This article was downloaded by: [UQ Library] On: 14 October 2014, At: 16:18 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
A novel multiplexed fluorescence polarization immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk a
a
b
a
a
a
a
Min Chen , Kai Wen , Xiaoqi Tao , Shuangyang Ding , Jie Xie , Xuezhi Yu , Jiancheng Li , Xi a
a
a
a
Xia , Yang Wang , Sanlei Xie & Haiyang Jiang a
College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
b
College of Food Science, Southwest University, Chongqing, 400715, PR China Accepted author version posted online: 13 Oct 2014.
To cite this article: Min Chen, Kai Wen, Xiaoqi Tao, Shuangyang Ding, Jie Xie, Xuezhi Yu, Jiancheng Li, Xi Xia, Yang Wang, Sanlei Xie & Haiyang Jiang (2014): A novel multiplexed fluorescence polarization immunoassay based on a recombinant bispecific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.976279 To link to this article: http://dx.doi.org/10.1080/19440049.2014.976279
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part A DOI: 10.1080/19440049.2014.976279
t
A novel multiplexed fluorescence polarization immunoassay based on
cr ip
a recombinant bi-specific single-chain diabody for simultaneous
us
Min Chen1, Kai Wen1, Xiaoqi Tao2, Shuangyang Ding1, Jie Xie1, Xuezhi Yu1,
College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR
M
1
an
Jiancheng Li1, Xi Xia1, Yang Wang1, Sanlei Xie1 & Haiyang Jiang1∗
China, 2College of Food Science, Southwest University, Chongqing, 400715, PR
ce pt ed
China ∗
Corresponding author: [email protected]
Abstract
Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarization
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
detection of fluoroquinolones and sulfonamides in milk
immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labeled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR) with different
1
excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits (MRLs) in milk samples. Recoveries for spiked milk
t
samples were from 76.4 to 128.4%, with a relative standard deviation lower than
cr ip
13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC–MS/MS. All three instances in which FQs and SAs were present
us
positive by the developed DB-FPIA, demonstrating that the method is suitable for
an
rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for
ce pt ed
samples.
M
fast multi-analyte screening of low-molecular weight chemical residues in food
Keywords ： Fluoroquinolones; Sulfonamides; Bispecific Single-chain diabody; Fluorescence polarization; Milk
Introduction
Recently, excessive residues of veterinary drugs have caused severe food safety
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
at concentrations near or above the assay limit of detection (LOD) were identified as
problems and have been a decisive factor favoring the growth of bacterial resistance (Franco et al., 1990; Smith et al., 1999).
To minimize the risk of antibiotic residues
on human health and on security of the entire ecosystem, procedures for the establishment of maximum residue limits (MRLs) of veterinary drugs in foodstuffs of animal origin are regulated by European Council (EC) Regulation no. 470/2009. 2
Although many sensitive analytical methods have been developed for monitoring the growing number of antibiotics, most of these methods are single-analyte assays; that is, one assay for one analyte. Therefore, the development of rapid and reliable
t
methods capable of simultaneous detection of multiple analytes in a complex sample
cr ip
has attracted much attention in the scientific community (Zhang et al., 2011; Tian et
al., 2012). Compared with the traditional single-analyte assay, the simultaneous
us
shortening analytical time, enhancing detection throughput, and decreasing sampling
an
volume.
Until now, a number of analytical methods have been developed for
M
multi-analyte detection of low-molecular weight chemicals, particularly methods based on LC–MS/MS (Chafer-Pericas et al., 2010). However, instrumental methods
ce pt ed
cannot fulfill the demand of rapid screening as they are time-consuming and costly, and sample preparations are demanding. Therefore, they are not suitable for monitoring drug residues in a large number of food samples. In comparison with instrumental methods, the microbial inhibition tests are much more affordable and easier to operate during the screening step, but they are time-consuming (about 20 h),
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
multiplexed immunoassays are capable of detecting several analytes simultaneously,
and the sensitivity is much lower (Okerman et al., 2007). Immunoassays have been
confirmed as effective and economical screening methods relying on their high sample throughput, sensitivity, and selectivity, as well as reliability and simplicity. New immunoassay research efforts are focusing on the development of multi-analyte-residue analysis and the design of user-friendly analytical devices for 3
continuous or on-site measurements. Recently, several multiplexed immunoassays have been developed to simultaneous detect various low-molecular weight chemical residues, including enzyme-linked immunosorbent assay (ELISA) (Adrian et al., 2008;
t
Jiang et al., 2013), fluorescence-linked immunosorbent assay (FLISA) (Peng et al.,
cr ip
2009; Zhu et al., 2011) and biosensors (Adrian et al., 2009). However, these reported multi-analyte immunoassays require more than two antibodies, which can complicate
us
of traditional heterogeneous immunoassays need to separate the free form from the
an
complex of the antigen and the antibody, and require multiple washing steps, and incubation steps, and therefore they are time-consuming.
M
Fluorescence polarization (FP) is a typical homogeneous technique that allows automated high-throughput, rapid and quantitative analysis of binding of a small
ce pt ed
fluorescent ligand to a larger protein using plan-polarized light to detect the change in effective molecular volume (Rossi & Taylor, 2011). Fluorescence polarization immunoassay (FPIA) is based on the competition of free (unlabeled) analyte and fluorescent-labeled antigen for antibody binding sites, which allows the determination of analytes within a short period of time. The advantage in determination speed makes
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
the detection system. In addition to lateral flow immunochromatographic tests, most
it more suitable for screening of a large number of samples. Throughout the past decades, this technique has been widely used in monitoring therapeutic drug levels in body fluids, small molecule drug discovery and high-throughput screening (Lea & Simeonov, 2011; Tian et al., 2012). FPIA has also been utilized for the measurement
of various small molecule analytes including toxins (Maragos et al., 2001; Nasir & 4
Jolley, 2002; Chun et al., 2009; Zezza et al., 2009), pesticides (Eremin et al., 2002; Xu et al., 2011), and veterinary drugs (Wang et al., 2007; Wang et al., 2008; Mi et al., 2013).
t
This paper introduces a novel multiplexed FPIA for the simultaneous screening
cr ip
of FQs and SAs. In our previous study, a recombinant bi-specific single-chain diabody
(scDb), exhibiting high affinity to 20 FQs and 14 SAs, was successfully constructed
us
(Chen et al., 2014). The scDb is capable of binding FQs and SAs simultaneously,
such
as
FPIA.
Then,
we
an
which are more suitable for detecting FQs and SAs in a homogeneous immunoassay, synthesized
two
fluorescent
tracers,
M
sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin -Texas Red (SAR-TR), whose wavelengths of excitation and emission are
ce pt ed
non-overlapping. Two tracers could bind scDb simultaneously and compete with unlabeled analyte for antibody binding. Therefore, a dual-binding fluorescence polarization immunoassay (DB-FPIA) based on the scDb for the rapid and simultaneous screening of FQs and SAs was developed. The method developed in this work combines the advantage of the FPIA and the broad sensitivity of scDb, which
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
and expressed based on the two single chain variable fragment antibodies (scFvs)
could be a potential tool for the rapid and simultaneous determination of FQs and SAs in field milk samples. According to current knowledge, it is the first report that a bi-specific scDb was used to develop a FPIA for simultaneous detection of multiple low molecular weight chemical residues from two different chemical classes.
5
Materials and methods Apparatus and buffers Microtiter plate reader—Sunrise microtiter plate reader (TECAN, Groedig, A SpectraMax
M5
microplate
reader
from
Molecular
Devices
t
Austria).
cr ip
(Downingtown, PA, USA) was used to measure fluorescence polarization (FP) and fluorescence intensity (FI) signal. Black microplates (96-well) with a nonbinding
us
Pre-coated silica gel 60GF254 glass plates (plate size = 10×10 cm; layer thickness =
an
0.15−0.2 mm, particle size = 2 µm) for thin-layer chromatography (TLC) were purchased from QingDao HaiYang Corp. (Shandong, China). PBS (0.01 M, pH 7.4)
M
was prepared by dissolving 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, and 3.63 g Na2HPO4·12H2O in 1 L purified water with 0.1% sodium azide and was used as the
ce pt ed
working buffer for all FPIA experiments. Working standard solutions of analytes in the range from 0.1 to 1000 ng mL−1 were prepared by dilution of stock solution with
assay buffer.
Reagents and Chemicals
The analytical standards of sulfathiazole (STZ), sulfameter (SFMT) sulfamethoxazole
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
surface for FPIA were obtained from Corning Life Sciences (New York, NY, USA).
(SMX), sulfachloropyridazine (SCP), sulfamonomethoxine(SMM) sulfadiazine (SDZ), sulfasalazine (SAZ), sulfathalidine (PST), sulfamethizole (SMT), sulfapyridine (SPY), sulfacetamide (SAA), sulfadimethoxine (SDM), sulfamerazine (SMZ) sulfamethazine (SHZ), sulfamethoxypyridazine (SMP), sulfaquinoxaline
(SQX), sulfanitran (SNI)
and ciprofloxacin (CIP), danofloxacin (DAN), difloxacin (DIF), enoxacin (ENO), 6
enrofloxacin (ENR), fleroxacin (FLER), amifloxacin (AMI), flumequine (FLU), levofloxacin (LEV), lomefloxacin hydrochloride (LOM), marbofloxacin (MAR), norfloxacin (NOR), ofloxacin (OFL), orbifloxacin (ORB), pazufloxacin (PAZ),
t
pefloxacin-d5 (PEF), prulifloxacin (PRU), sarafloxacin (SAR) and sparfloxacin (SPA)
cr ip
were purchased from VETRANAL® Fluka (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions (1 mg mL−1) of FQ and SA antibiotics were prepared by dissolving 5
us
was prepared by diluting the stock solution in PBS. N, N′-Dicyclohexylcarbodiimide
an
(DCC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate isomer I (FITC), Texas Red -NHS (TR-NHS) were purchased from Sigma-Aldrich (St. Louis, MO,
M
USA). All other chemicals and solvents were of analytical grade or better and were obtained from Beijing Chemical Reagent Co. (Beijing, P.R.C.).
ce pt ed
Synthesis of Fluorescent Conjugates (Tracers) SMP-FITC: Sulfamethoxypyridazine (SMP) (5 mg) was dissolved in 0.5 mL of
methanol. Triethylamine (50 µL, 7.2 mol L-1) and FITC (4 mg) were added with
mixing. After overnight reaction at room temperature, small portions (50 µL) of
reaction mixture were separated by thin-layer chromatography (TLC) using
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
mg of drug in 5 mL of 0.03 M sodium hydroxide. Working standard of each antibiotic
chloroform/methanol (4:1, v/v) as the eluent. The main yellow band at Rf 0.1 was
scraped from the plate and extracted with 1 mL of methanol. SAR-TR: Sarafloxacin (SAR) (2 mg) was dissolved in 50 µL (14.08 mol L-1) of N, N-dimethylformamide (DMF). Triethylamine (20 µL) and TR-NHS (2 mg) were added with mixing. After overnight reaction at room temperature, small portions 7
(50µL) of reaction mixture were separated by TLC using methanol / ethyl acetate / ammonia water (4:1:1, v/v/v) as the eluent. The new bright purple color band at Rf 0.8 was scraped from the plate, extracted with 1 mL of methanol, and stored in the dark at
t
4 °C.
cr ip
Assay protocol
Figure.1 shows the schematic diagram of the DB-FPIA procedure. Specific steps
us
of diluted scDb. Subsequently, 70 µL per well of standard solution or sample extract
an
was added, and the mixtures were shaken for 10 s in the microplate reader. After a short incubation period (5 min) at room temperature, the FP value was measured at
M
λex = 485 nm, λem = 530 nm (emission cutoff= 515 nm, G factor = 1.0) and λex = 585 nm, λem =620 nm (emission cutoff= 610 nm, G factor = 1.0), respectively. The
ce pt ed
integration time was set to 3 s for the polarization measurements. Over ten polarization measurements were taken each time, and they were then averaged for further data processing. The relative standard deviation was 2% for all measurements. Curve Fitting and Cross-Reactivity Determination To normalize the FP value, the ratio mP/mP0 (where mP0 is the maximum FP
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
as follows: 70 µL per well of tracer solution mixture was mixed with 70 µL per well
value of the inhibition curve and mP is the current value) resulting in relative units was used. The data were fitted with the four parameter logistic equation using Origin 8.0 software package (Microcal, Northampton, MA, USA). For standard curves, NOR and SMX were used as standards. IC50 is the standard concentration at 50% of specific binding. The cross-reactivity (CR) values were calculated according to the 8
following equation: CR (%) = [IC50 (NOR/SMX)/ IC50 (analyte)] ×100. Milk sample preparation Sample pre-treatment procedures were performed by a previously published
t
method (Wang et al., 2007). Briefly, aliquots (4 mL) milk samples was fortified with
cr ip
the appropriate FQs and SAs standard solution and then mixed with an equal volume
of 1.5% trichloroacetic acid (TCA).The mixtures were agitated on a shaker for 2 min
us
supernatants were diluted with assay buffer to fit the working range before
Analysis of field milk samples
an
measurement in FPIA.
M
Thirty milk samples were collected from retail outlets in Beijing. Each sample was divided into two portions: one was analyzed by the DB-FPIA and another was
ce pt ed
analyzed with HPLC-MS/MS (GB/T 20751–2006 and GB/T 20759–2006, China).
Results and discussion
Characterization of tracers
Monoclonal antibody-based FPIAs for detection of FQs and SAs have been
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
and then de-proteinised through centrifugation for 10 min (8000g at 4°C). The
developed in our laboratory (Wang et al., 2007; Wang et al., 2008; Mi et al., 2013). The previous work showed that higher sensitivity and faster dissociation rate were observed when using the heterogeneous and low cross-reactivities tracer, because it could be more easily replaced by competitors (Mi et al., 2013). Here，two haptens
(SAR and SMP) were selected for preparing fluorescent conjugates to perform a FPIA 9
because both of them have presented better sensitivity in our previous work (Wang et al., 2007; Mi et al., 2013). Subsequently, we synthesized two fluorescent tracers: SMP-FITC and SAR-TR. Because the tracer of SAR-TR (λex = 585 nm, λem =620
t
nm) exhibited significantly longer wavelength of excitation and emission than that of
cr ip
SMP-FITC (λex = 485 nm, λem = 530 nm), they can be used for simultaneous
determination of FQs and SAs. The appropriate two tracers could be preliminarily
us
saturating amounts of scDb, and a higher FP difference will result in a higher
an
signal-to-noise ratio. As shown in Fig. 2A, both SMP-FITC and SAR-TR exhibited sufficient binding with scDb, suggesting that they were successfully synthesized and
Assay optimization
M
could be used as a competition agent in the DB-FPIA.
ce pt ed
The amount of antibodies in the incubation solution was significant in the
competitive DB-FPIA. The scDb gave significant binding with both two tracers (at a fixed concentration 10 nM) at higher concentration and slowly decreased in FP values as antibodies became more diluted by PBS (Fig. 2A). As reported before (Wang et al.,
2008), a low tracer concentration will result in high sensitivity, but low precision of
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
independently characterized by observing the increase in FP signal after mixing with
FP signal. In addition, under the technical specifications of the instrument used in the present study, precision of
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
A novel multiplexed fluorescence polarization immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk a
a
b
a
a
a
a
Min Chen , Kai Wen , Xiaoqi Tao , Shuangyang Ding , Jie Xie , Xuezhi Yu , Jiancheng Li , Xi a
a
a
a
Xia , Yang Wang , Sanlei Xie & Haiyang Jiang a
College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
b
College of Food Science, Southwest University, Chongqing, 400715, PR China Accepted author version posted online: 13 Oct 2014.
To cite this article: Min Chen, Kai Wen, Xiaoqi Tao, Shuangyang Ding, Jie Xie, Xuezhi Yu, Jiancheng Li, Xi Xia, Yang Wang, Sanlei Xie & Haiyang Jiang (2014): A novel multiplexed fluorescence polarization immunoassay based on a recombinant bispecific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.976279 To link to this article: http://dx.doi.org/10.1080/19440049.2014.976279
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part A DOI: 10.1080/19440049.2014.976279
t
A novel multiplexed fluorescence polarization immunoassay based on
cr ip
a recombinant bi-specific single-chain diabody for simultaneous
us
Min Chen1, Kai Wen1, Xiaoqi Tao2, Shuangyang Ding1, Jie Xie1, Xuezhi Yu1,
College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR
M
1
an
Jiancheng Li1, Xi Xia1, Yang Wang1, Sanlei Xie1 & Haiyang Jiang1∗
China, 2College of Food Science, Southwest University, Chongqing, 400715, PR
ce pt ed
China ∗
Corresponding author: [email protected]
Abstract
Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarization
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
detection of fluoroquinolones and sulfonamides in milk
immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labeled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR) with different
1
excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits (MRLs) in milk samples. Recoveries for spiked milk
t
samples were from 76.4 to 128.4%, with a relative standard deviation lower than
cr ip
13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC–MS/MS. All three instances in which FQs and SAs were present
us
positive by the developed DB-FPIA, demonstrating that the method is suitable for
an
rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for
ce pt ed
samples.
M
fast multi-analyte screening of low-molecular weight chemical residues in food
Keywords ： Fluoroquinolones; Sulfonamides; Bispecific Single-chain diabody; Fluorescence polarization; Milk
Introduction
Recently, excessive residues of veterinary drugs have caused severe food safety
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
at concentrations near or above the assay limit of detection (LOD) were identified as
problems and have been a decisive factor favoring the growth of bacterial resistance (Franco et al., 1990; Smith et al., 1999).
To minimize the risk of antibiotic residues
on human health and on security of the entire ecosystem, procedures for the establishment of maximum residue limits (MRLs) of veterinary drugs in foodstuffs of animal origin are regulated by European Council (EC) Regulation no. 470/2009. 2
Although many sensitive analytical methods have been developed for monitoring the growing number of antibiotics, most of these methods are single-analyte assays; that is, one assay for one analyte. Therefore, the development of rapid and reliable
t
methods capable of simultaneous detection of multiple analytes in a complex sample
cr ip
has attracted much attention in the scientific community (Zhang et al., 2011; Tian et
al., 2012). Compared with the traditional single-analyte assay, the simultaneous
us
shortening analytical time, enhancing detection throughput, and decreasing sampling
an
volume.
Until now, a number of analytical methods have been developed for
M
multi-analyte detection of low-molecular weight chemicals, particularly methods based on LC–MS/MS (Chafer-Pericas et al., 2010). However, instrumental methods
ce pt ed
cannot fulfill the demand of rapid screening as they are time-consuming and costly, and sample preparations are demanding. Therefore, they are not suitable for monitoring drug residues in a large number of food samples. In comparison with instrumental methods, the microbial inhibition tests are much more affordable and easier to operate during the screening step, but they are time-consuming (about 20 h),
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
multiplexed immunoassays are capable of detecting several analytes simultaneously,
and the sensitivity is much lower (Okerman et al., 2007). Immunoassays have been
confirmed as effective and economical screening methods relying on their high sample throughput, sensitivity, and selectivity, as well as reliability and simplicity. New immunoassay research efforts are focusing on the development of multi-analyte-residue analysis and the design of user-friendly analytical devices for 3
continuous or on-site measurements. Recently, several multiplexed immunoassays have been developed to simultaneous detect various low-molecular weight chemical residues, including enzyme-linked immunosorbent assay (ELISA) (Adrian et al., 2008;
t
Jiang et al., 2013), fluorescence-linked immunosorbent assay (FLISA) (Peng et al.,
cr ip
2009; Zhu et al., 2011) and biosensors (Adrian et al., 2009). However, these reported multi-analyte immunoassays require more than two antibodies, which can complicate
us
of traditional heterogeneous immunoassays need to separate the free form from the
an
complex of the antigen and the antibody, and require multiple washing steps, and incubation steps, and therefore they are time-consuming.
M
Fluorescence polarization (FP) is a typical homogeneous technique that allows automated high-throughput, rapid and quantitative analysis of binding of a small
ce pt ed
fluorescent ligand to a larger protein using plan-polarized light to detect the change in effective molecular volume (Rossi & Taylor, 2011). Fluorescence polarization immunoassay (FPIA) is based on the competition of free (unlabeled) analyte and fluorescent-labeled antigen for antibody binding sites, which allows the determination of analytes within a short period of time. The advantage in determination speed makes
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
the detection system. In addition to lateral flow immunochromatographic tests, most
it more suitable for screening of a large number of samples. Throughout the past decades, this technique has been widely used in monitoring therapeutic drug levels in body fluids, small molecule drug discovery and high-throughput screening (Lea & Simeonov, 2011; Tian et al., 2012). FPIA has also been utilized for the measurement
of various small molecule analytes including toxins (Maragos et al., 2001; Nasir & 4
Jolley, 2002; Chun et al., 2009; Zezza et al., 2009), pesticides (Eremin et al., 2002; Xu et al., 2011), and veterinary drugs (Wang et al., 2007; Wang et al., 2008; Mi et al., 2013).
t
This paper introduces a novel multiplexed FPIA for the simultaneous screening
cr ip
of FQs and SAs. In our previous study, a recombinant bi-specific single-chain diabody
(scDb), exhibiting high affinity to 20 FQs and 14 SAs, was successfully constructed
us
(Chen et al., 2014). The scDb is capable of binding FQs and SAs simultaneously,
such
as
FPIA.
Then,
we
an
which are more suitable for detecting FQs and SAs in a homogeneous immunoassay, synthesized
two
fluorescent
tracers,
M
sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin -Texas Red (SAR-TR), whose wavelengths of excitation and emission are
ce pt ed
non-overlapping. Two tracers could bind scDb simultaneously and compete with unlabeled analyte for antibody binding. Therefore, a dual-binding fluorescence polarization immunoassay (DB-FPIA) based on the scDb for the rapid and simultaneous screening of FQs and SAs was developed. The method developed in this work combines the advantage of the FPIA and the broad sensitivity of scDb, which
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
and expressed based on the two single chain variable fragment antibodies (scFvs)
could be a potential tool for the rapid and simultaneous determination of FQs and SAs in field milk samples. According to current knowledge, it is the first report that a bi-specific scDb was used to develop a FPIA for simultaneous detection of multiple low molecular weight chemical residues from two different chemical classes.
5
Materials and methods Apparatus and buffers Microtiter plate reader—Sunrise microtiter plate reader (TECAN, Groedig, A SpectraMax
M5
microplate
reader
from
Molecular
Devices
t
Austria).
cr ip
(Downingtown, PA, USA) was used to measure fluorescence polarization (FP) and fluorescence intensity (FI) signal. Black microplates (96-well) with a nonbinding
us
Pre-coated silica gel 60GF254 glass plates (plate size = 10×10 cm; layer thickness =
an
0.15−0.2 mm, particle size = 2 µm) for thin-layer chromatography (TLC) were purchased from QingDao HaiYang Corp. (Shandong, China). PBS (0.01 M, pH 7.4)
M
was prepared by dissolving 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, and 3.63 g Na2HPO4·12H2O in 1 L purified water with 0.1% sodium azide and was used as the
ce pt ed
working buffer for all FPIA experiments. Working standard solutions of analytes in the range from 0.1 to 1000 ng mL−1 were prepared by dilution of stock solution with
assay buffer.
Reagents and Chemicals
The analytical standards of sulfathiazole (STZ), sulfameter (SFMT) sulfamethoxazole
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
surface for FPIA were obtained from Corning Life Sciences (New York, NY, USA).
(SMX), sulfachloropyridazine (SCP), sulfamonomethoxine(SMM) sulfadiazine (SDZ), sulfasalazine (SAZ), sulfathalidine (PST), sulfamethizole (SMT), sulfapyridine (SPY), sulfacetamide (SAA), sulfadimethoxine (SDM), sulfamerazine (SMZ) sulfamethazine (SHZ), sulfamethoxypyridazine (SMP), sulfaquinoxaline
(SQX), sulfanitran (SNI)
and ciprofloxacin (CIP), danofloxacin (DAN), difloxacin (DIF), enoxacin (ENO), 6
enrofloxacin (ENR), fleroxacin (FLER), amifloxacin (AMI), flumequine (FLU), levofloxacin (LEV), lomefloxacin hydrochloride (LOM), marbofloxacin (MAR), norfloxacin (NOR), ofloxacin (OFL), orbifloxacin (ORB), pazufloxacin (PAZ),
t
pefloxacin-d5 (PEF), prulifloxacin (PRU), sarafloxacin (SAR) and sparfloxacin (SPA)
cr ip
were purchased from VETRANAL® Fluka (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions (1 mg mL−1) of FQ and SA antibiotics were prepared by dissolving 5
us
was prepared by diluting the stock solution in PBS. N, N′-Dicyclohexylcarbodiimide
an
(DCC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate isomer I (FITC), Texas Red -NHS (TR-NHS) were purchased from Sigma-Aldrich (St. Louis, MO,
M
USA). All other chemicals and solvents were of analytical grade or better and were obtained from Beijing Chemical Reagent Co. (Beijing, P.R.C.).
ce pt ed
Synthesis of Fluorescent Conjugates (Tracers) SMP-FITC: Sulfamethoxypyridazine (SMP) (5 mg) was dissolved in 0.5 mL of
methanol. Triethylamine (50 µL, 7.2 mol L-1) and FITC (4 mg) were added with
mixing. After overnight reaction at room temperature, small portions (50 µL) of
reaction mixture were separated by thin-layer chromatography (TLC) using
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
mg of drug in 5 mL of 0.03 M sodium hydroxide. Working standard of each antibiotic
chloroform/methanol (4:1, v/v) as the eluent. The main yellow band at Rf 0.1 was
scraped from the plate and extracted with 1 mL of methanol. SAR-TR: Sarafloxacin (SAR) (2 mg) was dissolved in 50 µL (14.08 mol L-1) of N, N-dimethylformamide (DMF). Triethylamine (20 µL) and TR-NHS (2 mg) were added with mixing. After overnight reaction at room temperature, small portions 7
(50µL) of reaction mixture were separated by TLC using methanol / ethyl acetate / ammonia water (4:1:1, v/v/v) as the eluent. The new bright purple color band at Rf 0.8 was scraped from the plate, extracted with 1 mL of methanol, and stored in the dark at
t
4 °C.
cr ip
Assay protocol
Figure.1 shows the schematic diagram of the DB-FPIA procedure. Specific steps
us
of diluted scDb. Subsequently, 70 µL per well of standard solution or sample extract
an
was added, and the mixtures were shaken for 10 s in the microplate reader. After a short incubation period (5 min) at room temperature, the FP value was measured at
M
λex = 485 nm, λem = 530 nm (emission cutoff= 515 nm, G factor = 1.0) and λex = 585 nm, λem =620 nm (emission cutoff= 610 nm, G factor = 1.0), respectively. The
ce pt ed
integration time was set to 3 s for the polarization measurements. Over ten polarization measurements were taken each time, and they were then averaged for further data processing. The relative standard deviation was 2% for all measurements. Curve Fitting and Cross-Reactivity Determination To normalize the FP value, the ratio mP/mP0 (where mP0 is the maximum FP
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
as follows: 70 µL per well of tracer solution mixture was mixed with 70 µL per well
value of the inhibition curve and mP is the current value) resulting in relative units was used. The data were fitted with the four parameter logistic equation using Origin 8.0 software package (Microcal, Northampton, MA, USA). For standard curves, NOR and SMX were used as standards. IC50 is the standard concentration at 50% of specific binding. The cross-reactivity (CR) values were calculated according to the 8
following equation: CR (%) = [IC50 (NOR/SMX)/ IC50 (analyte)] ×100. Milk sample preparation Sample pre-treatment procedures were performed by a previously published
t
method (Wang et al., 2007). Briefly, aliquots (4 mL) milk samples was fortified with
cr ip
the appropriate FQs and SAs standard solution and then mixed with an equal volume
of 1.5% trichloroacetic acid (TCA).The mixtures were agitated on a shaker for 2 min
us
supernatants were diluted with assay buffer to fit the working range before
Analysis of field milk samples
an
measurement in FPIA.
M
Thirty milk samples were collected from retail outlets in Beijing. Each sample was divided into two portions: one was analyzed by the DB-FPIA and another was
ce pt ed
analyzed with HPLC-MS/MS (GB/T 20751–2006 and GB/T 20759–2006, China).
Results and discussion
Characterization of tracers
Monoclonal antibody-based FPIAs for detection of FQs and SAs have been
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
and then de-proteinised through centrifugation for 10 min (8000g at 4°C). The
developed in our laboratory (Wang et al., 2007; Wang et al., 2008; Mi et al., 2013). The previous work showed that higher sensitivity and faster dissociation rate were observed when using the heterogeneous and low cross-reactivities tracer, because it could be more easily replaced by competitors (Mi et al., 2013). Here，two haptens
(SAR and SMP) were selected for preparing fluorescent conjugates to perform a FPIA 9
because both of them have presented better sensitivity in our previous work (Wang et al., 2007; Mi et al., 2013). Subsequently, we synthesized two fluorescent tracers: SMP-FITC and SAR-TR. Because the tracer of SAR-TR (λex = 585 nm, λem =620
t
nm) exhibited significantly longer wavelength of excitation and emission than that of
cr ip
SMP-FITC (λex = 485 nm, λem = 530 nm), they can be used for simultaneous
determination of FQs and SAs. The appropriate two tracers could be preliminarily
us
saturating amounts of scDb, and a higher FP difference will result in a higher
an
signal-to-noise ratio. As shown in Fig. 2A, both SMP-FITC and SAR-TR exhibited sufficient binding with scDb, suggesting that they were successfully synthesized and
Assay optimization
M
could be used as a competition agent in the DB-FPIA.
ce pt ed
The amount of antibodies in the incubation solution was significant in the
competitive DB-FPIA. The scDb gave significant binding with both two tracers (at a fixed concentration 10 nM) at higher concentration and slowly decreased in FP values as antibodies became more diluted by PBS (Fig. 2A). As reported before (Wang et al.,
2008), a low tracer concentration will result in high sensitivity, but low precision of
Ac
Downloaded by [UQ Library] at 16:18 14 October 2014
independently characterized by observing the increase in FP signal after mixing with
FP signal. In addition, under the technical specifications of the instrument used in the present study, precision of
