Development of an Affimer-antibody combined immunological diagnosis kit for glypican-3

Chunmei Xie,Xuanyi Zhang,Weiwen Xu,Christian Tiede,Congrong Wang,Xiao Xu,Darren C Tomlinson,Michael J Mcpherson,Zhixiong Li

doi:10.1038/s41598-017-10083-w

Abstract

Glypican-3 (GPC3) is a promising new marker for hepatocellular carcinoma, but the reported values for serum GPC3 differ markedly between currently available kits. Here we isolated Affimer non-antibody binding proteins against GPC3 by phage display and developed a new sandwich chemiluminescence immunoassay (CLIA) combining an Affimer with a monoclonal antibody (Affimer-MAb CLIA). The proposed CLIA assay demonstrated a wide linear range 0.03–600 ng/mL) with a good linear correlation coefficient (0.9999), a high detection limitation (0.03 ng/mL) and specificity (0–0.002%) for detection of GPC3. The accuracy, hook effect and stability were demonstrated to be satisfactory. The mean level of GPC3 in serum was higher (>8.5 fold, P < 0.001) in hepatocellular carcinoma patients compared to healthy and other liver disease individuals. A poor correlation (correlation coefficients ranged from −0.286 to 0.478) was observed through pairwise comparison within different kits. However, only this newly developed CLIA test showed high specificity and correlated with the “gold standard” GPC3-immunohistochemistry. This study indicates that Affimer-MAb CLIA can be used to generate a sensitive immunodiagnostic kit, which offers the potential for a highly specific clinically-relevant detection system.

Highlights

Glypican-3 (GPC3) is a heparin sulfate proteoglycan molecule first identified by Pila and associates in patients with Simpson-Golabi-Behmel Syndrome in 19961
We report the development of a new sandwich chemiluminescence immunoassay (CLIA) that is much more sensitive than enzyme-linked immuno-sorbent assay (ELISA) and can be undertaken on an automatic system based on a combination of an Affimer and a monoclonal antibody (MAb) for measuring GPC3 in serum (Affimer-MAb CLIA)
Phage display library screening was performed against immobilized GPC3 protein

Summary

Introduction

Glypican-3 (GPC3) is a heparin sulfate proteoglycan molecule first identified by Pila and associates in patients with Simpson-Golabi-Behmel Syndrome in 19961. Highly related targets[14, 19] They have potential as an important alternative to antibodies for target molecule capture in diagnostic applications. The ability to use Affimers in sandwich ELISA’s has not yet been fully assessed and combining the use of Affimers with monoclonal antibodies in diagnostic kits may represent an important approach for altering their linear range, sensitivity and specificity. We report the development of a new sandwich chemiluminescence immunoassay (CLIA) that is much more sensitive than ELISA and can be undertaken on an automatic system based on a combination of an Affimer and a monoclonal antibody (MAb) for measuring GPC3 in serum (Affimer-MAb CLIA). The performance of the new proposed Affimer-MAb CLIA was assessed both with recombinant GPC3 protein and with clinical samples

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Conclusion