Anal Bioanal Chem DOI 10.1007/s00216-015-8693-3

RESEARCH PAPER

Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library Yang Xu 1 & Liang Xiong 1,2 & Yanping Li 1 & Yonghua Xiong 1 & Zhui Tu 1 & Jinheng Fu 1 & Bo Chen 1

Received: 27 January 2015 / Revised: 20 March 2015 / Accepted: 10 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Compared with peptide-based mimotope, antiidiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phagedisplayed VHH library. The affinity constant (KD) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to antiCIT McAb (68 nM). The developed VHH-based enzymelinked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10 % (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n=3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 =96.2 ng/ Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8693-3) contains supplementary material, which is available to authorized users. * Yanping Li [email protected] 1

State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, Number 235 Nanjing East Road, Nanchang 330047, Jiangxi, China

2

Department of Preventive Medicine, Gannan Medical College, Number 1 Yixueyuan Road, Ganzhou 341000, Jiangxi, China

mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISAwas evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library. Keywords Anti-idiotypic antibody . Citrinin . Single domain antibody . Hapten mimicry . Phage displayed library

Introduction Competitive immunoassays are methods used to determine mycotoxins in contaminated food products because such methods exhibit excellent specificity, feasibility, and higher sensitivity [1]. In these assays, competing antigen is a commonly used compound usually obtained by coupling mycotoxins with a carrier protein via chemical synthesis. For the optimum performance of assays, the binding affinity of competing antigen to antibodies should be slightly less than that of the target analytes to antibodies. Therefore, a library of analogs is commonly synthesized and analogs exhibiting the highest sensitivity and specificity to immunoassays are selected [2]. However, chemical synthesis is limited by many disadvantages, such as overuse of organic solvents and mycotoxin standards, potential toxic effect on operators, poor reproducibility, and complicated quality assessment of synthetics. As such, safe and renewable surrogates of mycotoxin haptens for immunoassays should be developed. Biosynthetic antigen is a novel hapten surrogate widely used in competitive immunoassays. In our previous study, phage-display random peptide libraries were successfully used to pan mycotoxin mimotopes, such as zearalenone

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(ZEN) [3], ochratoxin A (OTA) [4, 5], and fumonisin B1 (FB1) [6]. However, synthetic peptides can lose or reduce binding bioactivity to antibody because the primary activity of peptide-based mimotope partially depends on intact conformation with coat protein pIII of filamentous bacteriophages. Therefore, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic antigens [7–9] that can be used to mimic the epitopes of small molecular haptens because AIds contain accessible proteinaceous molecules with stable conformation [10–14]. According to the concept of AIds, AIds can be mainly classified into three subgroups [15]. The β subtype of AIds (β-AIds) can be considered as an internal image of antigens used in the paratope of a primary antibody; by contrast, αAIds and γ-AIds cannot mimic original antigens because these AIds recognize idiotopes distal from or close to the paratope of an antibody. Thus far, the most frequently reported structures of AIds are the monoclonal antibodies (McAbs, 150 kDa) [11, 16, 17], single chain fragment variable antibody (scFv, ~30 kDa) [10], and single variable domain antibody fragments (VHHs or so-called nanobodies, ~15 kDa) [12–14]. Among these structures, VHHs are the smallest intact antigen-binding fragments, which are derived from a subset of antibodies completely devoid of light chains in the camelid (camels, llamas, and alpacas) [18, 19]. Compared with conventional antibodies, such as McAb and scFv, VHHs contain only three hypervariable antigen-binding loops (H1–H3), in which H3 loop usually presents large convex paratopes and enters molecular cavities or crevices of target proteins [20, 21]; the paratope of conventional antibody usually forms a concave or a flat surface suited for binding of small molecules, peptides, or protein [22–24]. This architecture of VHHs shows a clear structural advantage over conventional antibodies in targeting clefts on antigenic binding sites of primary antibodies. However, to our knowledge, almost all of AIds, as well as anti-idiotypic nanobodies, are generated in vivo by animal immunization with heterologous idiotypic antibodies [10–14]. The main disadvantage of yielding AIds is that primary antibodies exhibit higher affinity to these AIds than to target analytes because of affinity-matured process of AIds in vivo during immune responses to primary antibodies; as a result, AIds show decreased sensitivity in competitive immunoassays. For example, Wang et al. [13] isolated alpaca AIds by subcutaneously immunizing a neutered male alpaca with anti-aflatoxin B1 (AFB1) McAb and successfully used AIds as surrogate antigens for AFB 1 immunoassay. Nevertheless, the sensitivity of developed AId-based enzyme-linked immunosorbent assay (ELISA) is lower than that of conventional ELISA; a similar conclusion was observed in our previous research [14]. In our opinion, diverse convex formation in VHHs can be potentially used to direct pan desired AIds from the alpaca naive VHH library. The affinity constant of selected AIds to

corresponding primary antibodies could be slightly lower than that of target analytes to primary antibodies because alpaca immunization was not performed. In the present study, as a proof of concept, citrinin (CIT) was used as a small molecule model and reference standard; this molecule, which has been detected in several food types, has shown potential nephrotoxicity, hepatotoxicity, immunotoxicity, and carcinogenicity [25, 26]. An anti-CIT-McAb designated as 4G6 was used as a primary antibody to select CIT AId from a naive alpaca VHH library (Fig. 1). An anti-idiotypic VHH was expressed; the properties of purified VHH, including cross-reaction to other anti-mycotoxin McAbs and affinity constant of VHH to primary antibody, were characterized. The selected anti-idiotypic VHH was used to develop a specific VHH-based ELISA (VELISA) for CIT analysis. Accuracy, precision, and limit of detection (LOD) of the proposed V-ELISA were evaluated by determining CIT-spiked wheat powder and rice samples. The proposed V-ELISA was further compared with conventional CIT ELISA to verify sensitivity and reliability by analyzing eight real Monascus-fermented rice samples.

Materials and methods Chemicals and reagents The anti-CIT-McAb 4G6 and other anti-mycotoxin McAb used in this study were previously prepared in our laboratory. Mycotoxin CIT, OTA, DON, ZEN, AFB1, and goat antimouse McAb-HRP were obtained from Sigma (St. Louis, MO, USA). Mouse anti-M13 McAb-HRP and His-Trap Ni2+-NTA Sepharose columns (1 mL) were purchased from GE Healthcare (Piscataway, NJ, USA). 3, 3′-5, 5′Tetramethylbenzidine (TMB) substrate was purchased from Bio Basic Inc. (Toronto, ON, Canada). All of the other reagents were of analytical grade or chromatographic grade and purchased from Sinopharm Chemical Corp. (Shanghai, China). Construction of a naive alpaca VHH phage display library A naive alpaca VHH phage display library was constructed according to our previous report [27]. In brief, total RNA was extracted from peripheral blood lymphocytes of two nonimmunized male alpacas and used to synthesize first-strand cDNA. The amplified VHH genes were ligated into phagemid pHEN1. Electroporation was then performed to transform the ligated DNA products into electrocompetent Escherichia coli TG1. Transforms were then scraped off the plates by 2× YT broth containing 20 % glycerol; approximately 0.5 mL of culture was used for phage preparation. The remaining culture was stored as 0.5 mL of aliquots at −80 °C. Library rescue was produced by infection with M13K07 helper phage. Phage titers were estimated by counting colony-forming units (cfu).

Anti-idiotypic nanobody as citrinin mimotope

Fig. 1 Outline of strategies to select the β-AId from a naive alpaca VHH library and to develop a friendly immunoassay for CIT based on VHH protein

Panning CIT β-AIds from the naive VHH library Microplate wells were coated with 100 μL of anti-CIT McAb 4G6 (100 μg/mL) solution at 4 °C overnight to select VHH that specifically recognizes the paratope of CIT-McAb. Nonspecific binding sites of the wells were blocked by incubation with 3 % BSA or CIT-ovalbumin (OVA) solution at 37 °C for 1 h. In the first round of panning, 100 μL of naive VHH library containing 1×1011 cfu phages were added to the well containing anti-CIT McAb 4G6 and incubated at 37 °C for 1 h. The phages were washed 10 times with PBST (containing 0.1 % Tween-20); the binding phages were eluted using 100 μL 0.2 M glycine-HCl (pH 2.2) and neutralized with 1 M Tris–HCl (pH 9.0). The elution solution was then amplified for subsequent rounds of panning. In the three subsequent rounds of panning, the concentrations of coating antibody were reduced to 70, 50, and 10 μg/mL, respectively. Meanwhile, the elution buffer was changed to 100 μL of CIT standard for competitive elution (5.0, 1.0, and 0.5 μg/mL in 10 % methanol, respectively). After four rounds of selection, 30 individual clones were randomly selected from 2× YT-GA (2 % glucose and 100 μg/mL ampicillin) plates and rescued on a small scale; their specific McAb binding activities were determined by phage ELISA. In brief, microplate wells were coated with McAb 4G6 (10 μg/mL) and blocked for 1 h at 37 °C with 4 % skim milk in PBS. After the wells were washed with PBST (containing 0.05 % Tween-20), a mixture of 50 μL of phage supernatant of each clone and 50 μL of 100 ng/mL CIT in 10 % methanol-PBS or pure dilution buffer was added and incubated at 37 °C for 1 h. After another round of washing was performed, 100 μL of 1:5000 dilution of antiM13 McAb-HRP was incubated in the wells at 37 °C for 30 min to detect the captured phage-VHHs; TMB substrate

(100 μL) was then added to the washed wells. The absorbance at 450 nm was determined using a microplate reader (Thermo Scientific, Waltham, MA, USA) after the reaction was stopped by adding 50 μL of 2 M H2SO4 per well. The phage VHH clones, which showed inhibitory binding to McAb by CIT standard, were identified as positive clones and sequenced. Expression of anti-idiotypic VHH The selected clone was subcloned into pET-25b (+) (Novagen, Billerica, MA, USA) and transformed into Rosetta (DE3) cells to express soluble VHHs. Protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside at OD600 = 0.6–0.8, and cultures were incubated overnight at 30 °C. The cells were ruptured by sonication and centrifuged at 8000×g for 20 min. Subsequently, 6× His-tagged VHHs were purified using a Ni2+-NTA metal-affinity column. Protein concentration was measured using a Nanodrop 1000 spectrophotometer (Thermo Scientific). Purity was verified by 15 % SDS-PAGE. Characterization of anti-idiotypic VHH The cross-activity with other anti-mycotoxin McAbs was examined by V-ELISA to determine the specific binding property of anti-idiotypic VHH. McAbs included anti-DONMcAb, anti-OTA-McAb, anti-FB1-McAb, and anti-AFB1McAb. The same serial dilutions of these McAbs were performed in PBS buffer. The simulation authenticity of positive anti-idiotypic VHH was identified using a conventional indirect competitive ELISA (ic-ELISA). In brief, the plates were coated with 0.17 μg of CIT-OVA in PBS by incubation overnight at 4 °C. The plates were then blocked with 4 % skim milk in

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PBS at 37 °C for 1 h. A mixture of 50 μL of McAb 4G6 and 50 μL of serial dilution of CIT or purified VHH fragments were added to the plate and incubated for 1 h at 37 °C. After the plates were washed, 100 μL of goat anti-mouse McAbHRP (1:2000) was added to detect the captured antibody. The absorbance at 450 nm was determined as described in a previous section. The binding kinetics of VHH or CIT-OVA to McAb was estimated by bio-layer interferometry using BLitz (ForteBio, Menlo Park, CA, USA) instrument. According to the manufacturer’s instructions, Ni-NTA biosensors (Catalog no. 18– 5101; ForteBio) were used to immobilize VHH with His-tag; amine reactive second-generation biosensors (Catalog no. 18– 5092; ForteBio) were used to immobilize CIT-OVA with amine-reactive chemical groups. Four readings at different McAb concentrations were used for determination. Data were globally fit using the built-in BLItz software to a 1:1 binding model to determine association constant (kon), dissociation constant (koff), and affinity constant (KD).

was centrifuged at 8000×g for 10 min. The supernatant was further diluted with PBS containing 10 % methanol, as appropriate for V-ELISA and/or ic-ELISA. Validation of V-ELISA The calibration curve of V-ELISA was generated by running standard CIT in PBS containing 10 % methanol to final concentrations of 0 (as negative control), 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 100.0, 200.0, 300.0, 400.0, and 500.0 ng/mL. The specificity of V-ELISA was evaluated by analyzing four common mycotoxins, including OTA, AFB1, DON, and ZEN. Accuracy and precision of the proposed assay were evaluated by analysis of recoveries and variation coefficients (CVs) of CIT-spiked wheat powder and rice samples. Each of the spiked samples was determined by V-ELISA in five replicates. Practicality and reliability of V-ELISA were determined by analyzing the eight real Monascus-fermented rice samples. The results obtained from V-ELISA were further compared with those of ic-ELISA.

V-ELISA for CIT Statistical analyses V-ELISA was developed to detect CIT in samples or standards to confirm the effectiveness of VHH as a coating antigen for CIT immunoassay. In brief, the wells were initially coated with purified VHH and blocked for 1.5 h at 37 °C with 5 % skim milk in PBS. After the wells were washed with PBST, 50 μL of McAb and 50 μL samples or CIT standard solution were added to the respective wells for 50 min at 37 °C. Subsequently, 100 μL of 1:2000 dilution of goat anti-mouse McAb-HRP was added to detect the captured antibody. The optimal concentrations of coating VHH and McAb (4G6) were determined by checkerboard titration. Preparation of cereal samples Wheat powder and rice samples, which were verified to be free of CIT by high-performance liquid chromatography method, were purchased from a local supermarket. Eight CIT-contaminated rice samples were prepared in laboratory scale. In brief, rice was soaked in pure water at room temperature overnight. Then, 15 g of pre-immersed rice was placed in a 250 mL Erlenmeyer flask and mixed well with 10 mL of pure water. The resulting mixture was sterilized at 121 °C for 30 min, and 1 mL of spore solution (approximately 1×106 spores/mL) of Monascus aurantiacus, which was identified to produce CIT on a rice culture medium, was inoculated. Eight rice samples were fermented at 28 °C under stationary conditions for 14 d. CIT-free cereal and Monascus-fermented rice sample were pretreated as follows: 1.0 g of samples was pulverized and mixed with 5 mL of PBS containing 10 % methanol. After ultrasonic extraction was performed for 20 min, the mixture

Paired t-test analysis was used to detect the consistency of two different detecting methods, and p