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Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Determination of sulphadimidine in animal feedstuffs by an enzyme‐linked immunoassay a
a
W. J. McCaughey , C. T. Elliott & S. R. H. Crooks
a
a
Veterinary Research Laboratories , Stormont, Belfast, BT4 3SD, UK Published online: 10 Jan 2009.
To cite this article: W. J. McCaughey , C. T. Elliott & S. R. H. Crooks (1990) Determination of sulphadimidine in animal feedstuffs by an enzyme‐linked immunoassay, Food Additives & Contaminants, 7:2, 259-264, DOI: 10.1080/02652039009373890 To link to this article: http://dx.doi.org/10.1080/02652039009373890
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, sublicensing, 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
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 2, 2 5 9 - 2 6 4
Determination of sulphadimidine in animal feedstuffs by an enzyme-linked immunoassay W. J. McCAUGHEY, C. T. ELLIOTT, and S. R. H. CROOKS Veterinary Research Laboratories, Stormont, Belfast BT4 3SD, UK (Received 15 July 1988;, revised December 1988; accepted 26 January 1989)
Downloaded by [ECU Libraries] at 09:13 24 April 2015
An enzymeimmunoassay technique for the determination of sulphadimidine in animal feedstuffs has been developed. The antibody showed limited cross-reactivity with other drugs, including sulphonamides, used as feed additives. Using spiked samples recoveries of 80-88% were obtained. The limit of detection of the assay was 70 ng/g. Keywords: sulphadimidine; animal feedstuffs; enzyme immunoassay
Introduction
The sulphonamides are commonly used in farm animal feedstuffs for prophylactic and therapeutic purposes (Saschenbrecher and Fish 1980). Sulphadimidine (SMT), which is quickly absorbed and relatively slowly excreted (Alexander 1976) is incorporated into pig feed either alone or in combination with other drugs (chlortetracycline, penicillin or tylosin) at rates of 100—160 g/ton (ABPI Compendium 1986). Under some modern management systems these drugs are considered essential to control the level of diseases, for example, atrophie rhinitis, pneumonias or scours. A maximum acceptable tissue residue concentration of 0-lmg/kg for sulphonamides in general has been set by the Veterinary Products Committee in the UK (Anon. 1987). The pharmacokinetics of the drug can be represented satisfactorily by a one-compartment model and a withdrawal period of 10 days has been recommended (Whipple 1977) but its use has resulted in residues in pigmeat (Van Houweling 1981, Bevill 1984, Schwartz 1985, Anon. 1987). Bevill (1984) considered that failure to observe the recommended preslaughter withdrawal interval was a primary cause of the residues. However, he also noted that the violations occurred where there was no evidence of the drug being incorporated in the feed. Several studies have shown that doses considerably less than the therapeutic or prophylactic levels will cause significant tissue residues (Jenny and Lloyd 1974, Bevill 1984). These may occur through contamination of feed during milling (Ashworth et al. 1986). Incorporation in feed may not be necessary as residues have been found in pigs kept in pens containing bedding contaminated with the excreta of pigs previously fed sulphadimidine (Samuelson et al. 1979). A programme of regular testing for sulphadimidine was established in Northern Ireland in 1986 to determine the extent of residues in pigmeat. Following each positive result the farm from which the pigs were supplied was visited to ascertain the cause of the residue. During these investigations a significant proportion of the farmers stated that they were not aware of the drug being in the ration. As a result 0265-203X/90 $3.00 ©1990 Taylor & Francis Ltd.
260
W. J. McCaughey et al.
an enzyme-linked immunoassay, previously developed to test for the drug in pig urine, was adapted to examine feed (to be published). This paper describes the development of the test. Materials and methods
Downloaded by [ECU Libraries] at 09:13 24 April 2015
Reagents Horseradish peroxidase (HRPO) (E.I.A. grade) was purchased from Boehringer, London. All other reagents (E.I.A. or AnalaR grade) were purchased from Sigma. Buffers Assay buffer, pH 7-0, 10 mM acetate containing 0-2% BSA in doubly distilled, de-ionised water; wash buffer, pH 7-2, 0-9% sodium chloride, 0-1% Tween 20; coating buffer, pH 6-0, 1 mM sodium acetate, 0-2% Tween 20; substrate buffer, pH 6-0, 20 mM citric acid, 40 mM disodium hydrogen phosphate, 0*5 mM 3,3'5,5'tetramethylbenzidine, 1 mM hydrogen peroxide. Antiserum A SMT human serum albumen (HSA) conjugate was produced by the method of Fleeker and Lovett (1985). A 55 mg mass of SMT was weighed into a glass vial, dissolved in 4 ml of 0.25 M sulphuric acid and cooled to 4°C. To this was added 19 mg of sodium nitrite in 1 ml of distilled water over a 5 min period. This was left for a further 5 min after which it was added dropwise to 100 mg of HSA dissolved in 4 ml of 1 M sodium carbonate. This mixture was stirred for 4 h at room temperature and then applied to a Sephadex G25 column to remove unreacted SMT. Each rabbit was primed by an initial injection of 2 mg of the derivative homogenised in 1 ml of 25:50 normal saline and complete Freunds' adjuvant. Booster injections were given at 14 day intervals and consisted of 2 mg in 1 ml of carrier in which the complete Freunds' adjuvant was replaced by incomplete Freunds' adjuvant. Test bleeds were taken at the time of each booster injection. The antibody used in this study was obtained after the seventh booster injection. Enzyme conjugate The sulphadimidine-HRPO conjugate was prepared by the carbodiimide method (Chard 1982). Sulphadimidine (5 mg) was dissolved in 200 jwl of pyridine and 200 ^1 of distilled water was added, followed by 10 mg of HRPO dissolved in 1 ml of distilled water. l-(3-Dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride (5 mg) was added and the solution stirred in the dark at 37 °C for 24 h. Unconjugated material was removed by filtration through a Sephadex G25 column and the resulting conjugate was stored at — 20 °C until use. Extraction procedure Extractions were performed by the method of Blanchflower and Rice (1988). Meal (10 g) was weighed into a flask to which 100 ml of an 80% methanohwater mixture were added. The mixture was heated at 65 °C in a water bath for 30 min. It was then shaken in an orbital shaker for 30 min, centrifuged at 1200 g for 10 min and 10 ml of the supernatant were removed, evaporated to dryness and resuspended in 10 ml of assay buffer.
Enzyme immunoassay for sulphadimidine in feedstuff s
261
Downloaded by [ECU Libraries] at 09:13 24 April 2015
Assay procedure The antibody was immobilised in the wells of the microtitration plates (Cel-Cult, Sterilin) by the addition of 100/tl of antibody diluted 1:1000 in coating buffer and incubated for 1 -5 h at 37 °C. Excess antibody was removed by inverting the plate and tapping gently on to tissue paper. Aliquots (25 \iS) of standard or sample were pipetted into plate wells in duplicate followed by 25 \A of assay buffer. Fifty microlitres of sulphadimidine-HRPO were diluted to 1:50000 in assay buffer and added to each well. The plates were incubated at 37 °C for 1-5 hours and then washed 12 times in wash buffer. Freshly prepared substrate (100 ^il) was pipetted into each well and the plates incubated for 12 min at 37 °C. The colour reaction was stopped by the addition of 25 \à of 2-5 M sulphuric acid. The optical density was measured at 450 and 690 nm (Titertek Multiskan MCC, Titersoft Program, Flow Laboratories). A standard curve was prepared and the unknowns and controls were assessed by interpolation. Results Figure 1 shows a typical standard curve for sulphadimidine determination with the presence of feed extract in the assay. There are no significant variations between this curve and that obtained using a buffer extract. The working range of the assay is 0-1-5*0 ng/well and the mid-point of the curve is 2-5 ng/well (640 ng/g meal). The specificity of the antibody used in the assay is shown in table 1. The cross-reactivities of the antibody were judged by preparing dose response curves to a
1.5 r
CO
w Ü
o
0.0
0
1 2 3 SULPHADIMIDINE CONCENTRATION (LOG n g / m l )
Figure 1. Standard curve for sulphadimidine with meal extract.
262
W. J. McCaughey et al.
wide range of sulphonamides and common veterinary antibiotics and applying these to the described ELISA test. The CR50% value (ng of SMT required to reduce colour production by 50%/ng of compound required to reduce colour production by 50% XI00) was used to evaluate the antibody specificity. For the sulphonamides tested, significant cross-reactivity was limited to sulphamerazine. There was no significant cross-reaction caused by other antibiotics commonly included in pig feed. The intra- and inter-assay coefficients of variation (tables 2 and 3) based on samples spiked with 0, 250, 500, 1000, 2500 and 5000 mg ranged from 9-2-15-0% (n= 14) and 11-7-15-0% (n = 6), respectively. The recovery of added sulphadimidine ranged from 80-8 to 87-6% when spikes were used. Analysis of known 14 sulphonamide-free meals gave values of 9 • 6 ± 13-7 ng/g (mean ± s.d.) from which the limit of detection was calculated at 50 ng/g. Downloaded by [ECU Libraries] at 09:13 24 April 2015
Table 1. The cross-reactivity of the antiserum to other sulphonamide and commonly used feed antibiotics. Cross-reactivity (CR 50%)
Compound
100 10 0-9 0-5 0-3 0-1
Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Determination of sulphadimidine in animal feedstuffs by an enzyme‐linked immunoassay a
a
W. J. McCaughey , C. T. Elliott & S. R. H. Crooks
a
a
Veterinary Research Laboratories , Stormont, Belfast, BT4 3SD, UK Published online: 10 Jan 2009.
To cite this article: W. J. McCaughey , C. T. Elliott & S. R. H. Crooks (1990) Determination of sulphadimidine in animal feedstuffs by an enzyme‐linked immunoassay, Food Additives & Contaminants, 7:2, 259-264, DOI: 10.1080/02652039009373890 To link to this article: http://dx.doi.org/10.1080/02652039009373890
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, sublicensing, 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
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 2, 2 5 9 - 2 6 4
Determination of sulphadimidine in animal feedstuffs by an enzyme-linked immunoassay W. J. McCAUGHEY, C. T. ELLIOTT, and S. R. H. CROOKS Veterinary Research Laboratories, Stormont, Belfast BT4 3SD, UK (Received 15 July 1988;, revised December 1988; accepted 26 January 1989)
Downloaded by [ECU Libraries] at 09:13 24 April 2015
An enzymeimmunoassay technique for the determination of sulphadimidine in animal feedstuffs has been developed. The antibody showed limited cross-reactivity with other drugs, including sulphonamides, used as feed additives. Using spiked samples recoveries of 80-88% were obtained. The limit of detection of the assay was 70 ng/g. Keywords: sulphadimidine; animal feedstuffs; enzyme immunoassay
Introduction
The sulphonamides are commonly used in farm animal feedstuffs for prophylactic and therapeutic purposes (Saschenbrecher and Fish 1980). Sulphadimidine (SMT), which is quickly absorbed and relatively slowly excreted (Alexander 1976) is incorporated into pig feed either alone or in combination with other drugs (chlortetracycline, penicillin or tylosin) at rates of 100—160 g/ton (ABPI Compendium 1986). Under some modern management systems these drugs are considered essential to control the level of diseases, for example, atrophie rhinitis, pneumonias or scours. A maximum acceptable tissue residue concentration of 0-lmg/kg for sulphonamides in general has been set by the Veterinary Products Committee in the UK (Anon. 1987). The pharmacokinetics of the drug can be represented satisfactorily by a one-compartment model and a withdrawal period of 10 days has been recommended (Whipple 1977) but its use has resulted in residues in pigmeat (Van Houweling 1981, Bevill 1984, Schwartz 1985, Anon. 1987). Bevill (1984) considered that failure to observe the recommended preslaughter withdrawal interval was a primary cause of the residues. However, he also noted that the violations occurred where there was no evidence of the drug being incorporated in the feed. Several studies have shown that doses considerably less than the therapeutic or prophylactic levels will cause significant tissue residues (Jenny and Lloyd 1974, Bevill 1984). These may occur through contamination of feed during milling (Ashworth et al. 1986). Incorporation in feed may not be necessary as residues have been found in pigs kept in pens containing bedding contaminated with the excreta of pigs previously fed sulphadimidine (Samuelson et al. 1979). A programme of regular testing for sulphadimidine was established in Northern Ireland in 1986 to determine the extent of residues in pigmeat. Following each positive result the farm from which the pigs were supplied was visited to ascertain the cause of the residue. During these investigations a significant proportion of the farmers stated that they were not aware of the drug being in the ration. As a result 0265-203X/90 $3.00 ©1990 Taylor & Francis Ltd.
260
W. J. McCaughey et al.
an enzyme-linked immunoassay, previously developed to test for the drug in pig urine, was adapted to examine feed (to be published). This paper describes the development of the test. Materials and methods
Downloaded by [ECU Libraries] at 09:13 24 April 2015
Reagents Horseradish peroxidase (HRPO) (E.I.A. grade) was purchased from Boehringer, London. All other reagents (E.I.A. or AnalaR grade) were purchased from Sigma. Buffers Assay buffer, pH 7-0, 10 mM acetate containing 0-2% BSA in doubly distilled, de-ionised water; wash buffer, pH 7-2, 0-9% sodium chloride, 0-1% Tween 20; coating buffer, pH 6-0, 1 mM sodium acetate, 0-2% Tween 20; substrate buffer, pH 6-0, 20 mM citric acid, 40 mM disodium hydrogen phosphate, 0*5 mM 3,3'5,5'tetramethylbenzidine, 1 mM hydrogen peroxide. Antiserum A SMT human serum albumen (HSA) conjugate was produced by the method of Fleeker and Lovett (1985). A 55 mg mass of SMT was weighed into a glass vial, dissolved in 4 ml of 0.25 M sulphuric acid and cooled to 4°C. To this was added 19 mg of sodium nitrite in 1 ml of distilled water over a 5 min period. This was left for a further 5 min after which it was added dropwise to 100 mg of HSA dissolved in 4 ml of 1 M sodium carbonate. This mixture was stirred for 4 h at room temperature and then applied to a Sephadex G25 column to remove unreacted SMT. Each rabbit was primed by an initial injection of 2 mg of the derivative homogenised in 1 ml of 25:50 normal saline and complete Freunds' adjuvant. Booster injections were given at 14 day intervals and consisted of 2 mg in 1 ml of carrier in which the complete Freunds' adjuvant was replaced by incomplete Freunds' adjuvant. Test bleeds were taken at the time of each booster injection. The antibody used in this study was obtained after the seventh booster injection. Enzyme conjugate The sulphadimidine-HRPO conjugate was prepared by the carbodiimide method (Chard 1982). Sulphadimidine (5 mg) was dissolved in 200 jwl of pyridine and 200 ^1 of distilled water was added, followed by 10 mg of HRPO dissolved in 1 ml of distilled water. l-(3-Dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride (5 mg) was added and the solution stirred in the dark at 37 °C for 24 h. Unconjugated material was removed by filtration through a Sephadex G25 column and the resulting conjugate was stored at — 20 °C until use. Extraction procedure Extractions were performed by the method of Blanchflower and Rice (1988). Meal (10 g) was weighed into a flask to which 100 ml of an 80% methanohwater mixture were added. The mixture was heated at 65 °C in a water bath for 30 min. It was then shaken in an orbital shaker for 30 min, centrifuged at 1200 g for 10 min and 10 ml of the supernatant were removed, evaporated to dryness and resuspended in 10 ml of assay buffer.
Enzyme immunoassay for sulphadimidine in feedstuff s
261
Downloaded by [ECU Libraries] at 09:13 24 April 2015
Assay procedure The antibody was immobilised in the wells of the microtitration plates (Cel-Cult, Sterilin) by the addition of 100/tl of antibody diluted 1:1000 in coating buffer and incubated for 1 -5 h at 37 °C. Excess antibody was removed by inverting the plate and tapping gently on to tissue paper. Aliquots (25 \iS) of standard or sample were pipetted into plate wells in duplicate followed by 25 \A of assay buffer. Fifty microlitres of sulphadimidine-HRPO were diluted to 1:50000 in assay buffer and added to each well. The plates were incubated at 37 °C for 1-5 hours and then washed 12 times in wash buffer. Freshly prepared substrate (100 ^il) was pipetted into each well and the plates incubated for 12 min at 37 °C. The colour reaction was stopped by the addition of 25 \à of 2-5 M sulphuric acid. The optical density was measured at 450 and 690 nm (Titertek Multiskan MCC, Titersoft Program, Flow Laboratories). A standard curve was prepared and the unknowns and controls were assessed by interpolation. Results Figure 1 shows a typical standard curve for sulphadimidine determination with the presence of feed extract in the assay. There are no significant variations between this curve and that obtained using a buffer extract. The working range of the assay is 0-1-5*0 ng/well and the mid-point of the curve is 2-5 ng/well (640 ng/g meal). The specificity of the antibody used in the assay is shown in table 1. The cross-reactivities of the antibody were judged by preparing dose response curves to a
1.5 r
CO
w Ü
o
0.0
0
1 2 3 SULPHADIMIDINE CONCENTRATION (LOG n g / m l )
Figure 1. Standard curve for sulphadimidine with meal extract.
262
W. J. McCaughey et al.
wide range of sulphonamides and common veterinary antibiotics and applying these to the described ELISA test. The CR50% value (ng of SMT required to reduce colour production by 50%/ng of compound required to reduce colour production by 50% XI00) was used to evaluate the antibody specificity. For the sulphonamides tested, significant cross-reactivity was limited to sulphamerazine. There was no significant cross-reaction caused by other antibiotics commonly included in pig feed. The intra- and inter-assay coefficients of variation (tables 2 and 3) based on samples spiked with 0, 250, 500, 1000, 2500 and 5000 mg ranged from 9-2-15-0% (n= 14) and 11-7-15-0% (n = 6), respectively. The recovery of added sulphadimidine ranged from 80-8 to 87-6% when spikes were used. Analysis of known 14 sulphonamide-free meals gave values of 9 • 6 ± 13-7 ng/g (mean ± s.d.) from which the limit of detection was calculated at 50 ng/g. Downloaded by [ECU Libraries] at 09:13 24 April 2015
Table 1. The cross-reactivity of the antiserum to other sulphonamide and commonly used feed antibiotics. Cross-reactivity (CR 50%)
Compound
100 10 0-9 0-5 0-3 0-1
