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Analytica Chimica Acta xxx (2014) xxx–xxx
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens Zhanhui Wang a,b , Huiyan Zhang a , Hengjia Ni a , Suxia Zhang a,b , Jianzhong Shen a,c, * a
College of Veterinary Medicine, China Agricultural University, Beijing 100193, People’s Republic of China Beijing Laboratory For Food Quality and Safety, Beijing 100193, People’s Republic of China c Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing 100193, People’s Republic of China b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new derivative of enrofloxacin was synthesized and used as coating hapten. The effect of coating hapten on the sensitivity and specificity of ELISA was studied. A highly sensitive and specific immunoassay for enrofloxacin was developed.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2014 Received in revised form 17 February 2014 Accepted 26 February 2014 Available online xxx
In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 mg L1 to as low as 0.07 mg L1. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. ã 2014 Elsevier B.V. All rights reserved.
Key words: Immunoassay Enrofloxacin Heterologous hapten Cross-reactivity Chicken muscle
1. Introduction Enrofloxacin (Fig. 1), one member of fluoroquinolones, is a highpotency effective against enteric Gram-negative bacteria, and it has been widely used only in veterinary clinical practice [1]. According to one WHO report in 1998, enrofloxacin is globally the most extensively approved antibiotic of the fluoroquinolones for livestock and aquiculture [2,3]. Thus, its residues may persist in edible tissue or product of food-producing animal, and also may result in the
* Corresponding author. Tel.: +86 10 6273 3289; fax: +86 10 6273 1032. E-mail address: [email protected] (J. Shen).
development of drug-resistant bacterial strains or allergies [4,5], for example, the USA FDA has withdrew the approval of enrofloxacin for poultry in 2005 with consideration of it causing the emergence of resistance in Campylobacter [3,6]. In order to protect consumers from potential contaminated food, the European Commission has established the maximum residue limit (MRL) for enrofloxacin at 100 mg kg1 in several edible animal tissues, while, Japan has set at 10 mg kg1 in chicken muscle [7,8]. In China, the species of animal, usage, dosage, and withdrawal period of enrofloxacin have been determined by the Ministry of Agriculture of the People’ Republic of China (No. 235, 2002). These have created an urgent need for sensitive, rapid, robust, and accurate analytical methods to monitor enrofloxacin residues in the food supply of animal origin.
http://dx.doi.org/10.1016/j.aca.2014.02.043 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Z. Wang, et al., Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.02.043
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Fig. 1. Chemical structures of fluoroquinolones.
Some analytical methods have been developed for the determination of fluoroquinolons residues, mostly including enrofloxacin, in tissues or product of food-producing animal such as liquid chromatography [9,10] and some methods are special for enrofloxacin [11–13]. Although these methods are mostly sensitive and accurate, they are generally expensive and time consuming and not suitable for routine monitoring a large amount of samples. Recently, alternative methods such as immunoassay based on antigen–antibody recognition have been reported for screening enrofloxacin in animal tissues [14,15]. Our group has reported the liposome immune lysis assay and quantum dot-based fluoroimmunoassay for enrofloxacine in carp and chicken muscle [16,17] and also genericELISA for 12 fluroquinolones in different food matrices [18]. Several immunoassays for the determination of enrofloxacine in edible animal tissues and aquatic product based on poly- or monoclonal antibodies are also described in the past decade [14,15,19,20]. However, the sensitivity of these methods was not very high, or the sample pretreatment steps were quite complicated and timeconsuming, thus they could not meet the requirement of strictly regulation for enrofloxacin. Normally, the heterologous enzyme immunoassay systems with different bridge length linkers between the hapten and carrier protein presents a good alternative to homologous assays in achieving higher sensitivity of ELISA [2,5], whereas, this strategy is not, so far, employed to develop ELISA for enrofloxacin to the author’s knowledge, partly due to the easy realization of enrofloxacin coupling to carrier protein by using its carboxyl group at position 3 (Fig. 1). In the paper we presented here, we have synthesized one new enrofloxacin derivative and used as coating hapten to develop ELISA for enrofloxacin in chicken muscle. When combining with
one new prepared polyclonal antibody, the developed heterologous ELISA in bridge length showed high sensitivity and specificity for enrofloxacin. 2. Experimental 2.1. Regents and apparatus Bovine serum albumin (BSA), ovalbumin (OVA), 4-aminobutanoic acid, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), isobutyl chloroformate, tributylamine, ofloxacin, lomefloxacin, enoxacin, danofloxacin, oxolinic acid, and marbofloxacin were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, sarafloxacin, and difloxacin were purchased from the China Institute of Veterinary Drug Control (Beijing, P.R. China). The peroxidase-conjugated Goat antiRabbit IgG were acquired from Huamei Biotech Co. (Beijing, P.R. China). Incomplete Freund’s adjuvant (IFA), complete Freund’s adjuvant (CFA) was obtained from Gibco BRL (Carlsbad, CA, USA). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Polystyrene microtiter plates were purchased from Costar (Costar Inc., Cambridge, MA, USA). The NanoDrop ND-1000 spectrophotometer was purchased from Gene Company Ltd. (Hong Kong, P.R. China). The ELISA plate reader was obtained from TECAN Inc. (Durham, NC, USA). Hapten was confirmed by high performance liquid chromatography–mass spectrometry (HPLC–MS/MS). Chromatography was performed on a Waters Alliance 2690 LC system (Waters Corp., Milford, MA, USA) and the Quattro LC triple-quadrupole mass spectrometer
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(Micro-mass, Manchester, UK) was connected to the LC system via an electrospray ionization (ESI) interface. 1H NMR spectra were obtained with a Bruker 300 spectrometer (Bruker, Daltonics, Billerica, MA, USA).
at 3500 g for 10 min. The supernatants were carefully collected, divided into aliquots and stored at 20 C until use.
2.2. Synthesis of hapten
Buffer and solution used in the ELISA method can refer to Electronic Supplementary Information. Polystyrene 96-well microtiter plates were coated with coating antigen (0.25 mg mL–1, 100 mL/ well) incubated at 4 C overnight. The plates were washed three times with washing buffer, and then blocked with blocking buffer (300 mL/well) at 37 C for 2 h. Antisera (1/10,000, 50 mL/well), various concentrations of analytes or buffer (50 mL/well), and goat anti-rabbit IgG-HRP (1/3000 in PBS, 100 mL/well) were simultaneously added and incubated at 37 C for 1 h. Following incubation the plates were washed three times with washing buffer. The substrate solution (100 mL/well) was added and incubated at 37 C for 30 min before adding 2 N H2SO4 (100 mL/well). The absorbance of each well was measured at 450 nm by the ELISA plate reader. The IC50 value, the assay dynamic range, and the limit of detection (LOD) serve as a criterion to evaluate the ELISA. These characteristics present the analyte concentrations that provide a tracer binding inhibition in OD values at 50%, between 20% and 80%, and at 90%. Curve fitting was performed using a four parameter logistic model as follow:
An amount of 5 mM enrofloxacin (1.8 g) was dissolved in 30 mL of oxalyl chloride and stirred for 5 h at room temperature. Then, the liquid phase was removed under reduced pressure. The residues were redissolved in 50 mL tetrahydrofuran and then 25 mM of 4aminobutyric acid (2.6 g) was added and then stirred overnight at room temperature. After 10 mL of pure water was added to the reactive mixture, and then was extracted by 15 mL of dichloromethane four times. The organic extracts were combined and dried over Na2SO4 (20 g) and removed under reduced pressure. Further purification by preparative TLC on silica gel plates with methanol/ tetrahydrofuran (3/1) containing 3% acetic acid as eluting solvents, gave 110 mg white yellow powder: 10.2% yield. The obtained hapten was named to be ENR-AA and NMR characterization can be found below: 1HNMR (300 MHz, MeOD), d (ppm) 8.78 (s, 1H, H-2 quinoline), 7.91 (d, 1H, J = 15.0 Hz, H-5 quinoline), 7.58 (d, 1H, J = 6.10 Hz, H-8 quinoline), 3.63 (m, 1H, CH cyclopropyl), 3.42 (t, 4H, CH 2 , H-1 and H-2 piperazine), 3.29 (t, 2H, N-CH 2 -CH2-CH2), 2.73 (T, 4H, H-3 and H-4 piperazine), 2.59–2.52 (q, 2H, CH3-CH 2 -), 1.48 (m, 2H, CH 2 cyclopropyl), 1.39–1.35 (m, 4H, CH 2 cyclopropyl and COOH-CH 2 -CH2), 1.19–1.14 (m, 5H, CH 3 -CH2, CH2-CH 2 -CH2). 2.3. Synthesis of protein conjugate The immunogens were prepared by conjugating enrofloxacin to BSA based on a mixed anhydride method [18]. Enrofloxacin (20 mg) was dissolved in 3 mL of dimethylformamide, and the solution was cooled at 4 C, and then 10 mL of triethylamine and 15 mL of isobutyl chloroformate were drop-wise added. The above solutionwas stirred for 20 min at room temperature before 30 mg BSA in 2 mL of carbonate buffer was added drop-wise followed by continuous stirring at room temperature for 6 h. Hapten densities of the immunogen were calculated by MALDI-TOF-MS by comparing the molecular weight of native BSA to that of conjugates. The coating antigens were prepared by conjugating enrofloxacin, pefloxacin, ofloxacin, norfloxacin and ENR-AA to OVA using Nhydroxysuccinimide ester method as following: the hapten (15 mg) was dissolved in 5 mL of dimethylformamide. The NHS (20 mg) and EDC (20 mg) were added to the hapten solution and stirred at room temperature overnight. OVA (30 mg) was dissolved in 5 mL of carbonate buffer and added drop-wise to the active NHS solution with continuous stirring, and further stirred at room temperature for 4 h. The coating antigens were dialyzed against PBS (pH 7.0) at 4 C for 72 h. The concentration of coating antigen was determinated by NanoDrop assay. 2.4. Production of polyclonal antibodies Three female New Zealand white rabbits were used to produce polyclonal antibodies. Immunogen (1 mg) for each rabbit was dissolved in 1 mL of 0.9% NaCl, and emulsified with 1 mL of CFA. The emulsion was injected intracutaneously in the primary immunization. For booster immunizations, immunogen (0.5 mg) for each rabbit was dissolved in 0.5 mL of the above buffer and emulsified with 0.5 mL of IFA. The emulsion was then injected subcutaneously. The booster immunizations were repeated every 2 weeks. Rabbits were bled through an ear vein 1 week after each booster injection (after the fourth injection). To obtain antisera, blood samples were stored to coagulate for 1 h at room temperature and overnight at 37 C, followed by centrifugation
2.5. ELISA methods
Y¼h
ðA DÞ 1 þ ðX=C ÞB þ D
i
where A = response at high asymptote, B = slope factor, C = concentration corresponding to 50% specific binding (IC50), D = response at low asymptote and X = calibration concentration. To normalize the OD value, the ratio B/B0 (where B0 is the maximum OD value of inhibition curve and B is the current value) resulting in relative units was used. 2.6. Cross-reactivity determination Stock solutions of different fluoroquinolones were prepared as described in Electronic Supplementary Information. Standard curves were prepared in assay buffer and each IC50 value was determined in the competitive experiment described above. The cross-reactivity values were calculated according to the following equation: " # IC50 ðenrofloxacin; mg L1 Þ 100% IC50 ðanalogs; mg L1 Þ 2.7. Matrix effect 2.7.1 Effect of pH values and acetonitrile concentration Physicochemical factors that are related to sample treatment including pH values and acetonitrile concentration were studied in the ELISA. Modification of ODmax (ODmax is the maximum absorbance of the inhibition curve) and IC50 parameters of the standard curves was evaluated under various conditions. 2.7.2 Effect of muscle matrix The chicken muscle matrix were serially diluted with assay buffer and used to prepare standard curves. The parallelism of the sigmoidal curves was compared to that prepared in the pure assay buffer in order to evaluate the extent of the interferences caused by the matrix. 2.8. Sample preparation Negative and positive chicken muscle was supplied by Veterinary Drug Safety Inspection &Testing Center of Ministry
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Agriculture (Beijing, P.R. China). Aliquots of chicken muscle homogenate (3 g) were fortified with 1000 mg L1 enrofloxacin standard solution to give tissue levels of 2, 5, 10 and 20 mg kg1 of enrofloxacin. An amount of 9 mL of 0.1 M sodium hydroxide–acetonitrile (1:9) was added and used as extract, then agitated on a shaker for 10 min. The samples were centrifuged at 3000 g for 5 min, and the supernatants (50 mL) were added to 950 mL of assay buffer, and 50 mL of solution was used in the ELISA. 3. Results and discussion 3.1. Synthesis of hapten and conjugate As a small molecule, enrofloxacin should be coupled to large carrier protein to generate specific antibody and use as coating antigen in competitive immunoassay. Hapten design is an important step not only for immunogen to produce a highly affinity antibody but also for coating antigen to afford high sensitivity of immunoassay. Generally, the preferential strategy of designing hapten is to avoid complicated chemical synthesis as much as possible. Unlike other fluoroquinolones, such as norfloxacin or ciprofloxacin, etc., enrofloxacin only comprises one active group, carboxyl group on position 3, which could be directly conjugated to carrier protein (Fig. 1). As far as can be determined from published literatures [14,15,19,20], the reported enrofloxacin antibody production have all relied on the use of carboxyl group of enrofloxacin in the preparation of immunogen and coating antigen/enzyme tracer by the mixed anhydride method or N-hydroxysuccinimide ester method [19,20]. These methods of preparing immunoreagent were effective and simple, thus, excellent antibodies and coating antigens have been obtained. Thus, in the present study, we prepared the immunogen ENR-BSA via directly conjugating enrofloxacin to bovine serum albumin by mixed anhydride and the homologous coating antigen ENR-OVA was prepared by N-hydroxysuccinimide ester method. The conjugation of ENR to BSA was estimated by measuring the hapten/protein molar ratio by MALDI-TOF-MS, showing the hapten/ protein ratios of 7.37.
According the previous reports, the heterologous coating hapten generally resulted in higher sensitivity than the homologous coating hapten in competitive ELISA for small molecule [21], i.e., heterology of coating antigen in the hapten structure, bridge length and site attachment could significantly increase the sensitivity of competitive ELISA [22]. In the case of ELISA for fluoroquinolones, structural heterology of coating hapten has mostly been employed for improving sensitivity, due to the readily commercial availability of structurally similar fluoroquinolone analogs [23–25]. However, both the most often employed conjugation procedure for enrofloxacin, mixed anhydride method and N-hydroxysuccinimide ester method, resulted in the formation of an amide linkage between hapten and carrier protein, thus, bridge recognition problem seem unavoidable in competitive ELISA for enrofloxacin. In order to ameliorate the potential problem and take advantage of heterologous bridge recognition benefit in ELISA, we have designed and synthesized one new enrofloxacin derivative that containing aminobutyric acid as bridge between enrofloxacin and OVA, designated as ENR-AA-OVA (Fig. 2). The introduction of the bridge was expected to decrease the affinity of antisera to the new coating antigen and increase the sensitivity of ELISA for enrofloxacin. Meanwhile, we prepared three structural heterologous coating antigens, NOR-OVA, PEF-OVA and OFL-OVA, to evaluate the effect of these coating antigens on the sensitivity and specificity of antisera. Previous studies reported that the ratio of hapten to carrier protein may have an important effect on sensitivity of ELISA [26,27]. To reduce the influence of hapten-protein ratio as low as possible, all coating antigens were synthesized by identical amount of materials and chemical route, which would guarantee to obtain similar ratio of hapten-carrier protein in the study. 3.2. Antisera screening Antisera titer was tested using a competitive ELISA format with homologous antigen coated ELISA. The dilution of antisera that generating the maximum of absorbance (ODmax) ranging from 1.5 to 1.8 and IC50 values were employed to evaluate the performance
Fig. 2. Synthesis procedure of hapten ENR-AA.
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Table 1 Screening of antisera from rabbits in homologous coating antigen ELISAa . Antisera
Rabbit no. 1 Rabbit no. 2 Rabbit no. 3 a
4th Injection
5th Injection
6th Injection
IC50 (mg L1)
Dilution
IC50 (mg L1)
Dilution
IC50 (mg L1)
Dilution
2.31 14.2 58.1
1/20,000 1/35,000 1/8000
1.24 11.2 60.7
1/40,000 1/50,000 1/15,000
1.52 10.6 71.4
1/40,000 1/40,000 1/15,000
The values represent the mean of three separate experiments each of which contained a minimum of three replicates.
of antisera. It can be observed in Table 1 that all rabbits injected with immunogens ENR-BSA produced relatively high titer sera (>1/ 8000) after the 4th injection (third boost). Among these antisera, rabbit no. 1 showed the lowest IC50 values and that of rabbit no. 2 showed the highest dilution (2.31 mg L1and 1/35,000, respectively). In the next bleeding, i.e., the 5th injection, the dilution and IC50 values of all antisera have been improved, whereas, the parameters of antisera of the 6th become slightly worse compared to the antisera of the 5th injection (Table 1). With consideration of both titer and IC50 values of three antisera, antisera no. 1 was selected for the following work. 3.3. Effect of heterologous coating hapten on ELISA sensitivity The high sensitivity is one of the most important goals for the analytical techniques, particularly for immunoassay that will be favorable for simplifying sample pretreatment procedure. As mentioned above, a strategy using a heterologous coating hapten could improve ELISA sensitivity in most cases. To achieve high sensitivity, five coating antigen were evaluated in competitive ELISA for enrofloxacin including homologous coating antigen (ENR-OVA), structural heterologous coating antigens (NOR-OVA, PEF-OVA and OFL-OVA), and bridge heterologous coating antigen (ENR-AA-OVA). The IC50 values of ELISA for enrofloxacin using both homologous and heterologous coating antigens were shown in Table 2. The data obtained from NOR-OVA were not given in Table 2 due to the antisera could not binding to NOR-OVA, providing low OD values (100,000 9842 32.34 12,014 6190 >100,000 >100,000
100 0.012 2.84 0.09 5.32 0.001 0.012 4.02 0.010 0.021 0.001 0.001
0.76 3454 32.9 1490 7.34 37,654 9500 73.7 36,102 6335 25,333 46,561
100 0.022 2.31 0.051 10.35 0.002 0.008 1.03 0.002 0.012 0.003 0.001
0.54 1687 10.8 1285 13.1 26,345 12,871 16.6 6754 3857 23,478 28,421
100 0.032 4.98 0.042 4.12 0.002 0.004 3.24 0.008 0.014 0.0023 0.0019
The values represent the mean of three separate experiments each of which contained a minimum of four replicates.
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Fig. 3. Calibration curves of ELISA based on ENR-AA-OVA and ENR-OVA for enrofloxacin. B is the absorbance of a tested enrofloxacin solution and B0 is the absorbance of buffer without enrofloxacin.
group must connect to the nitrogen atom instead of carbon atom of the piperazinyl ring. For example, the structure of lomefloxacin is similar to pefloxacin in all respects except for the methyl group position on the piperazinyl ring, and this different result in a 5000fold decrease in the IC50 value for lomefloxacin compared to pefloxacin (Table 2). The comparison of lomefloxacin and pefloxacin neglect the effect of substitution on position 8 that has limit influence on antibody recognition by comparing ofloxacin and pefloxacin. In addition, a comparison of pefloxacin to difloxacin, a cyclopropyl group in pefloxacin substituted by a fluorophenyl group in difloxacin, was conducted and the substitution produced a 45-fold decrease in the IC50 value for difloxacin compared with that of pefloxacin (Table 2). This was not surprising since the presence of the fluorophenyl group introduced a larger substituent on position 1 in difloxacin than in pefloxacin, and the aromatic ring as well as the electronegative fluorine would be expected to involve a number of binding interactions. The impotence of methyl group on piperazinyl ring to antisera recognition had been again observed by comparing the IC50 value for difloxacin (42.5 mg L1) to that of sarafloxacin (4132 mg L1). The combined effect of losing the ethyl group at the piperazinyl ring on position 7 and introduction of fluorophenyl group on position 1 can be evaluated by comparing the IC50 value for sarafloxacin to that of enrofloxacin. The specificity of the developed ELISA in the study was obviously unique comparing to previous reports [18]. The recognition profiles of antisera for structurally similar fluoroquinolones in the study were not easy to explain by only view of 2D-chemical structure, thus, we further investigated the recognition mechanism of antibody to fluoroquinolones at 3D level and quantitative manner [28]. The detailed results could be found in Supplementary materials.
experiments, the matrix effect of extract was expected to remove just by buffer dilution, mainly because of the highly sensitivity of the developed ELISA. The defined enrofloxacin extract solution containing acetonitrile and NaOH that may influence the analytical characteristics of ELISA. Before recovery study, the effects of these parameters were assessed by comparing IC50 and ODmax obtained under various concentrations of acetonitrile and pH values (The ODmax, reflecting the antibody recognition of the coating antigen in the absence of analyte, and the IC50, reflecting the antibody affinity for the enrofloxacin itself). To study the influence of pH in the assay system, competition curves for enrofloxacin were constructed at pH values ranging from 3 to 11. The relationship of these parameters as a function of pH was shown in Fig. 4. Deleterious effects on IC50 and ODmax were observed at lower or higher pH but no significant changes were observed in the pH range of 6–8. The results indicated that the assay sensitivity decreased at a more acidic or basic pH value. In the case of acetonitrile, the ODmax was significantly decreased as the concentration of acetonitrile increased. Acetonitrile concentration exceeding 10% (final concentration) were not tolerated by the ELISA (Fig. 4). The presence of 10% acetonitrile resulted in almost 50% reduction in ODmax. Therefore, pH values and final acetonitrile concentration should be carefully controlled for a robust ELISA procedure.
3.5. Matrix effect and recovery An important criterion for evaluation of a robust analytical procedure is the assessment of the matrix effect of sample, which should be minimized as much as possible by the sample preparation procedure [29]. In our previous reports, acetonitrile containing 10% 2 M NaOH has been proved an effective solvent for extracting enrofloxacin from animal tissues [16–18]. In these studies, clean-up step was necessary to reduce matrix effects prior to detection. The clean-up procedure, normally including usage of organic solvent, is time-consuming and harmful and would counteract the main advantage of the ELISA method in being a rapid and simple screening application. Thus, in the present
Fig. 4. Influence of (A) pH values and (B) acetonitrile concentration on the heterologous coating antigen ELISA for enrofloxacin. The left axes showed the maximum absorbance (ODmax) and the right axes showed the values of IC50 values expressed in mg L1. The data shown have been extracted from the four-parameter equation used to fit the standard curves. Standard curves were prepared using three well replicates.
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Acknowledgments This work is supported by the grants from Natural Science Foundation of China (U1301214, 31372475), Trans-Century Training Programme Foundation for the Talents by the Ministry of Education (NCET-12-0529). We thank Prof. Jing Shen of Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Core Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute for MALDI-TOF MS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.02.043. References
Fig. 5. Calibration curves of heterologous ELISA for enrofloxacin in PBS and PBS diluted extract. Parallelism was found between the standard curve prepared in PBS and the one prepared in 1/20 PBS diluted extract. Each value represents the average of at least three independent replicates.
In a separate experiment for evaluation of matrix interference, enrofloxacin standards were prepared in the diluted chicken muscle extract to obtain standard curves and shown in Fig. 5. The results demonstrated that the raw extract caused the most noticeable influence on the ELISA as evidenced by a more than100-fold increase of the IC50 values. Nevertheless, the IC50 values were almost not affected by a 20-fold dilution of raw extract. The possible reason may lie upon the fact that the dilution procedure had reduced the final concentration of acetonitrile (
Analytica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens Zhanhui Wang a,b , Huiyan Zhang a , Hengjia Ni a , Suxia Zhang a,b , Jianzhong Shen a,c, * a
College of Veterinary Medicine, China Agricultural University, Beijing 100193, People’s Republic of China Beijing Laboratory For Food Quality and Safety, Beijing 100193, People’s Republic of China c Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing 100193, People’s Republic of China b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new derivative of enrofloxacin was synthesized and used as coating hapten. The effect of coating hapten on the sensitivity and specificity of ELISA was studied. A highly sensitive and specific immunoassay for enrofloxacin was developed.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2014 Received in revised form 17 February 2014 Accepted 26 February 2014 Available online xxx
In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 mg L1 to as low as 0.07 mg L1. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. ã 2014 Elsevier B.V. All rights reserved.
Key words: Immunoassay Enrofloxacin Heterologous hapten Cross-reactivity Chicken muscle
1. Introduction Enrofloxacin (Fig. 1), one member of fluoroquinolones, is a highpotency effective against enteric Gram-negative bacteria, and it has been widely used only in veterinary clinical practice [1]. According to one WHO report in 1998, enrofloxacin is globally the most extensively approved antibiotic of the fluoroquinolones for livestock and aquiculture [2,3]. Thus, its residues may persist in edible tissue or product of food-producing animal, and also may result in the
* Corresponding author. Tel.: +86 10 6273 3289; fax: +86 10 6273 1032. E-mail address: [email protected] (J. Shen).
development of drug-resistant bacterial strains or allergies [4,5], for example, the USA FDA has withdrew the approval of enrofloxacin for poultry in 2005 with consideration of it causing the emergence of resistance in Campylobacter [3,6]. In order to protect consumers from potential contaminated food, the European Commission has established the maximum residue limit (MRL) for enrofloxacin at 100 mg kg1 in several edible animal tissues, while, Japan has set at 10 mg kg1 in chicken muscle [7,8]. In China, the species of animal, usage, dosage, and withdrawal period of enrofloxacin have been determined by the Ministry of Agriculture of the People’ Republic of China (No. 235, 2002). These have created an urgent need for sensitive, rapid, robust, and accurate analytical methods to monitor enrofloxacin residues in the food supply of animal origin.
http://dx.doi.org/10.1016/j.aca.2014.02.043 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Z. Wang, et al., Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.02.043
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Fig. 1. Chemical structures of fluoroquinolones.
Some analytical methods have been developed for the determination of fluoroquinolons residues, mostly including enrofloxacin, in tissues or product of food-producing animal such as liquid chromatography [9,10] and some methods are special for enrofloxacin [11–13]. Although these methods are mostly sensitive and accurate, they are generally expensive and time consuming and not suitable for routine monitoring a large amount of samples. Recently, alternative methods such as immunoassay based on antigen–antibody recognition have been reported for screening enrofloxacin in animal tissues [14,15]. Our group has reported the liposome immune lysis assay and quantum dot-based fluoroimmunoassay for enrofloxacine in carp and chicken muscle [16,17] and also genericELISA for 12 fluroquinolones in different food matrices [18]. Several immunoassays for the determination of enrofloxacine in edible animal tissues and aquatic product based on poly- or monoclonal antibodies are also described in the past decade [14,15,19,20]. However, the sensitivity of these methods was not very high, or the sample pretreatment steps were quite complicated and timeconsuming, thus they could not meet the requirement of strictly regulation for enrofloxacin. Normally, the heterologous enzyme immunoassay systems with different bridge length linkers between the hapten and carrier protein presents a good alternative to homologous assays in achieving higher sensitivity of ELISA [2,5], whereas, this strategy is not, so far, employed to develop ELISA for enrofloxacin to the author’s knowledge, partly due to the easy realization of enrofloxacin coupling to carrier protein by using its carboxyl group at position 3 (Fig. 1). In the paper we presented here, we have synthesized one new enrofloxacin derivative and used as coating hapten to develop ELISA for enrofloxacin in chicken muscle. When combining with
one new prepared polyclonal antibody, the developed heterologous ELISA in bridge length showed high sensitivity and specificity for enrofloxacin. 2. Experimental 2.1. Regents and apparatus Bovine serum albumin (BSA), ovalbumin (OVA), 4-aminobutanoic acid, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), isobutyl chloroformate, tributylamine, ofloxacin, lomefloxacin, enoxacin, danofloxacin, oxolinic acid, and marbofloxacin were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, sarafloxacin, and difloxacin were purchased from the China Institute of Veterinary Drug Control (Beijing, P.R. China). The peroxidase-conjugated Goat antiRabbit IgG were acquired from Huamei Biotech Co. (Beijing, P.R. China). Incomplete Freund’s adjuvant (IFA), complete Freund’s adjuvant (CFA) was obtained from Gibco BRL (Carlsbad, CA, USA). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Polystyrene microtiter plates were purchased from Costar (Costar Inc., Cambridge, MA, USA). The NanoDrop ND-1000 spectrophotometer was purchased from Gene Company Ltd. (Hong Kong, P.R. China). The ELISA plate reader was obtained from TECAN Inc. (Durham, NC, USA). Hapten was confirmed by high performance liquid chromatography–mass spectrometry (HPLC–MS/MS). Chromatography was performed on a Waters Alliance 2690 LC system (Waters Corp., Milford, MA, USA) and the Quattro LC triple-quadrupole mass spectrometer
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(Micro-mass, Manchester, UK) was connected to the LC system via an electrospray ionization (ESI) interface. 1H NMR spectra were obtained with a Bruker 300 spectrometer (Bruker, Daltonics, Billerica, MA, USA).
at 3500 g for 10 min. The supernatants were carefully collected, divided into aliquots and stored at 20 C until use.
2.2. Synthesis of hapten
Buffer and solution used in the ELISA method can refer to Electronic Supplementary Information. Polystyrene 96-well microtiter plates were coated with coating antigen (0.25 mg mL–1, 100 mL/ well) incubated at 4 C overnight. The plates were washed three times with washing buffer, and then blocked with blocking buffer (300 mL/well) at 37 C for 2 h. Antisera (1/10,000, 50 mL/well), various concentrations of analytes or buffer (50 mL/well), and goat anti-rabbit IgG-HRP (1/3000 in PBS, 100 mL/well) were simultaneously added and incubated at 37 C for 1 h. Following incubation the plates were washed three times with washing buffer. The substrate solution (100 mL/well) was added and incubated at 37 C for 30 min before adding 2 N H2SO4 (100 mL/well). The absorbance of each well was measured at 450 nm by the ELISA plate reader. The IC50 value, the assay dynamic range, and the limit of detection (LOD) serve as a criterion to evaluate the ELISA. These characteristics present the analyte concentrations that provide a tracer binding inhibition in OD values at 50%, between 20% and 80%, and at 90%. Curve fitting was performed using a four parameter logistic model as follow:
An amount of 5 mM enrofloxacin (1.8 g) was dissolved in 30 mL of oxalyl chloride and stirred for 5 h at room temperature. Then, the liquid phase was removed under reduced pressure. The residues were redissolved in 50 mL tetrahydrofuran and then 25 mM of 4aminobutyric acid (2.6 g) was added and then stirred overnight at room temperature. After 10 mL of pure water was added to the reactive mixture, and then was extracted by 15 mL of dichloromethane four times. The organic extracts were combined and dried over Na2SO4 (20 g) and removed under reduced pressure. Further purification by preparative TLC on silica gel plates with methanol/ tetrahydrofuran (3/1) containing 3% acetic acid as eluting solvents, gave 110 mg white yellow powder: 10.2% yield. The obtained hapten was named to be ENR-AA and NMR characterization can be found below: 1HNMR (300 MHz, MeOD), d (ppm) 8.78 (s, 1H, H-2 quinoline), 7.91 (d, 1H, J = 15.0 Hz, H-5 quinoline), 7.58 (d, 1H, J = 6.10 Hz, H-8 quinoline), 3.63 (m, 1H, CH cyclopropyl), 3.42 (t, 4H, CH 2 , H-1 and H-2 piperazine), 3.29 (t, 2H, N-CH 2 -CH2-CH2), 2.73 (T, 4H, H-3 and H-4 piperazine), 2.59–2.52 (q, 2H, CH3-CH 2 -), 1.48 (m, 2H, CH 2 cyclopropyl), 1.39–1.35 (m, 4H, CH 2 cyclopropyl and COOH-CH 2 -CH2), 1.19–1.14 (m, 5H, CH 3 -CH2, CH2-CH 2 -CH2). 2.3. Synthesis of protein conjugate The immunogens were prepared by conjugating enrofloxacin to BSA based on a mixed anhydride method [18]. Enrofloxacin (20 mg) was dissolved in 3 mL of dimethylformamide, and the solution was cooled at 4 C, and then 10 mL of triethylamine and 15 mL of isobutyl chloroformate were drop-wise added. The above solutionwas stirred for 20 min at room temperature before 30 mg BSA in 2 mL of carbonate buffer was added drop-wise followed by continuous stirring at room temperature for 6 h. Hapten densities of the immunogen were calculated by MALDI-TOF-MS by comparing the molecular weight of native BSA to that of conjugates. The coating antigens were prepared by conjugating enrofloxacin, pefloxacin, ofloxacin, norfloxacin and ENR-AA to OVA using Nhydroxysuccinimide ester method as following: the hapten (15 mg) was dissolved in 5 mL of dimethylformamide. The NHS (20 mg) and EDC (20 mg) were added to the hapten solution and stirred at room temperature overnight. OVA (30 mg) was dissolved in 5 mL of carbonate buffer and added drop-wise to the active NHS solution with continuous stirring, and further stirred at room temperature for 4 h. The coating antigens were dialyzed against PBS (pH 7.0) at 4 C for 72 h. The concentration of coating antigen was determinated by NanoDrop assay. 2.4. Production of polyclonal antibodies Three female New Zealand white rabbits were used to produce polyclonal antibodies. Immunogen (1 mg) for each rabbit was dissolved in 1 mL of 0.9% NaCl, and emulsified with 1 mL of CFA. The emulsion was injected intracutaneously in the primary immunization. For booster immunizations, immunogen (0.5 mg) for each rabbit was dissolved in 0.5 mL of the above buffer and emulsified with 0.5 mL of IFA. The emulsion was then injected subcutaneously. The booster immunizations were repeated every 2 weeks. Rabbits were bled through an ear vein 1 week after each booster injection (after the fourth injection). To obtain antisera, blood samples were stored to coagulate for 1 h at room temperature and overnight at 37 C, followed by centrifugation
2.5. ELISA methods
Y¼h
ðA DÞ 1 þ ðX=C ÞB þ D
i
where A = response at high asymptote, B = slope factor, C = concentration corresponding to 50% specific binding (IC50), D = response at low asymptote and X = calibration concentration. To normalize the OD value, the ratio B/B0 (where B0 is the maximum OD value of inhibition curve and B is the current value) resulting in relative units was used. 2.6. Cross-reactivity determination Stock solutions of different fluoroquinolones were prepared as described in Electronic Supplementary Information. Standard curves were prepared in assay buffer and each IC50 value was determined in the competitive experiment described above. The cross-reactivity values were calculated according to the following equation: " # IC50 ðenrofloxacin; mg L1 Þ 100% IC50 ðanalogs; mg L1 Þ 2.7. Matrix effect 2.7.1 Effect of pH values and acetonitrile concentration Physicochemical factors that are related to sample treatment including pH values and acetonitrile concentration were studied in the ELISA. Modification of ODmax (ODmax is the maximum absorbance of the inhibition curve) and IC50 parameters of the standard curves was evaluated under various conditions. 2.7.2 Effect of muscle matrix The chicken muscle matrix were serially diluted with assay buffer and used to prepare standard curves. The parallelism of the sigmoidal curves was compared to that prepared in the pure assay buffer in order to evaluate the extent of the interferences caused by the matrix. 2.8. Sample preparation Negative and positive chicken muscle was supplied by Veterinary Drug Safety Inspection &Testing Center of Ministry
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Agriculture (Beijing, P.R. China). Aliquots of chicken muscle homogenate (3 g) were fortified with 1000 mg L1 enrofloxacin standard solution to give tissue levels of 2, 5, 10 and 20 mg kg1 of enrofloxacin. An amount of 9 mL of 0.1 M sodium hydroxide–acetonitrile (1:9) was added and used as extract, then agitated on a shaker for 10 min. The samples were centrifuged at 3000 g for 5 min, and the supernatants (50 mL) were added to 950 mL of assay buffer, and 50 mL of solution was used in the ELISA. 3. Results and discussion 3.1. Synthesis of hapten and conjugate As a small molecule, enrofloxacin should be coupled to large carrier protein to generate specific antibody and use as coating antigen in competitive immunoassay. Hapten design is an important step not only for immunogen to produce a highly affinity antibody but also for coating antigen to afford high sensitivity of immunoassay. Generally, the preferential strategy of designing hapten is to avoid complicated chemical synthesis as much as possible. Unlike other fluoroquinolones, such as norfloxacin or ciprofloxacin, etc., enrofloxacin only comprises one active group, carboxyl group on position 3, which could be directly conjugated to carrier protein (Fig. 1). As far as can be determined from published literatures [14,15,19,20], the reported enrofloxacin antibody production have all relied on the use of carboxyl group of enrofloxacin in the preparation of immunogen and coating antigen/enzyme tracer by the mixed anhydride method or N-hydroxysuccinimide ester method [19,20]. These methods of preparing immunoreagent were effective and simple, thus, excellent antibodies and coating antigens have been obtained. Thus, in the present study, we prepared the immunogen ENR-BSA via directly conjugating enrofloxacin to bovine serum albumin by mixed anhydride and the homologous coating antigen ENR-OVA was prepared by N-hydroxysuccinimide ester method. The conjugation of ENR to BSA was estimated by measuring the hapten/protein molar ratio by MALDI-TOF-MS, showing the hapten/ protein ratios of 7.37.
According the previous reports, the heterologous coating hapten generally resulted in higher sensitivity than the homologous coating hapten in competitive ELISA for small molecule [21], i.e., heterology of coating antigen in the hapten structure, bridge length and site attachment could significantly increase the sensitivity of competitive ELISA [22]. In the case of ELISA for fluoroquinolones, structural heterology of coating hapten has mostly been employed for improving sensitivity, due to the readily commercial availability of structurally similar fluoroquinolone analogs [23–25]. However, both the most often employed conjugation procedure for enrofloxacin, mixed anhydride method and N-hydroxysuccinimide ester method, resulted in the formation of an amide linkage between hapten and carrier protein, thus, bridge recognition problem seem unavoidable in competitive ELISA for enrofloxacin. In order to ameliorate the potential problem and take advantage of heterologous bridge recognition benefit in ELISA, we have designed and synthesized one new enrofloxacin derivative that containing aminobutyric acid as bridge between enrofloxacin and OVA, designated as ENR-AA-OVA (Fig. 2). The introduction of the bridge was expected to decrease the affinity of antisera to the new coating antigen and increase the sensitivity of ELISA for enrofloxacin. Meanwhile, we prepared three structural heterologous coating antigens, NOR-OVA, PEF-OVA and OFL-OVA, to evaluate the effect of these coating antigens on the sensitivity and specificity of antisera. Previous studies reported that the ratio of hapten to carrier protein may have an important effect on sensitivity of ELISA [26,27]. To reduce the influence of hapten-protein ratio as low as possible, all coating antigens were synthesized by identical amount of materials and chemical route, which would guarantee to obtain similar ratio of hapten-carrier protein in the study. 3.2. Antisera screening Antisera titer was tested using a competitive ELISA format with homologous antigen coated ELISA. The dilution of antisera that generating the maximum of absorbance (ODmax) ranging from 1.5 to 1.8 and IC50 values were employed to evaluate the performance
Fig. 2. Synthesis procedure of hapten ENR-AA.
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Table 1 Screening of antisera from rabbits in homologous coating antigen ELISAa . Antisera
Rabbit no. 1 Rabbit no. 2 Rabbit no. 3 a
4th Injection
5th Injection
6th Injection
IC50 (mg L1)
Dilution
IC50 (mg L1)
Dilution
IC50 (mg L1)
Dilution
2.31 14.2 58.1
1/20,000 1/35,000 1/8000
1.24 11.2 60.7
1/40,000 1/50,000 1/15,000
1.52 10.6 71.4
1/40,000 1/40,000 1/15,000
The values represent the mean of three separate experiments each of which contained a minimum of three replicates.
of antisera. It can be observed in Table 1 that all rabbits injected with immunogens ENR-BSA produced relatively high titer sera (>1/ 8000) after the 4th injection (third boost). Among these antisera, rabbit no. 1 showed the lowest IC50 values and that of rabbit no. 2 showed the highest dilution (2.31 mg L1and 1/35,000, respectively). In the next bleeding, i.e., the 5th injection, the dilution and IC50 values of all antisera have been improved, whereas, the parameters of antisera of the 6th become slightly worse compared to the antisera of the 5th injection (Table 1). With consideration of both titer and IC50 values of three antisera, antisera no. 1 was selected for the following work. 3.3. Effect of heterologous coating hapten on ELISA sensitivity The high sensitivity is one of the most important goals for the analytical techniques, particularly for immunoassay that will be favorable for simplifying sample pretreatment procedure. As mentioned above, a strategy using a heterologous coating hapten could improve ELISA sensitivity in most cases. To achieve high sensitivity, five coating antigen were evaluated in competitive ELISA for enrofloxacin including homologous coating antigen (ENR-OVA), structural heterologous coating antigens (NOR-OVA, PEF-OVA and OFL-OVA), and bridge heterologous coating antigen (ENR-AA-OVA). The IC50 values of ELISA for enrofloxacin using both homologous and heterologous coating antigens were shown in Table 2. The data obtained from NOR-OVA were not given in Table 2 due to the antisera could not binding to NOR-OVA, providing low OD values (100,000 9842 32.34 12,014 6190 >100,000 >100,000
100 0.012 2.84 0.09 5.32 0.001 0.012 4.02 0.010 0.021 0.001 0.001
0.76 3454 32.9 1490 7.34 37,654 9500 73.7 36,102 6335 25,333 46,561
100 0.022 2.31 0.051 10.35 0.002 0.008 1.03 0.002 0.012 0.003 0.001
0.54 1687 10.8 1285 13.1 26,345 12,871 16.6 6754 3857 23,478 28,421
100 0.032 4.98 0.042 4.12 0.002 0.004 3.24 0.008 0.014 0.0023 0.0019
The values represent the mean of three separate experiments each of which contained a minimum of four replicates.
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Fig. 3. Calibration curves of ELISA based on ENR-AA-OVA and ENR-OVA for enrofloxacin. B is the absorbance of a tested enrofloxacin solution and B0 is the absorbance of buffer without enrofloxacin.
group must connect to the nitrogen atom instead of carbon atom of the piperazinyl ring. For example, the structure of lomefloxacin is similar to pefloxacin in all respects except for the methyl group position on the piperazinyl ring, and this different result in a 5000fold decrease in the IC50 value for lomefloxacin compared to pefloxacin (Table 2). The comparison of lomefloxacin and pefloxacin neglect the effect of substitution on position 8 that has limit influence on antibody recognition by comparing ofloxacin and pefloxacin. In addition, a comparison of pefloxacin to difloxacin, a cyclopropyl group in pefloxacin substituted by a fluorophenyl group in difloxacin, was conducted and the substitution produced a 45-fold decrease in the IC50 value for difloxacin compared with that of pefloxacin (Table 2). This was not surprising since the presence of the fluorophenyl group introduced a larger substituent on position 1 in difloxacin than in pefloxacin, and the aromatic ring as well as the electronegative fluorine would be expected to involve a number of binding interactions. The impotence of methyl group on piperazinyl ring to antisera recognition had been again observed by comparing the IC50 value for difloxacin (42.5 mg L1) to that of sarafloxacin (4132 mg L1). The combined effect of losing the ethyl group at the piperazinyl ring on position 7 and introduction of fluorophenyl group on position 1 can be evaluated by comparing the IC50 value for sarafloxacin to that of enrofloxacin. The specificity of the developed ELISA in the study was obviously unique comparing to previous reports [18]. The recognition profiles of antisera for structurally similar fluoroquinolones in the study were not easy to explain by only view of 2D-chemical structure, thus, we further investigated the recognition mechanism of antibody to fluoroquinolones at 3D level and quantitative manner [28]. The detailed results could be found in Supplementary materials.
experiments, the matrix effect of extract was expected to remove just by buffer dilution, mainly because of the highly sensitivity of the developed ELISA. The defined enrofloxacin extract solution containing acetonitrile and NaOH that may influence the analytical characteristics of ELISA. Before recovery study, the effects of these parameters were assessed by comparing IC50 and ODmax obtained under various concentrations of acetonitrile and pH values (The ODmax, reflecting the antibody recognition of the coating antigen in the absence of analyte, and the IC50, reflecting the antibody affinity for the enrofloxacin itself). To study the influence of pH in the assay system, competition curves for enrofloxacin were constructed at pH values ranging from 3 to 11. The relationship of these parameters as a function of pH was shown in Fig. 4. Deleterious effects on IC50 and ODmax were observed at lower or higher pH but no significant changes were observed in the pH range of 6–8. The results indicated that the assay sensitivity decreased at a more acidic or basic pH value. In the case of acetonitrile, the ODmax was significantly decreased as the concentration of acetonitrile increased. Acetonitrile concentration exceeding 10% (final concentration) were not tolerated by the ELISA (Fig. 4). The presence of 10% acetonitrile resulted in almost 50% reduction in ODmax. Therefore, pH values and final acetonitrile concentration should be carefully controlled for a robust ELISA procedure.
3.5. Matrix effect and recovery An important criterion for evaluation of a robust analytical procedure is the assessment of the matrix effect of sample, which should be minimized as much as possible by the sample preparation procedure [29]. In our previous reports, acetonitrile containing 10% 2 M NaOH has been proved an effective solvent for extracting enrofloxacin from animal tissues [16–18]. In these studies, clean-up step was necessary to reduce matrix effects prior to detection. The clean-up procedure, normally including usage of organic solvent, is time-consuming and harmful and would counteract the main advantage of the ELISA method in being a rapid and simple screening application. Thus, in the present
Fig. 4. Influence of (A) pH values and (B) acetonitrile concentration on the heterologous coating antigen ELISA for enrofloxacin. The left axes showed the maximum absorbance (ODmax) and the right axes showed the values of IC50 values expressed in mg L1. The data shown have been extracted from the four-parameter equation used to fit the standard curves. Standard curves were prepared using three well replicates.
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Acknowledgments This work is supported by the grants from Natural Science Foundation of China (U1301214, 31372475), Trans-Century Training Programme Foundation for the Talents by the Ministry of Education (NCET-12-0529). We thank Prof. Jing Shen of Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Core Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute for MALDI-TOF MS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.02.043. References
Fig. 5. Calibration curves of heterologous ELISA for enrofloxacin in PBS and PBS diluted extract. Parallelism was found between the standard curve prepared in PBS and the one prepared in 1/20 PBS diluted extract. Each value represents the average of at least three independent replicates.
In a separate experiment for evaluation of matrix interference, enrofloxacin standards were prepared in the diluted chicken muscle extract to obtain standard curves and shown in Fig. 5. The results demonstrated that the raw extract caused the most noticeable influence on the ELISA as evidenced by a more than100-fold increase of the IC50 values. Nevertheless, the IC50 values were almost not affected by a 20-fold dilution of raw extract. The possible reason may lie upon the fact that the dilution procedure had reduced the final concentration of acetonitrile (
