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. 2017 Aug 29;7(1):9608.
doi: 10.1038/s41598-017-10083-w.

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

Chunmei Xie  1 , Christian Tiede  2 , Xuanyi Zhang  3 , Congrong Wang  4 , Zhixiong Li  5 , Xiao Xu  3 , Michael J McPherson  2   6 , Darren C Tomlinson  7   8 , Weiwen Xu  9
Affiliations

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

Chunmei Xie et al. Sci Rep. .

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.

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Conflict of interest statement

D.C.T., and M.J.M. are co-inventors on the Adhiron patent. The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Standard curve and hook effect for the proposed Affimer-MAb CLIA assay (n = 3). (A) Standard curve for GPC3 and the intra-assay CV% for each concentration based on three independent replicates. (B) Hook effect for the proposed Affimer-MAb CLIA assay.
Figure 2
Figure 2
Cutoff value for the proposed Affimer-MAb CLIA assay. (A) Frequency distribution of GPC3 concentrations in a healthy population (n = 196). (B) The ROC curve of the proposed Affimer-MAb CLIA assay (n = 276). Green line represents the diagnostic reference line; blue line represents the ROC curve of GPC3.
Figure 3
Figure 3
Quantitative detection results of GPC3 in clinical samples. (A) GPC3 protein in serum from 80 HCC patients and other patients with liver diseases or other cancers measured using the new developed kit. (NP: normal population; HCC: hepatocellular carcinoma; ICC: intrahepatic cholangiocarcinoma; HB: hepatitis B; HC: hepatitis C; LC: liver cirrhosis). (B) Comparison results of the GPC3 concentration in 73 serum from HCC patients between the proposed new assay and dual-MAbs CLIA from DaRui (DaRui CLIA).
Figure 4
Figure 4
Comparison results between the four different kits. (A) Comparison between the proposed assay to dual-MAbs CLIA from DaRui(DaRui CLIA) (n = 25). (B) Comparison between the proposed assay to Abnova ELISA kit (n = 25). (C) Comparison between the proposed assay to R&D ELISA kit (n = 25). (D) Comparison of the DaRui CLIA kit to Abnova ELISA kit (n = 25). (E) Comparison of the Abnova ELISA kit to the R&D ELISA kit (n = 25). (F) Comparison of the DaRui CLIA kit to the R&D ELISA kit (n = 25).
Figure 5
Figure 5
The Correlation between serum AFP and GPC3. (A) GPC3 positive rate in AFP positive group (n = 50) and AFP negative group (n = 30) in HCC patients. (B) GPC3 level in AFP < 20 ng/mL group (n = 30), AFP ≥ 20 ng/mL group (n = 50) in HCC patients and in 196 normal people (NP).
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References

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