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Physics Procedia 25 (2012) 1829 – 1836

2012 International Conference on Solid State Devices and Materials Science

Development of an Heterologous Immunoassay for Ciprofloxacin Residue in Milk* Jiang Jinqing, Zhang Haitang, An Zhixing, Xu Zhiyong, Yang Xuefeng, Huang Huaguo and Wang Ziliang** College of Animal Science, Henan Institute of Science and Technology, Xinxiang, Henan, 453003, China **Corresponding author.

Abstract A heterologous immunoassay has been developed for the determination of Ciprofloxacin (CPFX) residues in milk. For this reason, carbodiimide active ester method was employed to synthesize the artificial antigen of CPFX-BSA, and mixed anhydride reaction was used to prepare the coating antigen of CPFX-OVA to pursue the heterologous sensitivity. Based on the square matrix titration, an icELISA method was developed for the quantitative detection of CPFX in cattle milk. The dynamic range was from 0.036 to 92.5 ng/mL, with LOD and IC50 value of 0.019 ng/mL and 1.8 ng/mL, respectively. Except for a high cross-reactivity (89.7%) to Enrofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 0.03 mol/L of HCl, or 10% of methanol was used in the assay buffer. 20-fold dilution in cattle milk gave an inhibition curve almost the same as that in PBS buffer. The regression equation for this assay was y = 0.9036 x + 1.4574, with a correlation coefficient (R2) of 0.9844. The results suggest the veracity of the heterologous immunoassay for detecting CPFX residue in milk. Keywords-Ciprofloxacin; Artificial antigen; polyclonal antibody; Indirect competitive ELISA; Heterologous; Milk

1.

Introduction

Fluoroquinolones are gaining widespread acceptance in veterinary medicine because of its wide spectrum of activity and favorable pharmacokinetic behavior [1]. Generally, fluoroquinolones are characterized by excellent tissue penetration, high bioavailability and long terminal half-life, in which enrofloxacin is used in beef breed cattle with respiratory and digestive diseases and in dairy cattle suffering from acute E.coli mastitis. However, enrofloxacin residue may persist in animal or human body, resulting in the development of drug-resistant bacterial strains or allergies [2]. Therefore, routine analysis and residual detection are required, and several immunoassay techniques have thus been developed for the determination of enrofloxacin [3-8]. Ciprofloxacin (CPFX) is a major metabolite of enrofloxacin, and is also one of the most widely used FQs for the treatment of urinary tract infections, respiratory tract infections and chronic bacterial prostatitis [9]. On the other hand, the CPFX residues in livestock products may cause serious public health problems. An EU Regulation has set an MRL of 30 ȝg/kg for the sum of CPFX and enrofloxacin in

1875-3892 © 2012 Published by Elsevier B.V. Selection and/or peer-review under responsibility of Garry Lee Open access under CC BY-NC-ND license. doi:10.1016/j.phpro.2012.03.318

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edible animal tissues [10]. Therefore, rapid and simple detection techniques are required to determine the CPFX residue level in food products. In this study, we have aimed to prepare the artificial antigen of CPFX and produce anti-CPFX polyclonal antibody (pAb). On the basis, we developed a quantitative enzyme immunoassay to detect the CPFX residue in cattle milk. The analytical performance of the developed method is reported here and its applicability to the analysis of CPFX residue is discussed. 2.

Materials and Methods

2.1 Materials and reagents Ciprofloxacin, Enrofloxacin, Sarafloxacin, Ofloxacin, Norfloxacin, and Danofloxacin were provided by Sigma (St. Louis, MO). BSA, OVA and GaRIgG-HRP were purchased from Sino-American Biotechnology Company (Shanghai, China). FCA, FIA and EDC were obtained from Pierce. NHS was from Japan, MSDS available. O-(Carboxymethyl) hydroxylamine hemihydrochloride, Succinic anhydride, were supplied by Sigma while Dialysis bag (8000-14000 Da) was from Solarbio Company. TMB, phenacetin, urea peroxide were obtained from Sigma Company. All other solvents and reagents were of analytical grade or higher, unless otherwise stated. A spectrophotometric microtitre reader, MULTISKAN MK3 (Thermo company, USA) was used for absorbance measurements. 2.2 Synthesis of immunogen and coating antigen The immunogen of CPFX-BSA was prepared by carbodiimide active ester method as a modified previous paper [11]. A total of 7.72 mg of CPFX, 40 mg of EDC, and 10 mg of NHS were added to 0.5 mL DMF in order. The mixture solution was incubated for 24 h at room temperature in dark. Then 2.5 mL of PBS (0.01 mol/L, pH 7.4) with 64 mg BSA was added slowly to the mixture solution with stirring, followed by 4-h incubation at room temperature. Finally, the reaction mixture was dialyzed under stirring against PBS for 6 d with repeated changes of the PBS solution to remove the unconjugated hapten. The solution was stored at -20ć. A CPFX-OVA conjugate was prepared based on the mixed anhydride reaction described by Bucknall et al. [12]. 2.3 Immunization Three Female New Zealand white rabbits were subcutaneously immunized at four sites in the back with CPFX-BSA conjugate. FCA was employed in the first immunization and FIA was used in the subsequent boost injections. Rabbits were immunized every 28 days with 500 ȝg of immunogen, and blood samples were taken for ELISA identification from the marginal vein of the ear 10 days after each immunization. Ten days after the final boost, all rabbits were exsanguinated by heart puncture and the serum was purified with saturated ammonium sulfate (SAS) precipitation method. 2.4 Buffers The ELISA buffers used were as follows: phosphate buffered saline (PBS) consisted of NaCl (137 mM), Na2HPO4.12H2O (10 mM), KCl (2.68 mM) and KH2PO4 (1.47 mM), pH 7.4; Carbonate buffer saline (CBS) contained Na2CO3 (15 mM), NaHCO3 (35 mM), pH 9.6; Washing buffer consisted of PBS containing 0.05% Tween-20; Blocking buffer contained BSA (1%, w/v) in PBS buffer. The stopping solution was 2 M H2SO4.


2.5 Development of ELISA standard curves Indirect and indirect competitive ELISA procedures were performed according to Jiang et al. [13]. The antibody titer was defined as the reciprocal of the dilution that resulted in an absorbance value that was twice that of the background. With the icELISA format, analytes that do not react with the antibody would produce absorbance near 100%; conversely, analytes that do react with the antibody would decrease in percentage of absorbance. All samples were run in triplicates, and competition curves were obtained by plotting absorbance against the logarithm of analyte concentrations, which were fitted to a four-parameter logistic equation. 2.6 Characteristics and optimization of icELISA Sensitivity was evaluated according to the inhibition rate, and the data were calculated using the IC50 values, which represented the concentration of CPFX that produced 50% inhibition of antiserum binding to the hapten conjugate. The detection of limit (LOD) was defined as the lowest concentration that exhibits a signal of 15% inhibition [14]. The dynamic range for the icELISA was calculated as the concentration of the analyte providing a 20–80% inhibition rate (IC20–IC80 values) of the maximum signal. Specificity was defined as the ability of structurally related chemicals to bind to the specific antibody. The cross-reactivity was calculated as: (IC50 of CPFX)/ (IC50 of competitors) ×100. The lower the CR is, the higher the specificity of CPFX pAb is. CPFX stock solution was prepared in PBS containing different concentrations of HCl or methanol. To optimize the schedule, the standards were prepared using 0.01, 0.02, 0.03, 0.04, and 0.05 mM of HCl, or 5%, 10%, 20%, 30%, and 40% of methanol. The effects on icELISA standard curves were evaluated by Amax and IC50 values. 2.7 Matrix effects in authentic cattle milk samples A total of 2 mL of milk samples were centrifuged at 4ć with a speed of 10000 rpm for 30 min, and the floated fat was discarded. In order to reduce the backgrounds and assess residual matrix interference, extracted milk samples were diluted in PBS (total 2, 5, 10 and 20 –fold dilution) before they were applied to the microtiter plate. Typical experimental response curves were plotted by the absorbance values, against the concentration of CPFX, where B is the absorbance of the well containing CPFX and B0 is the absorbance of the well without competitor. Diluted curves were compared with that generated from the PBS buffer to determine the appropriate milk dilution. Under the optimal dilution programme, recovery studies were determined by spiking a pool of negative milk samples with different CPFX concentrations based on the icELISA calibration curve. The concentration measured and concentration fortified was compared to validate the effectiveness of the developed immunoassay. 3.

Results And Discussions

3.1 Immunogen and coating antigen Synthesis CPFX with a molecular weight of 331.4, is too small to be immunogenic, and must be conjugated to a carrier protein before immunization to elicit an immune response. Among protein carriers, BSA and OVA are two of the most preferred ones which were used for synthesizing immunogen and coating antigen respectively. The synthetic pathways for complete antigens and coating antigen are presented in Fig. 1 and 2. It is said that heterology is a proper strategy for the improvement of assay sensitivity in immunoassays [15], which has also been demonstrated in our following experimental data. Heterologous

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system in competitive ELISA is termed to indicate the differences in hapten structure, linker attachment site, or bridge character, which usually results in weaker recognition of antibodies to coating antigen compared to target compound. When designing the antigen, we attempted to exploit the previously described principles. Carbodiimide active ester method were employed in immunogen synthesis while mixed anhydride reaction was used for coating antigen of CPFX-OVA, which pursued the difference in bridge length. O O F

O COOH NH2(SH)-BSA

N

N

EDC

HN

F

N

C-NH-BSA N

HN

Fig. 1. Synthesis procedure for CPFX immunogen through carbodiimide active ester method. O F

N

O COOH

N

F

(CH3)3COOCCl N

HN

COOCOO(CH3)3 N

HN O F

COOCONH-OVA

NH2-OVA N

N

HN

Fig. 2. Synthesis procedure for CPFX coating antigen through the mixed-anhydride technique.

3.2 Establishment of the indirect competitive ELISA Three rabbits were immunized with the CPFX-BSA conjugate following the standard protocols described above, while CPFX-OVA were coated onto ELISA plates to determine the titer and inhibition level of antisera. Checkerboard titrations were performed, taking into account the optimal dilutions. The optimal reagent concentrations were determined when the maximum absorbance (Amax) was around 1.0, and the dose-response curve of inhibition ratio versus the logarithm of CPFX concentration pursued the lowest IC50 values. From the checkerboard assays (data not shown), four representative standard inhibition curves are shown in Fig. 3. Based on the results, a competitive curve was obtained with the icELISA format (Fig. 4). As can be seen, the optimum concentration of coating antigen was 2 ȝg/mL and pAb was 1:5000 dilutions. This assay allowed the detection of CPFX (20-80% inhibition of color development) from 0.036 to 92.5 ng/mL, with an IC50 value of 1.8 ng/mL. The limit of detection (LOD) of the assay was 0.019 ng/mL.

Jiang Jinqing et al. / Physics Procedia 25 (2012) 1829 – 1836 100 A

80

B/B0 (%)

B C

60

D 40 20 0 0.001

0.1

10

1000

100000

Concentration of CPFX (ng/mL)

Fig. 3. Determining optimal parameter of coating antigen and CPFX pAb by indirect competitive ELISA. (A) coating antigen 2 ȝg/mL, pAb 1:5000; (B) coating antigen 2 ȝg/mL, pAb 1:10000; (C) coating antigen 1 ȝg/mL, pAb 1:5000; (D) coating antigen 1ȝg/mL, pAb 1:10000.

Inhibition rate (%)

100 80 60 40 20 0 -3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

Ciprofloxacin [Log(ng/mL)]

Fig. 4. Optimized standard heterologous icELISA inhibition curve for CPFX. Data were obtained by averaging three independent curves, each run in triplicate. CPFX-OVA (2 ȝg/mL) as coating antigen was prepared in CBS (pH 9.6), purified anti-serum produced by CPFX-BSA as immunogen was diluted 1:5000 in PBS (pH 7.4), CPFX was prepared in PBS, containing 10% methanol, GaRIgGHRP was diluted 1:1000 in incubation buffer. The regression curve was y=17.614x+45.369, R2=0.9812.

3.3 Specificity Specificity is inherent to all immunoassays, which was evaluated by determination of the crossreactivity based on the IC50 values. The cross-reactivity rate for each compound is presented in Table 1. Of all the cross-reacting analogues, this assay exhibited a high cross-reactivity (89.7%) to Enrofloxacin, but negligible cross-reactivity to the other compounds tested. It proves that the immunoassay is highly specific for CPFX. Table 1. Cross-reactivities of related functional analogues in the CPFX immunoassay analogues

IC50 ( ng/mL )

Ciprofloxacin

1.8

100

Enrofloxacin Sarafloxacin

2.01

89.7

˚3600

˘0.05

CR (%)

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Jiang Jinqing et al. / Physics Procedia 25 (2012) 1829 – 1836 Ofloxacin

˚3600

˘0.05

Norfloxacin

˚18000

˘0.01

Danofloxacin

˚18000

˘0.01

3.4 Preparation of CPFX stock solution CPFX is difficultly dissolved in water; therefore suitable hydrotropy solvents are needed. HCl or methanol is two commonly used agents for this, which are also applied in this study. The effects of these parameters were estimated by running standard curves under various conditions. The maximum absorbance (Amax, the absorbance value at zero concentration of CPFX) and half-maximum inhibition concentration (IC50, the value represents the concentration of CPFX that produce 50% inhibition of antibody binding to the hapten conjugate) were calculated, and the results are shown in Fig. 5. Based on the results, 0.03 mM HCl or 10% methanol is suitable for this study. 5 IC50 (ng/mL)

Absorbance at 450 nm

1.0

0.8

3 2 1

0.02 0.04 HCl (mM)

0.6

0.4

0.2

0.0 -3 10

HCl (mM) 0.01 0.02 0.03 0.04 0.05

10

0.06

A

-2

10

-1

0

1

10 10 CPFX (ng/mL)

10

IC50 (ng/mL)

1.0


4

0.8

2

10

3

10

4

4 3 2 0

10 20 30 40 50 Methanol (%)

0.6

0.4

0.2

0.0 -3 10

Methanol (%) 5 10 20 30 40

10

-2

10

B

-1

0

1

10 10 10 CPFX (ng/mL)

2

3

10

4

10

10

5

Fig. 5. Effects of HCl in assay buffer (A) and methanol concentrations (B). Each solid symbol represents the mean of three replicates. Insets indicate the fluctuation of IC50 values.

3.5 Matrixl effects It has been known that various substances existing in complex matrixes can affect antigen-antibody interaction. Dietary components, higher ionic strengths and the pH parameters also strongly suppress the IC50 value and the maximum absorbance. A comparison between calibration plots for CPFX prepared in PBS and those prepared in different dilutions of milk gave clear evidence of matrix effect (Fig. 6). It was found that the dilution 1:20 in cattle milk gives the inhibition curve almost the same as that of PBS buffer.



1.0

0.8

0.6

0.4

0.2

0.0 1E-3

0.01

0.1

1 10 CPFX (ng/mL)

100

1000

Fig. 6. CPFX standard curves in the diluted milk samples.Ƶ in the PBS buffer, Ʒ 2-fold dilution, ƹ 5-fold dilution, ƾ 10-fold dilutionƽ 20-fold dilution. Each point represents the average of three separate assays in triplicate.

3.6 Validation of the heterologous icELISA Under the 20-fold dilution, the accuracy of the analysis was studied by the comparative detection of fortified CPFX in milk samples at different concentrations, and the measurement correlations were shown in Fig. 7. We can find that the data spots were nearly distributed on both sides of the trendline, the regression equation for this assay was y = 0.9036 x + 1.4574, with a correlation coefficient (R2) of 0.9844. This indicates that an excellent correlation was found, and the results also suggest the veracity of the icELISA method for detecting CPFX residue in milk. Concentration determined (ng/mL

60 y = 0.9036x + 1.4574

50

2

R = 0.9844

40 30 20 10 10

20

30

40

50

60

Concentration spiked (ng/mL)

Fig. 7. Correlations between concentration spiked and concentration determined in cattle milk samples fortified with CPFX.

Acknowledgment This work was supported by Henan Innovation Project for University Prominent Research Talents (2010HASTIT026).

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