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Preparation of a chicken scFv to analyze gentamicin residue in animal derived food products Cui Li, Jinxin He, Hao Ren, Xiao-ying Zhang, Enqi Du, and Xinping Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00426 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 18, 2016
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Analytical Chemistry
Figure 1. Agarose gel electrophoresis of the amplified antibody variable fragments. (A) Chicken spleen total RNA extraction. (B) PCR amplification of VH and VL. Lane 1 and 2: PCR products of VL, about 370bp. Lane 3 and 4: PCR products of VH, about 420bp. (C) Amplification of scFv by overlap PCR, about 750bp. 40x29mm (300 x 300 DPI)
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Figure 2. Phage-ELISA. (A) The enrichment of specific scFv in each library after four rounds panning. (B) Binding activity of scFv antibodies to Gent. S-1—S-33: scFv antibodies from randomly selected clones from the 4th panning; C: blank control; N: negative control, BSA; P: positive control, IgY. 228x385mm (300 x 300 DPI)
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Analytical Chemistry
Figure 3. Analysis of S-1 and S-5 by SDS-PAGE. (A) Lane 1 and 2: the S-1 induced and non-induced bacterium; lane 3 and 4: the S-5 induced and non-induced bacterium; lane 5 and 6: the pET-30a induced and non-induced bacterium. (B) Lane 1 and 2: the pellet and supernatant after bacterium cell disruption of S-1, lane 3 and lane 4: the pellet and supernatant after bacterium cell disruption of S-5; (C) Lane1 and 2: the denatured protein (scFv) of S-1 and S-5; lane M: unstained protein marker. 400x299mm (300 x 300 DPI)
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Figure 4. Standard curves of the competitive ELISA for Gent. X-axis logarithm concentration of Gent, Y-axis represents the inhibition rate (B/B0). B0 and B are the absorbance values obtained from binding at zero and certain concentrations of Gent standard. 282x211mm (300 x 300 DPI)
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for TOC only 84x47mm (300 x 300 DPI)
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84x47mm (300 x 300 DPI)
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Analytical Chemistry
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Preparation of a chicken scFv to analyze gentamicin residue
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in animal derived food products
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Cui Li, Jinxin He, Hao Ren, Xiaoying Zhang*, Enqi Du, Xinping Li
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College of Veterinary Medicine, Northwest A&F University, Yangling, 712100,
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Shaanxi, China;
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*Corresponding authors
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Dr. X.Y. Zhang, College of Veterinary Medicine, Northwest Agriculture and Forestry
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University, Yangling, 712100, China Tel./Fax: +86 29 8709 1239. E-mail address:
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[email protected].
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Abstract: Chicken is an ideal model for simplified recombinant antibody library
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generation. It has been rarely been reported to apply chicken single-chain variable
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fragments (scFvs) in immunoassays for the detection of antibiotic and chemical
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contaminants in animal food products. In this study, the scFvs (S-1 and S-5) were
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isolated from a phage display library derived from a hyperimmunized chicken. The
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checker board titration revealed that the optimum concentrations of S-1 and S-5 were
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0.78µg/mL and 0.44µg/mL respectively, to obtain OD450 around 1.0 at 5µg/mL of
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Gent-OVA coating concentration. Both S-1 and S-5 exhibited negligible cross
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reactivity with kanamycin and amikacin. The 50% inhibitory concentration (IC50) of
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S-1 and S-5 were 12.418ng/mL and 14.674ng/mL respectively. In the indirect
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competitive ELISA (ic-ELISA), the limits of detection for S-1 and S-5 were
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0.147ng/mL and 0.219ng/mL respectively. The mean recovery for Gent ranged from
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60.91% to 118.09% with 10.35% relative standard deviation (RSD) between the
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intra-assay and the inter-assay. These results indicate the chicken scFv based
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ic-ELISA method is suitable for the detection of Gent residue in animal derived edible
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tissues and milk.
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Keywords: single-chain variable fragments (scFvs), gentamicin, ic-ELISA, chicken
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IgY
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Introduction
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Gentamicin (Gent) is an aminoglycoside antibiotic commonly used to treat
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Gram-negative bacterial infection1. It has been extensively used for treatment and
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growth promotion in livestock management. The overuse of Gent in animal husbandry
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could result into accumulation of its residues in animal derived foods2,3. The presence
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of Gent residues in food has been considered as high risk for consumption due to its
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adverse effects such as loss of hearing or of vestibular function (ototoxicity) and
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impairment of renal function (nephrotoxicity)4,5. Besides, the overuse of gentamicin
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promotes the emergence of antibiotic-resistant2. In order to monitor and control the
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antibiotic abuse, the regulatory authorities in EU and China have established the
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maximum residue limits (MRL) of gentamycin in food stuffs (Table 1) 6,7.
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Table 1 MRL Values Established by the EU and China for Gentamicin of Veterinary Use (µg/kg)
Species of animal
Tissue
European Union (EU )
China
muscle
50
100
liver
200
2000
kidney
750
5000
fat
50
100
Bovine
milk
100
200
Chicken
edible tissue
N
100
Pig/bovine
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N represents not established.
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Numerous methods have been developed for the detection of gentamycin such as
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chromatography8, LC-ES-tandem MS9, microbiological growth-inhibition assay10,
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ELISA11, radioimmunoassay12, immunochromatographic assay13. The first three
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methods are laborious, time-consuming, or in the need of sophisticated equipment.
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Rests of the three methods (immunoassays) are widespread rapid screening techniques
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because of specificity, sensitivity, low cost, and suitability for screening. ELISA is the
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most commonly available method which is rapid, sensitive and simple for analysis of
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various antibiotic residue or chemical contaminants. However, production of
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polyclonal antibody has the limitations in terms of its standardization and
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reproducibility, which make the recombinant antibody fragments (scFv, Fab) become
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a credible alternatives. In consideration of the complex production process of
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recombinant antibodies from mammal, chicken is an ideal model for simplified
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recombinant antibody library generation owning to its unique mechanism in the
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generation of antibody diversity. Meanwhile, avian has only one isotype of light chain
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(lambda), as compared to two isotypes of light chain (kappa and lambda) in mammal.
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Hence, the present study was aimed to develop a simple and quick method to prepare
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recombinant antibody against Gent and develop a rapid detection method with high
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sensitivity for analyzing gentamicin residue in animal-derived food.
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Materials and methods
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Chicken immunization
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Two seven-weeks old White Leghorns chicken were first immunized with 250µg
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of Gent-BSA with Freund’s complete adjuvant (FCA; Sigma-Aldrich Co., St. MO,
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USA), then four booster injections with Freund’s incomplete adjuvant (FIA;
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Sigma-Aldrich Co., St. MO, USA) were given at two weeks interval. Glutaraldehyde
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method was employed to synthesize Gent-BSA as per the previously described
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method11. Briefly, the mixture of 10.94mg of Gent and 15mg of BSA in 3mL of
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phosphate buffer saline (PBS, 0.01M, pH 7.4) was stirred with 50µL of 25%
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glutaraldehyde at 25°C for 16 h and centrifuged for 10min at 5000g. Then the
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supernatant was dialyzed in PBS (0.01 M, pH 7.4) at 4°C for 72h and the dialysate
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was renewed every 8h. At last, the Gent-BSA was obtained. A total of 1mL
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immunogen contains 250µg of Gent-BSA in 500µL phosphate buffer saline (PBS)
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with an equal volume of FCA or FIA was administered to each chicken
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intramuscularly in four different sites of pectoral muscles.
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RNA extraction and cDNA synthesis
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On 7th day after the final immunization, the chickens were euthanized,
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exsanguinated and the spleens were harvested. Total RNA was extracted from spleens
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using RNAsimple Total RNA Kit (TIANGEN Biotech (Beijing) Co., Ltd). First-strand
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cDNA was synthesized using HiScript Q Select RT SuperMix for qPCR (+gDNA
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wiper) (Vazyme Biotech Co., Ltd).
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RT-PCR and overlap PCR
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First-strand cDNA (derived from the RNA extract obtained from the
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Gent-immunized chicken spleen) was used to amplify heavy and light chain variable
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fragment (VH and VL) genes using primers HF-Sfi I&HR-Linker for the VH region
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or LF-Linker &LR-Not I for the VL region (Table 2) as described previously14. In
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order to obtain the scFv fragments, the VH and VL which both contained the
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sequence of a peptide linker were assembled by a second round of PCR (overlap PCR)
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with the primers HF-Sif I&LR-Not I.
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Table 2 Oligonucleotide primer sequences Primer
Oligonucleotide sequence
HF-SifI
ATGTCTATGGCCCAGCCGGCCGTGACGTTGGACG
HR-Linker
CAGAGCCACCTCCGCCTGAACCGCCTCCACCGGAGGAGACGATGAC
LF-Linker
TTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGCGCTGACTCAGCCGTCCT
LR-NotI
AGTTACTGGAGCGGCCGCACCTAGGACGGTCAGGG
Construction of scFv original library
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The scFv fragments were digested with restriction enzymes Sfi I&Not I and
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ligated with Sfi I/Not I digested pCANTAB5E vector using the T4 DNA Ligase
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(Thermo Fisher Scientific Inc.). The ligated products were transformed into E. coli
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TG1 cells. Serial dilutions of 10-1-10-6 were plated onto SOB plates (2% Tryptone,
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0.5% Yeast Extract, 0.05% NaCl, 2.5mM KCl, 10mM MgCl2 and 1.5% Agar powder)
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containing 100µg/mL ampicillin and 2% glucose (SOBAG) to determine the library
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size. After inoculation, the SOBAG plates were incubated overnight at 30°C. The
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next day 10 clones were randomly chosen and examined for scFv inserts by
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colony-pick PCR. Then the colonies were scraped into 20mL 2YT (1.6% Tryptone, 1%
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Yeast Extract and 0.5% NaCl), named the original antibody library and stored at -80℃
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containing 20% glycerol.
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Analysis and Sequencing the diversity of original antibody library
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To analyze the diversity of original antibody library, ten clones which were
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randomly chosen were BstO I (Promega Corporation) digested. Then the results of
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BstO I restriction analysis were observed through agarose gel electrophoresis. The recombinant plasmids with scFv gene were sent to Invitrogen Corporation to sequence. Panning of phage displayed antibody libraries
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The original antibody library was panned using a solid-phase protocol to select
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scFvs with higher affinities. Briefly, for first three rounds of selection, 10µg of
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Gent-OVA (the method of the synthesis of Gent-OVA was same as Gent-BSA) in the
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Carbonate Buffer solution (CBS 0.05M, pH 9.6) was coated in each well of Corning
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Maxisorb ELISA plate (1 x 8 Stripwell™) (Corning Inc., USA). In the last round of
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selection, 5µg of Gent-OVA in the CBS was coated in each well. The plates were
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incubated overnight at 4°C and next day unbound sites were blocked by 150µL (per
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well) of 5% skimmed milk in PBS (PBSM) for 2h at 37°C. After washing, the PBSM
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was replaced by 170μL of freshly prepared phage from the starting library of each
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round of selection and the plates were incubated at 37°C for 2h to allow phage
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binding. The unbound phages were discarded and the wells were firstly washed with
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0.5% Tween–PBS (PBST) and PBS. The wells were washed five times in the first
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round of selection, then 10 times for second and third round; and 15 times in the last
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round. The bound phage were eluted by applying 150μL/well of HCl-glycine (0.2M,
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pH 2.2) with gently shaking for 15min, the lower pH of the eluted phage was
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neutralized with 150μL/well of Tris-HCl (1 M, pH 7.4). After each round of panning,
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all the eluted phages were added to 8mL 2YT and cultured in shaker incubator at
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30°C with 250rpm for 1h, and then the bacteria solution was taken out from shaker
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incubator and stored at -80℃ with 20% glycerol, named second, third and fourth
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antibody library respectively. 100µL of serially diluted (10-5-10-10) Phage-infected
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log-phase bacterial cells from each panning were plated out onto each SOBAG plate
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and cultured at 30°C for 16h. Then, the bacterial colonies were counted to determine
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each library size after screening..
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Phage ELISA
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Phage-scFv suspensions from each round of selection against Gent-OVA were
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tested using phage ELISA. In detail, taking out 50µL bacteria solution from each
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antibody library was inoculated into 5mL of 2YT and cultured at 30°C for 3h with
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shaking at 250rpm. Aliquots (50µL) were transferred into 5mL of 2YT containing
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100μg/ml ampicillin, 2% glucose (2YT-AG) and 4×1012PFU of M13KO7 phages
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were added. Culture medium was incubated at 37°C for 2.5h at 150rpm and then
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centrifuged at 1,000g for 15min. The precipitate was resuspended in 400μL of
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2YT-AK and cultured at 37°C overnight with shaking at 250rpm. The bacterial culture
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solution was centrifuged and the phage-containing supernatant was collected for
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phage ELISA analysis.
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Corning Maxisorb ELISA plate was coated with 10μg of Gent-OVA per well in
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100μl of CBS overnight at 4°C and then blocked with 150μL of PBSM for 2 h at 37°C.
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Phage-scFvs were added to each well for incubating 2h at 37°C. The bound
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phage-scFv was detected with horseradish peroxidase (HRP)-conjugated anti-M13
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antibody (Amersham Pharmacia Biotech). The color was developed using TMB
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(Promega Biotech Co., Ltd) for 10 min and the absorbance was read at 450 nm.
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Expression and purification of the anti-Gent scFv protein
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The target gene and pET-30a vector were digested by the Bgl II and Hind III
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restriction enzyme (TaKaRa Biotechnology (Dalian) Co., Ltd.). After this, the scFv
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fragments were inserted into pET-30a vector using the T4 DNA Ligase. Then the
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positive recombinant plasmid was used to transform into E. coli strain BL21 (DE3)
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and cultured in the Luria–Bertani (LB) medium (1% Tryptone, 0.5% Yeast Extract
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and 1% NaCl) containing 100µg/mL kanamycin at 37°C. When the OD600 reached 0.7,
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expression of the recombinant fusion protein was induced by 0.5mmol/L isopropyl
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β-D-1-thiogalactopyranoside (IPTG) for 10h at 20°C.
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Then the bacteria cells were collected by centrifugation at 12000g (America,
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Beckman Avanti I-26 XPI) for 2min and re-suspended in 50mL PBS. Subsequently,
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the pellets were sonicated (37%W, working 2s and pause 1s, total 20min). After the
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liquid was centrifuged at 10000g for 1min and then the pellet and supernatant was
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collected respectively. At last the pellet and supernatant was used to analyze the
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solubility of the protein by SDS-PAGE.
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Denaturation and renaturation of the anti-Gent scFv protein
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The inclusion bodies were washed three times with PBS containing 0.1% Triton
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X-100 and 2M urea at 3h interval. Subsequently, the inclusion bodies were solubilized
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in 10mL 8M urea solutions by slowly stirring at 4°C overnight. To remove the
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insoluble ingredient, the solubilized solution was centrifuged at 12000rpm for15min.
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Finally, the solution containing the denatured scFv was dialyzed in PBS (pH 7.4) at
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4°C over a period of 48h to make the urea been removed as much as possible from the
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protein solutions.
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Indirect competitive ELISA (ic-ELISA)
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The checkerboard titration was used to determine the optimum concentration of
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scFv and coating antigen for the ic-ELISA. The ic-ELISA was performed as described
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previously15. Briefly, microtiter plate was coated with the optimum concentration of
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Gent-OVA by overnight incubation at 4°C. The plate was washed two times with
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PBST and two times with PBS; then, unbound sites were blocked by incubation with
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5% PBSM at 37°C for 2 h. After washing, 100µL/well of scFv previously diluted with
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PBS and a series of Gent standard concentrations (200, 100, 80, 50, 20, 10, 5, 2, 1 and
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0.1ng/mL) was mixed, respectively. After incubation for 2h, the plates were washed
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two times with PBST and two times with PBS, and 100µL/well of mouse anti-His
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HRP-conjugated IgG (1/3000 dilution in PBS) was added. After incubation at 37°C
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for 1h, the plates were washed three times with PBST and two times with PBS. Then
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the TMB was added and incubated for 10min in the dark at room temperature and the
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absorbance was read at 450nm.
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A four-parameter logistic equation was used to fit the immunoassay data.
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Calculations were performed using OrginPro 7.5 software (Origin Lab Corp.,
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Northampton, MA, USA). The IC50 value, assay dynamic range, and limit of detection
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(LOD) served as criteria to evaluate the ic-ELISA.
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Cross-reactivity analysis
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The specificity of the scFvs under optimized conditions was evaluated by
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measuring cross-reactivity (CR) with a group of structurally related compounds,
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including kanamycin, amikacin. In order to calculate the CR values, the IC50 values of
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these compounds were compared with that of Gent. The equation is as follows:
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CR(%)=[IC50(Gent)/IC50(analyte)]×100%.
223 224
Sample Preparation
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Milk, pork, beef and chicken samples were obtained from local market. For
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recovery study, Gent (1000µg/mL, prepared in PBS) was added into each sample to
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produce spiked concentration of 0, 50, 100 and 200μg/kg. It was performed as follows:
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an aliquot of tissue sample (1g wet mass) was homogenized and transferred to a 50mL
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polypropylene centrifuge tube. 5mL 5% trichloroacetic acid and 10mL of 0.2M PBS
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was mixed with tissue sample. The mixture was then incubated for 30min at 60°C.
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Subsequently, the suspension was centrifuged at 4000g for 15min at room temperature.
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The supernatant was separated and 100μL of which was diluted with 900μL water.
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Fifty microliter aliquots of the dilution were then pipetted into the microtiter plate for
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analysis. After the Gent of different concentrations (0, 50, 100 and 200μg/L) was
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added into milk sample, the milk samples were defatted by centrifugation at 4500g for
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20min at 4°C. Then, after adding 60µL sodium nitroprusside (0.36mol/L) and 60µL
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zinc sulfate (1.04mol/L) into 2mL of the defatted milk samples, the samples were
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vortexed for 1min and then centrifuged at 4000g for 15 min at 4°C. The supernatant
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was removed and diluted 10 times with PBS for analysis. The recoveries were
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calculated on the basis of the standard curve constructed by ic-ELISA.
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Results and Discussion
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Construction of chicken scFv
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Antibodies are ubiquitous tools in biotechnology. To date, different types of
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antibodies, such as polyclonal antibody (pAb)15,16, monoclonal antibody (mAb),
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recombinant antibodies (scFv and nanobody)17-19, specific to various antibiotics have
246
been produced. Alarmingly, there is an increasing concern among scientific
247
community that many antibodies could be unreliable reagents and the results of many
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biomedical experiments cannot be reproduced due to the fact that traditional antibodies
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have to rely on animals or cell lines, which can lead to the quality differentiation of
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different batches of antibodies20,21. Instead, recombinant antibodies, especially the
251
scFv , with exact gene sequences are widely used in different biomedical purposes
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with significant advantages on reproducibility over pAb or mAbs22.
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Mice derived scFv was widely used initially. Because of the complex production
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process of mice derived scFv, chicken derived scFv is served as an ideal recombinant
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antibody to detect Gent in animal derived food products in this work. Chicken scFvs
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are easier to make and chicken antibodies are ideal to use in the mammalian milieu as
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they cross react less14. Chicken scFv combines both the high specificity and
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homogeneity of the monoclonal antibodies as well as unique features of chicken
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antibodies14,23. As a technical merit, in most cases, only one pair of primer is needed
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to produce chicken scFv because both the heavy and light chain loci in chickens
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consist of single functional variable region genes (V) and joining (J) region genes as
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well as multiple diversity (D) segments that are very similar in the heavy chain locus
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in mammal24. Using one pair primer not only saves time and resources, but also it can
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reduce rare transcript loss due to the difference in the primer efficiencies25.
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In this work, total RNA was isolated from spleen of immunized chickens (Figure
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1A). After the synthesis of cDNA, The VH and VL fragments of the expected size
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(about 420bp and 370bp) were amplified by PCR (Figure 1B). The scFv was
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assembled with the VH, VL by overlap PCR using the primers as described above
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(Figure 1C).
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Figure 1 is here
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In the process of producing specific scFv, the diversity of the phage-antibody
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library is of great importance. However, there is no need to concern the diversity in
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the production of chicken antibodies, chicken possess a diversity generating process
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that is distinctly unique compared to mammals26. Chicken use a gene conversion
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strategy to create a diverse antibody repertoire, it makes the genes undergo a single
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V(D)J recombination event, which were followed by gene conversion utilizing
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multiple upstream V-region pseudogenes14,26. In this work, the results of PCR
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indicated that positive rate of insertion of phage clones could reach at 90% (Figure
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S-1, see Supporting Information section). According to different length of the
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fragments after the BstO I digesting and the sequence alignment with chicken
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germline gene (Supporting Information section), the diversity of antibody library
282
reaches about 80%, which could meet the subsequent antibody screening work.
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Panning of phage displayed antibody libraries
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In order to obtain highly specific antibodies from the scFv original library (the
285
size of original library was 2.27×107), we applied a strategy where the washing steps
286
were progressively increased while the concentrations of coated Gent-OVA were
287
decreased from 10µg/well to 5µg/well (Table 3)27. After four rounds of panning,
288
phage-ELISA had been done to evaluate the enrichment of specific scFv in each
289
library. The result indicated that the degree of enrichment of the specific scFv
290
increased gradually except the fourth round panning (Figure 2A), it could be triggered
291
by the sudden decrease of the amount of coating antigen. The size of anti-Gent scFv
292
antibody library of each panning was displayed using output (Table 3). Result
293
indicates that, our panning strategy was effective in isolation of positive scFv clones.
294
Thirty-three phage clones randomly chosen from the fourth round of panning were
295
analyzed by phage ELISA (Figure 2B). A total of 12 clones were sequenced in our
296
study, and they have been supplemented in the Supporting Information section.
297
Table 3 The library size and phage titer of each panning Coated
Coating
Rounds
298 299
Output Input
Output/Input
antigen
concentration/well
(titer)
1
Gent-OVA
10µg
4×1011
3.7×105
9.25×10-7
2
Gent-OVA
10µg
4×1011
6.7×106
1.675×10-5
3
Gent-OVA
10µg
4×1011
3.71×109
9.275×10-3
4
Gent-OVA
5µg
4×1011
1.65×109
4.125×10-3
Figure 2 is here Expression, purification, and identification of the anti-Gent scFv
300
Among the 33 phage clones, two clones (S-1 and S-5) were used to subsequent
301
expression. A 35kDa band was detected by SDS-PAGE whereas this band appeared in
302
both the induced and non-induced cell culture (Figure 3A). The analysis of solubility
303
indicated that expressed scFv protein existed as inclusion bodies (Figure 3B). Then
304
the inclusion bodies were washed using 2 M urea at 4°C. Subsequently, the inclusion
305
bodies were solubilized by slowly diluting the purified protein in 8M urea at 4°C
306
overnight. Finally, the denatured/purified protein was renatured by dialysis in PBS
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Page 18 of 27
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(pH 7.4) at 4°C over a period of 48 h and the urea was as much as possible to remove
308
from the protein. The purity of protein solution was confirmed by SDS-PAGE (Figure
309
3C), and then the purified protein was stored at −20°C. Figure 3 is here
310 311
Indirect competitive ELISA (ic-ELISA) for constructing the standard curve
312
The purified S-1 and S-5 were evaluated for their performance by ic-ELISA. The
313
optimum concentrations of S-1 and S-5 were 0.78µg/mL and 0.44µg/mL respectively
314
to obtain OD450 around 1.0 at 5µg/mL of Gent-OVA coating concentration by the
315
checkerboard titration. Under these optimal conditions, the regression curve equations
316
of
317
y=0.76066-0.23479x (R2=0.97475) respectively as shown in Figure 4. The IC50 values
318
of the assay established with S-1 and S-5 were 12.418ng/mL and 14.674ng/mL
319
respectively. The IC50 demonstrated that, the scFv could be used to detect the Gent.
320
The linear range of the assay established with S-1 and S-5 were 0.147 to
321
204.165ng/mL and 0.219 to 208.459ng/mL respectively.
the
the
S-1
S-5
were
y=0.74407-0.23033x
(R2=0.9849)
and
Figure 4 is here
322 323
and
Cross-Reactivity
324
Owning to the advantage of antibody fragments, scFv could better recognize
325
some antigen-antibody binding sites which are partially obscured from antibody
326
recognition by the associated the three-dimensional carbohydrate structures of antigen,
327
as compared to macromolecular antibodies28. Thus, the cross-reactivity of the scFvs
328
with kanamycin and amikacin was tested by ic-ELISA. Result indicated that, the S-1
329
and S-5 showed very low cross-reactivity with other aminoglycosides (kanamycin and
330
amikacin), indicating that the scFv is highly specific for Gent (Table 4).
331
Table 4 Cross-reactivity of anti-Gent scFvs with various aminoglycoside antibiotics
Analytes
ScFvs
Cross-reactivity (%)
S-1
100
S-5
100
S-1
