emori, Koji Imamura, Yoshiaki Omura, Michimasa Kishimoto. Improved lectin ELISA for glycosylation analysis of biomarkers using PS-tag-fused single-chain Fv. Journal of immunological methods. 2012 Nov 30;385(1-2):15-22

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PMID: 22884622

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