Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein

Talanta 147 (2016) 523–530

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Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein Jing Chen a,b, Qing-hua He a,b,n, Yang Xu a,b,n, Jin-heng Fu a, Yan-ping Li a, Zhui Tu a, Dan Wang a,b, Mei Shu a,b, Yu-lou Qiu a,b, Hong-wei Yang a,b, Yuan-yuan Liu b a b

State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 31 July 2015 Received in revised form 8 October 2015 Accepted 11 October 2015 Available online 21 October 2015

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48 ng mL
1, and the half of saturation concentration (SC50) value was 6.68 70.56 ng mL
1. These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005 ng mL
1, and the linear range was 0.01–10,000 ng mL
1. These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. & 2015 Elsevier B.V. All rights reserved.

Keywords: Nanobody Cancer biomarker Immunoassay Alpha-fetoprotein

1. Introduction Cancer biomarkers, particularly refer to those substances associated with genetic mutations or epigenetic alterations, often offer a quantitative way to determine when individuals are predisposed to particular types of cancers [1]. Currently, there are a number of biomarkers known to be related with certain states of human health and diseases. These include carcinoembryonic antigen (CEA) for colon and rectal cancers, cancer antigen 125 (CA125) for ovarian cancer, prostate-specific antigen (PSA) for prostate cancer, neuron-specific enolase (NSE) for samll cell lung cancer, and alpha-fetoprotein (AFP) as an indication of liver cancer [2]. At present, numerous immunoassay-based methods such as radioimmunoassay, chemilumniescence immunoassay, enzyme-linked immunosorbent assay (ELISA), nanoparcticle-based immmunosensor, electrochemical immunosensor have been opened up widespread used for cancer biomarkers diagnostic because of sensitivity, rapidly, high-throughput, and, in some case, capability n Corresponding authors at: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China. E-mail addresses: [email protected] (Q.-h. He), [email protected] (Y. Xu).

http://dx.doi.org/10.1016/j.talanta.2015.10.027 0039-9140/& 2015 Elsevier B.V. All rights reserved.

of working on-site [3–8]. Although there are many types of immunoassay, the core of immunoassay is based on specific interaction between antigen and antibody. For an immunoassay to be applied to a real sample, it should have high sensitivity and reliability in the matrix in which it is detected. These properties are largely dependent on the availability of antibody with high affinity and specificity to their target analyte along with a high stability in the matrix [9]. Up to now, many researchers have focused their efforts on developing polyclonal, monoclonal antibody, artificial antibody such as aptamer, molecular imprinted polymer, and recombinant antibody [10–12]. Compared with traditional antibodies, recombinant antibodies such as Fab, scFV, diabodies and minibodies have many attractive attributes that include the low production costs and the relative ease of securing bacterial clones [13,14]. However, these constructed antibodies often have lower affinity and are less stable than the intact antibody [9]. Nanobody, which is from the serum of camelidae species, is a functional heavy-chain antibody lacking light chain. This unique antibody interacts with antigen by the virtue of only single variable domain of the heavy chain, referred to as VHH, which is considered to be the smallest intact antigen-binding fragment that can be produced from a functional immunoglobulin. The nanobody provides many

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advantages over the other conventional antibody, in particular, ease of expression in various expression system, high thermal stability, excellent solubility, high affinity, and its well-established immunoglobulin fold allows straightforward design of tailored derivatives [15–17]. Obviously, these beneficial properties stimulated researchers to employ nanobody to develop numerous applications, make nanobody as novel agents for cancer therapy, high affinity binders with macromolecules and small molecules, tracers for noninvasive in vivo molecular imaging, and probes in novel biosensors [18–22]. At present, most reports about diagnostic application of nanobody are related to virus, environmental biomarkers and biotoxins, while the development of nanobody for the quantitative detection of cancer biomarkers has been surprisingly slow [2]. AFP is a plasma protein that is produced in early fetal life by the liver and by a variety of tumors including hepatocellular carcinoma, hepatoblastoma, and nonsemiomatous germ cell tumors of ovary and testis [23]. At present, numerous of immunoassays for AFP have been developed [24–26]. However, for conventional antibody-based immunoassay, the improvement of reproducibility and stability under harsh field-deployable condition is still an important challenge [17,27]. The aim of this study is to prepare nanobody specifically bind to AFP by biopanning from a phage display nanobody library. These prepared nanobodies are clonable, thermally stable and can be applied in both phage displayed and soluble expressed form for immunoassay. In addition, in this study, both sandwich ELISA and immuno-PCR assay setup with nanobodies for ultrasensitive detection of AFP were developed. To our

best knowledge, the application of nanobody in immunoassay for AFP has not been documented up to now.

2. Materials and methods 2.1. Chemicals and reagents Biomarker AFP, NSE, CA-125 and CEA were obtained from LincBio Science Co. (Shanghai, China). Anti-AFP monoclonal antibodies (MAbs), horseradish peroxidase (HRP) conjugated anti-AFP MAbs were purchased from Key Biotech Co. (Beijing, China). Ni-affinity chromatography and HRP-conjugated anti-M13 phage antibodies were obtained from GE Healthcare (Piscataway, NJ). Bovine serum albumin (BSA), ovalbumin (OVA), Freund's complete/incomplete adjuvant was purchased from Sigma-Chemical Co. (St. Louis, MO). Enzyme Sfi I, Not I, Noc I, T4, PrimeScript ™RT-PCR Kit, RNAiso plus Kit, Prime STAR HS DNA polymerase, chloromycetin, isopropylthioβ-D-galactoside (IPTG) were obtained from Takara Dalian (Dalian, China). pHEN 1 vector was kindly donated by Dr. Ma Weijun (Shanghai Jiao Tong University, China). pET25b ( þ) vector and Escherichia coli Rosetta were prepared in our laboratory. Primer AlpVh-LD, AlpVHHR1, AlpVHH-R2, AlpVh-F1 [28], forward primer Phage-F1(5′–3′:CAG TTG CAG CTC GTG GAG) and reverse primer Phage-R1(5′–3′:TGA GGA GAC GGT GAC) were synthesized by Invitrogen (Shanghai, China). All organic solvents and inorganic chemicals were reagent grade.

Fig. 1. The schematic presentation of the construction of nanobody phage display library, biopanning of nanobodies specific to AFP and its application in immunoassay for AFP.

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2.2. Immunization and nanobody library construction A three-year old health male alpaca was immunized with 200 μg of AFP mixed with an equal volume of Freund's complete adjuvant for the first time, and three additional injections with Freund's incomplete adjuvant were given at 2-week intervals. After the last injection, peripheral blood lymphocytes (PBLs) were isolated from blood samples for library construction. The schematic illustration of strategies to construct library was presented in Fig. 1, total mRNA was extracted from the isolated lymphocytes and 60 μg mRNA was used to synthesize the cDNA with oligodT primers by using Primer Script TMRT-PCR Kit. Then, a two-step nested PCR was used to amplify VHH gene fragments. The first step PCR was performed using the synthetic cDNA as template with the primers AlpVh-LD and AlpVHH-R1. The PCR products were analyzed by agarose gel electrophoresis and reextracted the 700 bp fragments to use as the template for the second PCR. The primers AlpVh-F1 and AlpVHH-R2 were used to amplify the nanobody repertoire and the final products of ∼400 bp were extracted by agarose gel purification. Then, the purified PCR products were digested by restriction enzyme Sfi I and Not I, and sub-cloned into phage-display phagemid pHEN1. Afterward, the constructed phagemid were transfected into electrocompetent E. coli TG1 cells by electroporation (25 μF, 200 Ω, 2.5 kV). The transformed cells were mixed with helper phage M13KO7 and cultured at 37 °C overnight. The capacity of constructed library was measured by counting the colonies numbers and DNA sequencing. 2.3. Biopanning and characterization of nanobody against AFP Biopanning of anti-AFP nanobody from constructed phage displayed nanobody library was carried out as described by Kim with some modification [9]. Briefly, in the first round of biopanning, microplate wells were coated with 100 mL of AFP (100 m g mL
1) in 0.01 M PBS (pH 7.4) overnight at 4 °C and washed six times with PBST (PBS containing 0.1% Tween 20). After blocked with 3% OVA-PBS for 1 h at 37 °C and washed six times, 100 mL of constructed phage displayed nanobody library (1.0  1011 colonyform units per mL, cfu mL
1) was added into AFP coated wells and incubated for 1 h at 37 °C with gently shaking. The unbound phages were washed away with PBST for 10 times. Then, the bound phages were eluted with 100 μL of elution buffer [0.2 M Glycine-HCl (pH 2.2), 1 mg mL
1 BSA] for 10 min at 37 °C, and immediately neutralized with 15 μL of 1 M Tris–HCl (pH 9.1). For the next two rounds of biopanning, in all cases, the number of inputted phages remained the same (1.0  1011 cfu mL
1), while the concentration of coated AFP was gradually reduced to 75, 50 m g mL
1 in the second and third round of biopanning, respectively. After three rounds of panning-elution selection, eluted phages were used to infect E. coli TG1 cells for subsequently amplification and titration. Individual plaques from the third round eluted tittering LB/ Amp plate were randomly picked up and used to infect E. coli TG1 cells with helper phage M13KO7 for phage amplification and isolation. Phage ELISA was carried out to identify phages that can specifically bind AFP [29]. Afterward, ELISA positive phage clones were used for sequencing with M13R-48 (5′-CAG GAA ACA GCT ATG ACC-3′) sequencing primer. 2.4. Expression of anti-AFP nanobody Plasmid DNAs from the ELISA positive phage clones were extracted and digested by restriction enzyme Not I and Noc I, respectively. The digested products were ligated to expression vector pET-25( þ ) and transformed into E. coli Rosetta competent cells.

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For expression, the transformed E. coli Rosetta cells were cultured in LB medium (containing 100 μg mL
1 ampicillin and chloramphenicol) at 37 °C by shaking (250 rpm) to OD600∼0.6. The culture was then induced with 0.1 mM IPTG and shaken at 30 °C for 7 h. After that, the periplasmic proteins were extracted by ultrasonic broken. The expressed soluble nanobodies containing 6xHis tag were purified with Ni-chelating affinity chromatography following manufacture's instruction. Nanobodies were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of temperature, pH and ionic strength were considered to evaluate the stability of nanobody. N-18 nanobody and anti-AFP MAbs were incubate for 10 min at 37, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 °C in water-bath, 100 mL of AFP(5 mg/mL, diluted in PBS, pH ¼7.4) was added into microwells (Costar, 42592) and incubate at 4 °C for overnight. After blocking with 5% non-fat dry milk in PBS at 37 °C for 2 h and washing with 0.05% PBST. 100 mL N-18 nanobody and anti-AFP MAbs after heat treatment (1 mg/mL, diluted in PBS, pH ¼7.4) were added to the wells and incubated at 37 °C for 1 h. Following washing 6 times with PBST, 100 mL HRP conjugated anti-His antibody and HRP conjugated anti-IgG antibody (1:2000, diluted in PBS, pH ¼7.4) were transferred to the wells respectively and incubated for 30 min at 37 °C. The peroxidase activity was finally developed with 100 μL of TMB substrate, and the optical density at 450 nm was detected on microplate reader (Thermo Scientific). N-18 nanobody and anti-AFP MAbs were diluted in pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10 PBS (10 mM) and 0, 10, 20, 40, 60, 80 mM PBS(pH 7.4) respectively. Other steps were same with heat stability measurement above to assess nanobody stability.The activity of N-18 incubated at 37 °C, pH 7.0 and 10 mM PBS was regarded as 100% respectively. 2.5. Double nanobodies based sandwich ELISA for AFP The double Nanobodies sandwich ELISA was performed based on pairing expressed soluble nanobody with phage displayed nanobodies. 100 mL of capture antibody (expressed soluble nanobodies, diluted in PBS, pH ¼7.4) was added to microwells (Costar, 42592) and incubated at 37 °C for 1 h. After blocking with 5% nonfat dry milk in PBS at 37 °C for 2 h and washing with 0.05% PBST, 100 mL of each serial concentration of AFP (diluted in PBS) or sample extract was added to the wells and incubated at 37 °C for 1 h. Following washing 6 times with PBST, 100 mL of detection antibody (phage displayed nanobodies) were incubated in the wells at 37 °C for 30 min and then thoroughly washed with PBST. The HRP conjugated anti-M13 phage antibody (GE Healthcare, 1:5000, diluted in PBS) was transferred to the wells and incubated for 30 min at 37 °C. The peroxidase activity was finally developed with 100 μL of TMB substrate, and the optical density at 450 nm was detected on microplate reader (Thermo Scientific). To measure the optimized dilution of immunoassay reagents, a checkboard assay was conducted by using a different dilution of capture antibody and detection antibody in advance. 2.6. Double nanobodies mediated Immuno-PCR The procedure of double nanbodies mediated real-time immuno-PCR was similar to that of double nanobodies based sandwich ELISA except that the detecting step. Firstly, expressed soluble nanobodies against AFP were coated in 96-well PCR plate which was previously treated with 20 μL of 0.8% glutaraldehyde in Milli-Q water for 5 h at 37 °C. After incubation with AFP antigen and phage displayed nanobodies, the wells were washed with PBST for 6 times and with Milli-Q water for 6 times to remove nonspecifically bound phages. The bound phages were used as DNA template for real-time quantitative PCR (qPCR). qPCR was

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performed directly in a 96-well PCR plate using the 7900HT FastReal-Time PCR system. Each PCR reaction consisted of SYBR* PremixEx Taq II (Tli RNaseH Plus), primer (forward primer: 5′-CAG TTG CAG CTC GTG GAG-3′; reverse primer: 5′-TGA GGA GAC GGT GAC-3′), phages and distilled water in a final volume of 20 μL. The dissociation-curve analysis was performed at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The thermal cycle conditions included 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. In addition, negative control that contained all the qPCR reagents except the DNA template was included to verify the quality of amplification. The amplicon encoding part of the phage DNA was detected by fluorescence detector. AFP concentration and CT of the PCR-fluorescence were inverse correlation.

2.7. Method validation with serum samples The recovery of nanobodies mediated immunoassay was assessed by spiking health human serum samples (confirmed by commercial ELISA kits) with various concentration of standard AFP antigen. The serum samples were obtained from the local Center for Disease Control (Nanchang, China) and the assay was carried out in three replicates on the same day for within-assay precision evaluation.

3. Results and discussion 3.1. Library construction and selection of AFP specific nanobodies An ELISA test of the serum prepared from immunized alpaca revealed an increase in reactivity toward AFP antigen after forth immunization from the beginning of the immunization (Figure S1). The total RNA was extracted from alpaca blood and VHH gene fragement ( 400 bp) were obtained by nest-PCR using a set of sense primers specific for the heavy chain variable genes (Figure S2). Phage displayed nanobody library was constructed by subcloning the amplified VHH genes into phagemid vector pHEN1 which was then electroporated into electrocompetent E.coli TG1 cells. After calculating the phage clones, the result showed that a library about 1.0  108 cfu mL
1 individual transformant were obtained, and PCR analysis of 50 randomly picked colonies from the library indicated that about 90% of the colonies contained phagemid with an insert of expected size of a VHH gene. After biopanning from constructed nanobody library, the enrichment of output phage clones was observed after the first round of panning-elution selection. The phage clones with affinity to AFP were enriched from an initial 7.5  104 to 3.4  106 and 7.8  109 (cfu mL
1) in the following second and third round of panning-elution. Ninety five phage clones randomly chosen from the third round of panning were analyzed by phage ELISA. Among them, 22 phage clones were identified to be specific binding to AFP antigen (Fig. 2A). The DNA sequencing results showed that the 20 positive phages were virtual 2 unique sequences (P-5 and P-18).

Fig. 2. (A) Phage ELISA for identifying the positive phage clones binding to AFP; (B) Two kinds of different amino acid sequences of phage displayed nanobodies against AFP (P-5 and P-18).

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As presented in Fig. 2B, the framework regions are highly conserved among these two sequences. However, the amino acids in CDR3 regions show great variation. Furthermore, to the best of our knowledge, this is the first report for identification of nanobodies which specifically bind to AFP antigen. 3.2. Subcloning and nanobody expression For the expression of selected nanobody, AFP binders (P-5 and P-18) were subcloned into pET25b(þ ) expression vector and two expression plasminds of nanobody were constructed and confirmed by DNA sequencing, respectively (Figure S3). The nanobody is directed to the periplasm of E.coli Rosetta cells and carries a Histag to facilitate purification by immobilized Ni-chelating affinity chromatography. Before nanobody was expressed by IPTG inducing, an orthogonal experiment was carried out to analyze effects of IPTG concentration, induction temperature and time. The results showed that the optimal nanobody expression was induced with 0.1 mM IPTG at 30 °C for 7 h and an average yield of about 10 mg protein was obtained per liter of culture grown in shake flasks. SDS-PAGE analysis of these two purified nanobodies to be high quality with more than 90% purity and no contaminants or degradation products were detected in the gel (Fig. 3A). The purified nanobody, corresponding to the above selected phage displayed nanobody (P-5 and P-18), were named as N-5 and N-18,

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respectively. The stability of nanobody (N-18) was presented in Fig. 3. The tested results showed that the activity was still maintained as 89% after incubation for 10 min at 60 °C. Even after incubation at 95 °C, N-5 activity was maintained nearly 67% (Fig. 3B). However,the activity of anti-AFP MAbs was declined significantly. Meanwhile, N-18 maintained a higher activity in different pH and ionic strength than anti-AFP MAbs (Fig. 3C and D). These data ensure the application of nanobody under harsh conditions. 3.3. Nanobodies based sandwich ELISA for AFP A sandwich ELISA for AFP was developed by pairing expressed soluble nanobody with phage displayed nanobody, which was used as capture and detection antibody, respectively. The optimal concentrations of capture antibody (N-5 and N-18) and detection antibody (P-5 and P-18) were firstly determined by a checkboard procedure. In addition, considering the ionic strength and pH could affect the performance of assay, the influence of ionic strength (5, 10, 20, 40 mM) and pH (5.0, 6.0, 7.4, 8.0, 9.0) were evaluated (Figure S4). However, there is no obvious variation in different ionic strength and pH. Taking account of the SC50 and signal density, the better performance was obtained at 10 mM PBS assay buffer in pH 7.4. Under the optimal conditions (the reagent of N-5, N-18, P-5 and P-18 with concentration of 2.0 μg mL
1,

Fig. 3. The stability analysis of nanobody N-18. (A) SDS-PAGE analysis of expressed nanobodies: Lane 1: affinity-purified N-5; Lane 2: affinity-purified N-18; M: protein marker; (B) Thermal stability analysis of nanobody N-18 at 37, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 °C for 10 min in water-bath, respectively;(C) Stability analysis of nanobody N-18 in pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10 PBS(10 mM), respectively; (D) The stability analysis of nanobody N-18 diluted in 0, 10, 20, 40, 60, 80 mM PBS(pH 7.4). Data are represented as an average7standard deviation of three replicates.

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1.8 μg mL
1, 5.8  109 (cfu mL
1), 7.0  109 (cfu mL
1) was selected as the working condition, respectively), the SC50 value of sandwich ELISA based on “N-5: P-18” and “N-18: P-5” pair was 8.11 70.46 ng mL
1 and 6.687 0.56 ng mL
1, respectively (Fig. 4A and B), which was 8-fold more sensitive than that measured for conventional anti-AFP MAb based sandwich ELISA (its SC50 value was 44.6 70.91 ng mL
1, Fig. 4C). Some literatures have been reported that nanobody has a better affinity than conventional IgG antibody [30–32].The limit detection of immunoassay setup with “N-5: P-18”, “N-18: P-5” pair, was estimated from the mean (plus 3 standard deviations ) of the 10 blank samles, was 0.65 ng mL
1 and 0.48 ng mL
1, respectively. In order to demonstrate the specificity of the nanobodies based sandwich ELISA, three of other biomarkers (CAE, NSE, CA-125) were also chosen to perform sandwich ELISA as described above. As shown in Fig. 4A and B, both “N-5: P-18” and “N-18: P-5” pair based immunoassay had strongly binding ability to AFP antigen, but no cross-reactivity with other cancer biomarkers. 3.4. Double nanobodies mediated immuno-PCR for AFP In order to evaluate the analytical performance of double nanobodies mediated real-time immuno-PCR (“N-18: P-5” pair revealed more sensitive than “N-5: P-18” pair, so “N-18: P-5” pair was used in this following studies). We first prepared series of standard AFP solutions with concentrations ranging from 0.001 ng mL
1 to 10, 000 ng mL
1 and detected each of them in triplicate. After the cut-off fluorescence intensity value was set in the exponential amplification region automatically using the machine, the cycle threshold number (Ct) could be obtained for each concentration of AFP (Fig. 5A). By plotting these averaged Ct values against the logarithmic value of each concentration, a linear calibration curve (Y¼ 32.95136
3.22969x, Y¼Ct value, x ¼log (AFP) in mL
1, r2 ¼0.99226) was obtained in the whole concentration range with the coefficient of variation being between 12.7% and 27.7%. By setting Ct 73SD from the blank (0 ng mL
1) as the cutoff value, we observed that the limit of detection of this assay was 5.0 pg mL
1, and the dynamic range of this assay was 0.01–10,000 ng mL
1 (Fig. 5B). According to the previous reports, compared with the obtained data from conventional antibody based immunosensors for AFP [3,8,24,26], this established nanobody-based immuno-PCR assay exhibited a wider work range than anti-AFP MAb based immunoassays for AFP. Furthermore, we also validated this method by detecting the recoveries of spiked serum samples. Eighteen serum samples were collected from health persons. All of the samples were previously analyzed by commercial ELISA kit for AFP and were added with AFP at different concentration (from 0.1 to 1000 ng mL
1) and assayed by double nanobodies mediated immuno-PCR in triplicate. The recoveries of AFP from human serum samples were shown in Table 1. This result indicated that double nanobodies mediated immuno-PCR could effectively detect AFP in real samples.

4. Conclusion

Fig. 4. The standard curves of sandwich ELISA for AFP based on different types of antibody against AFP. Each value is the average of 3 experimental replicates7 standard deviation (A) The capture and detection antibody is N-18 and P-5, respectively; (B) The capture and detection antibody is N-5a nd P-18, respectively; (C) The capture and detection antibody is anti-AFP MAbs and HRP cojugated antiAFP MAbs, respectively.

In this study, we have successfully developed a phage displayed nanobody library constructed from AFP-immunized alpaca animal. Two nanobodies specific to AFP were selected by biopanning from this library. These nanobodies exhibited high thermal stability, excellent solubility, and high affinity to AFP and were applied to both sandwich ELISA and immuno-PCR assay for sensitive detection of AFP. These nanobodies based immunoassays for AFP have excellent analytical performance with high sensitivity, good reproducibility and satisfactory stability. It is particularly worth mentioning that nanobody mediated immuno-PCR assay showed a

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wide working range of 0.01 to 10,000 ng mL
1 and a low limit detetion value (0.005 ng mL
1). To our best knowledge, it has not been reported that the application of nanobodies in immunoassay for AFP. Furthermore, this concept of double Nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

Acknowledgments This work was supported financially by Grants from the National Natural Science Funds (Grants NSFC-31360386, NSFC31201360 and NSFC-31171696), the Jiangxi Province Key Technology R&D Program (Grant 20141BBG70090), major program of Natural Science Foudation of Jiangxi, China (20143ACB21008), the Natural Science Foundation of Jiangxi, China (Grant 20132BAB214005) and by a Grant of the Education Department of Jiangxi Province (Grant GJJ13095)

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2015.10. 027.

Reference

Fig. 5. Quantitative detection of standard AFP solution by nanobodies mediated immuno-PCR. (A) Real-time PCR amplification curves. Curves represent serial dilution of AFP from 0.001 ng mL
1 to 10,000 ng mL
1. (B) Calibration plot of log AFP concentration versus threshold cycles. The results obtained from three individual experiments were averaged.

Table 1 Recovery of AFP detected with nanobody mediated immuno-PCR. Serum number

commercial ELISA kit (n¼ 3) (ng mL
1)

AFP added (ng mL
1)

AFP detected with nanobody mediated Immuno-PCR (n¼ 3) (ng mL
1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0.337 0.15 1.217 0.22 0.89 7 0.14 5.337 0.35 3.38 7 0.28 4.89 7 0.11 10.55 7 0.20 5.29 7 0.23 4.420 7 0.41 9.03 7 0.58 4.29 7 0.88 3.29 7 0.26 15.59 7 0.25 4.98 7 0.27 20.337 1.12 14.25 7 0.68 13.75 7 0.42 9.89 7 0.32

0.1 1 10 100 1000 100,000 0.1 1 10 100 1000 10,000 0.1 1 10 100 1000 10,000

0.447 0.07 2.29 7 0.23 10.63 7 0.86 102.117 17.21 1099.23 7 132.50 9980..23 7 80.47 10.107 0.11 6.317 0.13 8.830 7 1.329 119.317 27.33 1089.08 7 109.84 10110.52 7 102.09 15.62 7 0.73 4.117 0.23 29.65 7 0.92 120.157 12.72 1099.247 132.51 10330.537 40.34

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