Immunoassays for Residue Analysis - ACS Publications - American

Chapter 37

Analysis of Sulfamethazine in Milk by an Immunosensor Assay Based on Surface Plasmon Resonance

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch037

Åse Sternesjö, Catarina Mellgren, and Lennart Björck Department of Food Science, Swedish University of Agricultural Sciences, P.O. Box 7051, S-750 07 Uppsala, Sweden

A recently developed immunosensor assay was applied in the analysis of sulfamethazine residues in milk from individual cows, herds and car tankers. Previously frozen milk samples were defatted by centrifugation whereas fresh milk required no sample preparations. The average relative standard deviation and the limit of detection of the assay were less than 2% and 1 μg/kg, respectively. The correlation with a conventional HPLC method was found to be high in analysis of incurred samples from a sulfamethazine-treated cow. In addition, the method was used to analyse 330 tanker milk samples, of which one was found to contain sulfamethazine at the low ppb level by both the immunosensor assay and HPLC.

Residues of antibiotics and chemotherapeutics occur in all types of food of animal origin as a consequence of their widespread use in animal husbandry. To comply with the dairy farmers wish for profitability, the veterinarian often resorts to antimicrobial substances to maintain or restore good health of an animal. In many infections the response to therapy is poor, and new, improved antimicrobial products are continuously introduced for treatment of resistant organisms. Meanwhile, public concern over food safety has never been stronger and consumers are increasingly aware of the health consequences of the food they eat. Although most industrial countries already have control systems for antimicrobials in milk, higher standards of quality assessment are being introduced. In the European Community (EC), Commission Regulations No. 675/92, 3093/93 and 3426/93 (1-3) indicate maximum residue levels (MRLs) for a variety of pharmacologically active substances in animal derived foods. The MRLs that concern antibiotic substances in milk are shown in Table I. There is still no common control system for residues in milk in the EC, but an integrated system, where two levels of control can be distinguished, has been discussed. For screening of farm milk samples at milk grading laboratories, microbial 0097-6156/96/0621-0463$15.00/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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inhibitor assays are likely to remain the methods of choice. Positive results may be confirmed to various degrees, and for detection of certain substances more specific methods can be applied. For a number of substances, however, the methods currently in use have too low sensitivities to comply with new regulations. The inhibitor control is therefore insufficient and a complementary control, likely to be performed by national food authorities, will be required. Table I. Maximum Residue Levels (MRLs) of Antibiotic Substances in M i l k


Pharmacologically active substance

MEL llz/kz

Antibiotics Penicillins Benzyl penicillin 4 Ampicillin 4 Amoxicillin 4 Oxacillin 30 Cloxacillin 30 Dicloxacillin 30 Tetracyclines 100 Macrolides Spiramycin 150 Tylosin 50 rnotherapeutics Sulfonamides 100 Diarninopyrirnidine derivate Trimethoprim 50 Other chemotherapeutics Dapson Sum of all substances belonging to the family a

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Status

Final Final Final Final Final Final Temporary until January 1996 Temporary until July 1995 Temporary until July 1995 Temporary until January 1996 Temporary until January 1996 Use of prohibited

In the development of effective, future control strategies, fast screening procedures suitable for automation are of great importance. For many years biosensors have been applied to a range of biological systems. The technology has, however, been slow to penetrate the area of food analysis. Due to recent advances in both biotechnology and electronics, the development of biosensors that enable direct measurement of chemical components has accelerated (5). This paper describes the development of an immunosensor assay for detection of sulfamethazine (SMZ), a sulpha drug used in dairy cows. Classification of Biosensors A biosensor may generally be defined as an analytical device composed of two parts: a biological or biologically-derived element that detects the analyte integrated with a physicochemical element (transducer) which converts this detection event into an electronic signal. The biological component usually consists either of a biocatalyst or a bioreceptor. For biocatalysts, e.g. enzymes, recognition and binding of a substance is followed by a chemical reaction (6). Bioreceptors, e.g. antibodies, differ in that the

In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


37. STERNESJÔ ET AL.

Analysis of Sulfamethazine in Milk

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binding is non-catalytic and essentially irreversible. Based on the mode of signal transduction, almost all biosensors fall into four categories, i.e. optical, mass, electrical and thermal (5). The merits of each system depend on the application, and have to be evaluated in comparison with other techniques currently on the market. Compared with biocatalyst sensors, bioreceptor sensors were slower to find acceptance (6), possibly because of the initial limited availability of receptor molecules and difficulties in monitoring binding events. Today, the detection of substances such as hormones and drugs can be achieved at very low concentrations and the development of suitable monitoring methods for immunosensors has proceeded rapidly. Optical biosensors are receiving considerable attention and are said to hold considerable potential for on-line quality and safety monitoring in the food industry. Surface plasmon resonance (SPR) is the principle for detection in many optical biosensors, such as the BIAcore®, an immunosensor instrument developed by Pharmacia Biosensor AB, Uppsala, Sweden. This paper describes the application of BIA (Biospecific Interaction Analysis) in real-time measurements of SMZ in milk. Surface Plasmon Resonance Detection

At an interface between two transparent media of different refractive index, e.g. glass and water, light coming from the side of the higher refractive index will be partly reflected and partly refracted. Above a specific angle of incidence no light will be refracted and total internal reflection is observed. Although the light is totally reflected, an electromagnetic field component of the light called the evanescent wave will penetrate into the medium with the lower refractive index. If the interface between the two media is coated with a thin metal film and the light is monochromatic and plane polarised, the evanescent wave will interact with free oscillating electrons, plasmons, in the metal film surface. Light energy will thereby be lost to the metal film and the reflected light intensity will decrease. This phenomenon is called surface plasmon resonance (SPR) and occurs only at a sharply defined angle of incidence. The SPR angle is dependent on the refractive index in the close vicinity of the surface and the refractive index is, in turn, a linear function of the mass concentration. When a macromolecule binds to the surface, the mass, and thereby the refractive index on the sensor surface, changes, causing a shift in the SPR angle that can be used for biosensing purposes (7). By continuously monitoring the SPR angle, expressed as resonance units (RU) by the BIAcore software, and plotting the value against time, a sensorgram is obtained. Sensor Surface

The sensor chip consists of a glass substrate on to which a thin gold film has been deposited. The gold film is covered with a carboxymethylated dextran hydrogel covalently attached through a linker layer (8). This dextrangel provides a hydrophilic environment suitable for studies of biospecific interactions and enhances the immobilisation capacity of the surface. There are four independent flow cells on each sensor chip surface, thereby enabling four different measurements to be performed, using different immobilised ligands if desired.


IMMUNOASSAYS FOR RESIDUE ANALYSIS

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Assay Principle To observe biomolecular interactions in the BIAcore instrument, one interaction partner is immobilised on a sensor chip and the other is injected over the sensor surface. The detector response is proportional to the mass of bound analyte, and for macromolecular analytes the response arising from the analyte can be measured directly. For detection of low molecular weight components, e.g. antibiotic substances, a different approach is required. In our assay, SMZ is immobilised to the sensor surface (9). Polyclonal anti-SMZ antibodies are allowed to react with free antigen in the milk sample. The milk sample (35 pL) is then injected over the sensor surface and free antibodies bind to immobilised SMZ and produce a signal which is in inverse proportion to the concentration of free antigen. After each measurement, the surface is regenerated by injection of dilute NaOH and HC1. Detennination of Sulfamethazine in Milk To evaluate the above-described assay, S M Z has been analysed in spiked milk samples and samples from a S M Z treated cow. The method also has been used to analyse different categories of milk samples, i.e. samples from individual cows, herds and tankers, to study variations in response due to variations in milk composition, within In addition, 330 tanker milk samples from the Schleswig-Holstein area in Germany were screened for SMZ residues by the biosensor assay. The precision and limit of detection (LOD) of the assay was determined by analysis of S M Z standards in both raw and defatted milk. Standard curves were constructed by analysis of samples spiked with known concentrations of SMZ. Using the recommended value of 3 standard deviations, the L O D was graphically determined to be 0.3-0.4 ppb (Figure 1). The average relative standard deviation in skim and raw milk were 1.9% and 3.4%, respectively. The relative standard deviations between defatted milk samples from individual cows (n=24), herds (n=39) and tankers (n=40), were 4.4%, 2.4% and 2.2%, respectively. Milk samples from a S M Z treated cow (Selectavet sulfadimidine 33%) were collected up to nine days after the last injection (Institute for Hygiene, Federal Dairy Research Centre, Kiel, Germany). For a validation of the BIA assay, the milk samples also were analysed by liquid chromatography (10). The high correlation between the two methods is illustrated in Figure 2. Because SMZ is no longer registered for use in lactating cows in Sweden, tanker milk samples were randomly collected in Schleswig-Holstein (Institute for Hygiene, Federal Dairy Research Centre, Kiel, Germany), frozen and sent to Uppsala for analysis by the biosensor assay. In total 330 samples were analysed, of which one sample was positive. The sample was returned to Kiel, where H P L C analysis confirmed the presence of very low concentrations of SMZ (0.9-2.1 ppb). Figure 3 shows the typical response for a limited number of analyzed samples. Discussion and Conclusion The aim of the present study was to investigate the applicability of B I A in determination of veterinary drug residues in milk. The developed assay detects SMZ



SULFAMETHAZINE (PPB) Figure 1. Standard curve for SMZ in raw and defatted milk. The response is expressed as percentage of the response of a negative control sample and the error bars indicate three average standard deviations (n=10). A= negative control milk (defatted), B= positive control milk (raw), C= positive control milk (defatted). The arrows indicate the limit of detection (LOD) in raw and defatted milk respectively. Reproduced with permission from reference 9.




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Figure 3. Typical response in the analysis of tanker milk samples from Schleswig-Holstein. Included in the graph are two positive (1.4 ppb SMZ) and two negative (0 ppb SMZ) control milk samples as well as one incurred tanker milk sample. A= negative tanker milk samples, B= incurred tanker milk sample, C= negative control milk samples, D= positive control milk samples.



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IMMUNOASSAYS F O R RESIDUE ANALYSIS

concentrations of less than 1 ppb in raw milk with a high repeatability on the individual cow, herd and tanker milk levels. Frozen and thawed milk samples were centrifuged to remove the milk fat in order to avoid the risk that it would interfere with the assay. The small sample volume required, and the possibility to perform the analysis of fresh milk without any preparative steps, makes the technique most promising for future milk quality control. In addition, the assay has been used to analyze S M Z in other matrixes than milk, i.e. pork and beef. Preliminary results indicate the potential of the assay, once the extraction procedures have been optimised. It is foreseen that assays for other veterinary drug residues can be developed in analogy with the BIA assay presented here. At present, a method for determination of enrofloxacin, a quinolone derivate used for treatment of Gram negative pathogens in mastitis therapy in Sweden, is under evaluation. This assay has approximately the same sensitivity as the SMZ assay, and requires no preparation of the milk sample. There also is great interest in looking at other types of substances, e.g. hormones and viruses. Because the method is based on general principles, provided that specific antibodies or other receptors are available, B I A technology may be applied in determination of a number of foreign substances of interest in food. Acknowledgments The authors would like to thank everyone involved in the project at the Pharmacia Biosensor A B , Uppsala, Sweden, for their support, and Drs. Gertraud Suhren and Philipp Hammer, Institute for Hygiene, Federal Dairy Research Centre, Kiel, Germany, for collaboration and for supplying antibodies. The Swedish Dairies Association is gratefully acknowledged for providing financial support. Literature cited 1. Commission Regulation (EEC) No 675/92. Off. J. Eur. Communities 1992, L73/8 of 19 March 1992. 2. Commission Regulation (EEC) No 3093/92. Off. J. Eur. Communities 1992, L311/18 of 28 October 1992. 3. Commission Regulation (EEC) No 3426/93. Off. J. Eur. Communities 1993, L312/15 of 15 December 1993. 4. Suhren, G.; Hammer, P.; Heeschen, W. Kiel. Milchwirtsch. Forschungsber. 1994, 46, 237-248. 5. Deshpande, S. S.; Rocco, R. M. Food Technol. 1994, 48, 146-150. 6. Brooks, S. L.; Turner, A. P. F. Meas. Control 1987, 20, 37-43. 7. Liedberg, B.; Nylander, C.; Lundström, I. Sens. Actuators 1983, 4, 299-304. 8. Löfås, S.; Johnsson, Β. J. Chem. Soc. Chem. Commun. 1990, 21, 1526-1528. 9. Sternesjö, Å.; Mellgren, C.; Björck, L. Anal. Biochem. 1995, 175-181. 10. Suhren, G.; Heeschen, W. Anal. Chim. Acta 1993, 275, 329-333. RECEIVED October 11, 1995


Immunoassays for Residue Analysis - ACS Publications - American

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