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Development of an Affimer-Antibody Combined Immunological Diagnosis www.nature.com/scientificreports OPEN Development of an Afmer- antibody combined immunological diagnosis kit for glypican-3 Received: 15 March 2017 Chunmei Xie1, Christian Tiede2, Xuanyi Zhang4, Congrong Wang5, Zhixiong Li6, Xiao Xu4, Accepted: 27 July 2017 Michael J. McPherson2,3, Darren C. Tomlinson 2,3 & Weiwen Xu1 Published: xx xx xxxx Glypican-3 (GPC3) is a promising new marker for hepatocellular carcinoma, but the reported values for serum GPC3 difer markedly between currently available kits. Here we isolated Afmer non-antibody binding proteins against GPC3 by phage display and developed a new sandwich chemiluminescence immunoassay (CLIA) combining an Afmer with a monoclonal antibody (Afmer-MAb CLIA). The proposed CLIA assay demonstrated a wide linear range 0.03–600 ng/mL) with a good linear correlation coefcient (0.9999), a high detection limitation (0.03 ng/mL) and specifcity (0–0.002%) for detection of GPC3. The accuracy, hook efect 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 coefcients ranged from −0.286 to 0.478) was observed through pairwise comparison within diferent kits. However, only this newly developed CLIA test showed high specifcity and correlated with the “gold standard” GPC3- immunohistochemistry. This study indicates that Afmer-MAb CLIA can be used to generate a sensitive immunodiagnostic kit, which ofers the potential for a highly specifc clinically-relevant detection system. Glypican-3 (GPC3) is a heparin sulfate proteoglycan molecule frst identifed by Pila and associates in patients with Simpson-Golabi-Behmel Syndrome in 19961. Recent studies2–5 show that GPC3 is associated with the devel- opment and presence of hepatocellular carcinoma (HCC), and might serve as an auxiliary diagnostic marker for the disease. As a result, a GPC3 immunohistochemistry (IHC) kit has been developed and approved by China Food and Drug Administration (CFDA) for use in diagnosing HCC6. Moreover, numerous studies7–10 have detected GPC3 in the peripheral blood of HCC patients, and have reported that its concentration in serum indi- rectly refects the number of GPC3 positive cells present in vivo11. It has been reported that GPC3 is a serological marker, with the highest expression level of those markers currently found in alpha fetal protein (AFP) negative HCC12. Te capability to detect GPC3 glycoprotein could assist in the early diagnosis of HCC and help to estab- lish a clinical prognosis for HCC patients. While there is support for the diagnostic value of detecting GPC3 in serum10, 13, the current GPC3 detection kits remain of value only for research purposes rather than of clinical value. Afmers are non-antibody binding proteins that constrain two 9-aa variable regions for molecular recogni- tion. Afmers can mimic many important functions of antibodies, are highly stable and can be produced rela- tively cheaply, reproducibly and in high quantities using E. coli recombinant protein expression systems. Large phage display libraries have been generated14 and screened against hundreds of target proteins, peptides and small molecules to isolate Afmers for diferent applications, including diagnostics15–18. It has previously been shown that Afmer reagents can be isolated that are specifc for a protein target and can discriminate between even 1State Key Laboratory of Organ Failure Research, Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, China. 2BioScreening Technology Group, School of Molecular and Cellular, Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. 3Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. 4First Afliated Hospital of Zhejiang University, Hangzhou, 310003, China. 5Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. 6R&D center, DaRui Biotechnology Co., Ltd, Guangzhou, 510655, China. Chunmei Xie and Christian Tiede contributed equally to this work. Correspondence and requests for materials should be addressed to D.C.T. (email: [email protected]) or W.X. (email: xu_ [email protected]) SCIENTIFIC REPORTS | 7: 9608 | DOI:10.1038/s41598-017-10083-w 1 www.nature.com/scientificreports/ Figure 1. Standard curve and hook efect for the proposed Afmer-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 efect for the proposed Afmer-MAb CLIA assay. highly related targets14, 19. Tus they have potential as an important alternative to antibodies for target molecule capture in diagnostic applications. Te ability to use Afmers in sandwich ELISA’s has not yet been fully assessed and combining the use of Afmers with monoclonal antibodies in diagnostic kits may represent an important approach for altering their linear range, sensitivity and specifcity. Here, 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 Afmer and a monoclonal antibody (MAb) for measuring GPC3 in serum (Afmer-MAb CLIA). Te performance of the new proposed Afmer-MAb CLIA was assessed both with recombinant GPC3 protein and with clinical samples. Results Screening and generation of Afmers against GPC3. Phage display library screening was performed against immobilized GPC3 protein. Tirty two Afmer clones from the third panning round were tested for their ability to bind GPC3 by phage ELISA. Twenty nine showed binding to the target (see Supplementary Fig. S1) and DNA sequencing revealed eleven unique Afmers. Clones representative of the six most frequently identifed sequences were initially assessed by CLIA for their ability to capture or detect GPC3. Screening of the paired core materials for CLIA. Six Afmers (GPC3-1, GPC3-4, GPC3-6, GPC3-21, GPC3-22, and GPC3-25) and 2 MAbs (8G6 and 7D11, prepared in our previous work13) were assessed for their ability to be used as reagents. Te signal to noise ratio (SNR) was calculated using the formula: SNR = (lumines- cence value of 300 ng/mL standard)/(luminescence value of 0 ng/mL standard). Te most efcient capture reagent was Afmer-GPC3-22, which showed the highest SNR (see Supplementary Table S1) when used in combination with all the other Afmers and MAb reagents. In fact the highest SNR was observed when Afmer-GPC3-22 was used in combination with MAb-7D11. Tus, Afmer-GPC3-22 as capture reagent and MAb-7D11 as detection reagent were prepared for the subsequent experiments for CLIA kit development. Development of the CLIA. Using the Afmer as a capture reagent and the MAb for detection, a CLIA was developed (see Supplementary Fig. S2 for the schematic illustration). All the key parameters were optimized and six reference concentration points were set at 0, 2.5, 25, 50, 300 and 600 ng/mL. Trough optimization, the results were as follows: Te proportion of Afmer-GPC3-22 to magnetic beads was 1:80 (w/w). Te ratio of MAb to NHS-LC-Biotin was 1:10 (w/w). Te volume of acridinium ester (AE)-labeled streptavidin (SA) mixture used was 100 μl. Te pH of the dilution bufer was 7.8. Under these optimized conditions a calibration curve for the proposed CLIA was developed (Fig. 1). Te working curve equation (Fig. 1A) was Y = 3.95 + 1.03X with the lin- ear correlation coefcient of 0.9999, and the coefcient of variation (CV) < 10%(n = 3). Analytic performance of the proposed CLIA assay. Initially the sensitivity and linear range were assessed. Te lower limit of detection (LLD) of the assay was 0.03 ng/mL when the luminescence value (M + 2 SD) of 0 ng/mL standard (n = 20) was substituted into a standard curve equation. A biologically relevant concentra- tion range from 0 to 1200 ng/mL was used to evaluate the linear range of the CLIA. Te results show a wide linear range from 0.03 to 600 ng/mL and no hook efect was observed up to 1,200 ng/mL of GPC3 (Fig. 1B). To evaluate cross-reactivity, several proteins known to be upregulated in HCC were selected, including alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carcino embryonic antigen19-9 (CA19-9), hepatitis B sur- face antigen (HBsAg) and Hepatitis C virus–core antigen (HCV-cAg). Te cross-reactivity was calculated using the formula: cross-reactivity (%) = (measured concentration of GPC3)/(actual concentration of interferent). Te cross-reactivity against these protein was low (0% to 0.002%) and had negligible efect on the simultaneous assay for GPC3 in human serum, which indicates our proposed assay is highly selective for GPC3. SCIENTIFIC REPORTS | 7: 9608 | DOI:10.1038/s41598-017-10083-w 2 www.nature.com/scientificreports/ Intra-assay precision (n = 20) Inter-assay precision (n = 10 * 3) Sample concentration Mean ± SD Mean ± SD (ng/mL) (ng/mL) CV (%) (ng/mL) CV (%) 1 0.92 ± 0.07 7.60 0.89 ± 0.08 8.98 5 4.29 ± 0.26 6.06 5.01 ± 0.40 7.98 50 44.07 ± 2.80 6.35 46.89 ± 3.35 7.14 Table 1. Precision of the proposed assay. SD: standard deviation; CV: coefcient of variation. CV = (SD/Mean) ×100%. dual-MAbs CLIA kit Proposed assay Detect limitation 0.05 ng/mL 0.03 ng/mL Linearity 0.9999 0.99999 Linear range 0–500 ng/mL 0.03–600 ng/mL Specifcity 0.015–0.021% 0–0.002% Accuracy 94.50–106.80% 91.80–104.53% Intra-assay 4.76–5.42% 6.06–7.60% Precision Inter-assay 6.41% 7.14–8.98% Stability sealed 12 months 12 months (2–8 °C) uncovered 30 days 14 days Table 2.
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