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TECHNICAL REPORTS

Organic Compounds in the Environment

Enzyme-Linked Immunosorbent Assay for Ultratrace Determination of Antibiotics in Aqueous Samples

^a Department of Veterinary Diagnostic Medicine, University of Minnesota, St. Paul, MN 55108
^b Department of Biological Systems Engineering, University of Wisconsin, Madison, WI 53706

^* Corresponding author (kkumar{at}soils.umn.edu).

Received for publication March 20, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Two commercially available enzyme-linked immunosorbent assay (ELISA) kits that are commonly used for tylosin or tetracycline residues in meat and milk were adapted for ultratrace analysis of these antibiotics in surface and ground waters. These two antibiotics are commonly fed to swine, turkeys, and cattle at subtherapeutic doses for growth promotion purposes. Both ELISA techniques were found to be highly sensitive and selective for the respective antibiotics with detection limits of 0.10 and 0.05 µg L^–1 for tylosin and tetracycline, respectively. The recovery of both tylosin and tetracycline from spiked samples of lake waters, runoff samples, soil saturation extracts, and nanopure water was close to 100%. Tetracycline ELISA was highly specific for tetracycline and chlortetracycline but not for other forms of tetracycline (oxytetracycline, demeclocycline, and doxycycline). Analysis of a few liquid swine manure samples by liquid chromatography–mass spectrometry (LC–MS) showed lower concentrations for chlortetracycline as compared with concentrations obtained using ELISA. However, the concentrations of tylosin from ELISA were comparable with that of LC–MS. The lower concentrations of chlortetracycline obtained by LC–MS in manure samples indicate the presence of other similar or transformed compounds that were detected by ELISA but not determined by LC–MS. These results indicate that both ELISA kits can be useful tools for low-cost screening of tylosin, tetracycline, and chlortetracycline in environmental waters. Furthermore, both ELISA procedures are rapid, portable, and easily adaptable for testing of multiple samples simultaneously.

Abbreviations: ELISA, enzyme-linked immunosorbent assay • LC–MS, liquid chromatography–mass spectrometry • OD, optical density

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
SINCE THEIR DISCOVERY, antibiotics have been instrumental in treating infectious diseases that were previously known to kill humans and animals. However, it has now become clear that widespread use of antibiotics is not without problems (Halling-Sørensen et al., 1998; Jørgensen and Halling-Sørensen, 2000). The major concern is the development of antibiotic-resistant microorganisms, which are difficult to treat with existing antibiotics (Ford, 1994; Herron et al., 1997). Increasingly more microorganisms are becoming resistant to multiple antibiotics (Goldburg, 1999).

According to one estimate, more than 22.7 million kg of antibiotics are currently being produced each year in the USA (Environmental Media Services, 2000). Although most of these antibiotics are used for treatment of infections in humans and animals, a significant portion is used as a supplement in animal feed to promote growth of food-producing animals. According to Environmental Media Services (2000), more than 40% of the antibiotics produced in the U.S. are used as feed supplements. The use of antibiotics in animal feed helps increase the animal's ability to absorb feed and reach market weight on time. It may also counteract the effects of crowded living conditions and poor hygiene in intensive animal agriculture systems (Environmental Media Services, 2000).

Antibiotics commonly used as feed additive for animals include chlortetracycline, bacitracin, bambermycins, erythromycin, lincomycin, monensin, oleandomycin, oxytetracycline, penicillin, tylosin, and virginiamycin (Church and Pond, 1982). The antibiotic dose varies from 1 to 100 g Mg^–1 of feed depending upon the type and size of the animal and the type of antibiotic. Most of the antibiotics added to animal feed are excreted in urine or feces. In some cases, as much as 80% of the antibiotic administered orally may pass through the animal unchanged (Levy, 1992).

Once excreted in urine and feces, antibiotics may enter surface and/or ground waters through nonpoint-source pollution from manure-applied lands. Land application of manure is a common practice in many parts of the USA. In the northern tier of the country, manure is applied even during winter over snow. Manure is applied to land because of its value in supplying nutrients to crops as well as a means of disposing unwanted waste. Recently, the United States Geologic Survey (Koplin et al., 2002) reported the presence of several antibiotics in 139 streams across 30 states in the United States. However, the contribution of agricultural runoff as compared with wastewater (from sewage treatment plants) in terms of antibiotics is unclear.

The conventional trace analysis methods for tylosin and tetracycline depend on high performance liquid chromatography (HPLC) or liquid chromatography–mass spectrometry (LC–MS) (Cooper et al., 1998; Loke et al., 2000; Sacher et al., 2001). However, these methods are expensive and time consuming. Recently, enzyme-linked immunosorbent assays (ELISA) have been shown to be useful for analyzing antibiotic residues in milk, meat, fish, eggs, and honey (Lee et al., 2001; De Wasch et al., 2001; Draisci et al., 2001; Mascher et al., 2001). This technique has only recently been introduced into environmental chemistry research for the determination of herbicides in surface and ground water samples (Thurman et al., 1990; Aga and Thurman, 1993). Meyer et al. (2000) developed a radioimmunoassay procedure for quantification of tetracycline antibiotics in wastewaters; however, the concentrations these authors measured using this procedure were substantially less than those measured using LC–MS. The advantages of ELISA compared with HPLC and LC–MS have been recognized by the USEPA (1992) and guidelines for evaluating ELISA kits are being developed.

This study examined the use of ELISA method for measuring the concentration of tylosin- and tetracycline-class antibiotics in water samples. These two antibiotics are routinely fed to animals and are often present in manure (Kumar et al., 2002). The specific objective of this study was to evaluate two commercially available ELISA kits (used to test antibiotic residues in milk, meat, or honey samples) for measuring tylosin and tetracycline concentrations in runoff and percolating waters from manure-applied fields and surface water bodies. We used soil saturation extracts to simulate percolating waters.

	MATERIALS AND METHODS

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Reagents
Pure antibiotic salts of tylosin tartrate, tetracycline hydrochloride, chlortetracycline hydrochloride, oxytetracycline hydrochloride, doxycycline hydrochloride, and demecocycline hydrochloride were purchased from Sigma Chemicals (St Louis, MO). A tylosin one-step ELISA kit was obtained from International Diagnostic Systems Corporation (St. Joseph, MI) and Ridascreen tetracycline kits were obtained from R-Biopharm AG (Darmstadt, Germany).

Enzyme-Linked Immunosorbent Assay Basis
Tylosin
The tylosin test is based on solid phase immunoassay technology. Tylosin-containing standards or water samples are added to microtiter wells coated with high affinity capture antibody to tylosin. The tylosin enzyme conjugate competes with tylosin in the sample for binding sites on the capture antibody. After a wash step, a substrate is added that reacts to any bound enzyme, creating a blue color. Tylosin in samples blocks the binding of enzyme conjugate to the capture antibody, resulting in little or no color development depending on the amount of tylosin in the sample. Results are quantified by measuring optical density (OD) (at 450 nm) of both standards and samples after stopping the reaction with a stop solution in a microplate reader. The OD is inversely proportional to tylosin concentration in the sample.

Tetracycline
The basis of the tetracycline ELISA test is similar to that of tylosin except that it is an antigen–antibody reaction. The microtiter wells coated with tetracycline–protein conjugate are mixed with tetracycline containing standards or water samples. Free tetracycline and immobilized tetracycline compete for the tetracycline antibody binding sites (competitive enzyme immunoassay). Any unbound antibody is then removed in a washing step and enzyme-labeled secondary antibody (which was directed against the anti-tetracycline antibody) is added. After removing unbound enzyme-labeled antibodies with a wash step, enzyme substrate (urea peroxide) and chromogen (tetramethyl-benzidine) are added to the wells and incubated. Bound enzyme conjugate converts the colorless chromogen into a blue product. The addition of the stop reagent leads to a color change from blue to yellow. Results are quantified by measuring OD of both standards and samples at 450 nm in a microplate reader. The OD is inversely proportional to tetracycline concentration in the sample.

Analytical Procedures
Tylosin
Twenty microliters each of six standards ranging from 0 to 20 µg of tylosin L^–1 and a sample diluent as a negative control were added in microtiter wells of the ELISA plate. The samples were diluted with sample diluent buffer (supplied with the kit) in the ratio of 1:1 before adding them to the microtiter wells. One hundred microliters of reconstituted tylosin enzyme was added to each well and incubated on a plate shaker for 10 min at room temperature. The solution in the plate wells was dumped out by inverting and blotting on paper towels. Using a multichannel pipette, 400 µL of wash solution was then added and the wells were inverted immediately and tamped on paper towels three or four times to remove excess moisture. The wash step was repeated three times. If bubbles were present, a clean pipette tip was used to break them. The wells were not allowed to dry and 150 µL of one-piece substrate was added to each well. The substrate was allowed to react at room temperature by placing on a plate shaker for 10 min. A medium blue color developed in the wells with the negative control and samples with no tylosin. The reaction was stopped by adding 150 µL of stop solution (supplied with kit) to each well.

Using a microplate reader and a 450-nm filter, the OD of each well was read. Optical density is inversely related to tylosin concentration. Using a standard curve and appropriate sample dilution factor, the concentration of tylosin in water samples was determined. Analysis of each water sample, standards, and control was replicated three times.

Tetracycline
Fifty microliters each of prepared water samples (1:1 dilution with same buffer used to make standards) and standards were added in microtiter wells of the ELISA plate. Fifty microliters of anti-tetracycline antibody solution was then added to each well. The plates were incubated on a plate shaker for 1 h at room temperature. The solution in the wells was discarded and the microplate was tapped three or four times on blotting paper to ensure complete removal of solution from wells. The wells were filled with 250 µL of washing buffer. The liquid was again poured out and the wash step was repeated three times. One hundred microliters of enzyme conjugate was then added to each well, mixed gently on a plate shaker, and incubated at room temperature for 15 min. The liquid in the wells was discarded and the wash step was repeated three times. Next, 50 µL of substrate and 50 µL of chromogen were added to each well. Samples were mixed on a plate shaker and then incubated again for 15 min. Finally, 100 µL of stop solution was added and the samples were mixed on a plate shaker. The OD was measured at 450 nm. Analysis of each sample was replicated three times.

Verification of Enzyme-Linked Immunosorbent Assay Selectivity
Selectivity is the ability of a test to determine that negative samples are truly negative. The selectivity test was performed on saturation extracts of four different soils, surface water from two different lakes, runoff water from two agricultural fields that had no history of manure application, and a sample of nanopure water. The soils used in this study were: Seelyeville muck (euic, frigid Typic Haplosaprists) from Randall, MN; Hubbard loamy sand (sandy, mixed, frigid Entic Hapludolls) from Becker, MN; Webster clay loam (fine-loamy, mixed, superactive, mesic Typic Endoaquolls) from Lamberton, MN; and Rozetta silt loam (fine-silty, mixed, superactive, mesic Typic Hapludalfs) from Lancaster, WI. All samples except nanopure water were filtered through 0.7-mm glass-fiber filters (GF/F; Whatman, Maidstone, UK) before any testing. In addition to the above samples, a set of samples to be tested for tylosin were spiked with 5 µg L^–1 of tetracycline and another set to be tested for tetracycline was spiked with 5 µg L^–1 of tylosin. These samples were spiked to test the cross-reactivity of these antibiotics. All selectivity tests were performed in triplicate.

Enzyme-Linked Immunosorbent Assay Sensitivity
The same matrices used to test ELISA selectivity were also spiked with three or four different concentrations (above the detection limits) to test the sensitivity of the ELISA kits. These tests were also performed in triplicate.

Liquid Manure Sample Preparation for Enzyme-Linked Immunosorbent Assay and Liquid Chromatography–Mass Spectrometry
Four swine manure samples were obtained from the hog farmers in Minnesota. Twenty milliliters of liquid manure was prepared for extraction by adding 50 µL of 40% H₂SO₄, 30 mL nanopure water, and a 1-g scoop of disodium ethylenediaminetetracetate (Na₂EDTA). The samples were placed on an orbital shaker for 4 h at 100 rpm. After shaking the samples were filtered using 0.7-µm glass-fiber filters. A small aliquot of the filtered sample was further diluted (1:100 to 1:250) with dilution buffer supplied with ELISA kits. The diluted samples were analyzed using the ELISA procedure described earlier. For LC–MS analysis, the rest of the filtered sample was processed using solid-phase extraction (SPE) with 60-mg hydrophilic–lipophilic (HLB) cartridges from Waters (Milford, MA) using the procedure described by Lindsey et al. (2001). The eluted samples from HLB cartridges were then analyzed by LC–MS.

Liquid Chromatography–Mass Spectrometry Analysis
The procedure for the confirmation of the chlortetracycline and tylosin using LC–MS was adapted from a method described by Zhu et al. (2001). Isocratic separation was achieved using a 150- x 4.6-mm C₁₈ column. Ion source and trap conditions were adjusted and programmed to achieve the most stable and intense product ions that provided maximum sensitivity for each compound. The conditions were the following: ESI source voltage = 4.03 kV, vacuum = 1.02 x 10^–5 Torr, lens voltage = 25.73 V, and multiplier voltage = 973.5 V. The LC–MS was programmed to monitor ions from m/z 100 to 1100.

	RESULTS

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Enzyme-Linked Immunosorbent Assay Sensitivity
There was substantial variability (data not reported) in OD (absorbance units) between the negative control (dilution buffer) supplied with the kits and different background matrices (surface water, runoff water, nanopure water, and the soil saturation extracts). This variability reduced to 5 to 7% when dilution buffer was added to various background matrices in 1:1 volume and the OD was expressed as inhibition value (normalized difference in OD of the sample with respect to the negative control):

The data in Table 1 show that the coefficient of variation in OD values for the tylosin and tetracycline ELISA tests varied from 10 to 15 and 6 to 9%, respectively. However, when inhibition was calculated this variation reduced to 7 to 8 and 4% for tylosin and tetracycline, respectively. The 35 blank samples from 6 different matrices had an inhibition value of 7% with a standard deviation of 0.5 for tylosin ELISA and 5% inhibition value with a standard deviation of 0.2 for tetracycline ELISA (Table 1). Taking into consideration the recommendations of the Subcommittee on Analysis (American Chemical Society Committee on Environmental Improvement and Subcommittee on Environmental Analytical Chemistry, 1980), the limits of detection were set at three standard deviations above the blank. In this case the detection limits corresponded to 9% inhibition for tylosin and 6% inhibition for tetracycline. These numbers correspond to detection limits of 0.10 µg L^–1 for tylosin and 0.05 µg L^–1 for tetracycline in water samples. Because of the reduction in variability on dilution buffer addition, all water samples were mixed with dilution buffer (1:1) before analysis.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Relationship between percent inhibition versus (a) log tylosin concentration and (b) log tetracycline concentration.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Liquid chromatography–mass spectrometry (LC–MS) chromatograms of a standard mixture of chlortetracycline (m/z 479, RT = 7.8) and tylosin (m/z 916, RT = 12.9) compared with different swine manure samples.

	DISCUSSION

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The ELISA tests were highly selective for the respective antibiotic. The recoveries of both tylosin and tetracycline in spiked samples at various concentrations and background matrices were close to 100%. These results show that these kits can be adapted to quantify tylosin and tetracycline concentrations in water and manure samples. Furthermore, the tetracycline ELISA test is specific for tetracycline and chlortetracycline only. Samples containing oxytetracycline, doxycycline, and demecocycline cannot be quantified using the tetracycline ELISA kit.

In general, the ELISA values for chlortetracycline in manure samples were higher than values obtained using LC–MS analysis (Table 6). This may be due to the presence of transformed or decay products of chlortetracycline in manure that reacted with ELISA antibody thus resulting in higher concentrations of chlortetracycline as compared with LC–MS. The concentrations of tylosin in manure samples obtained with ELISA and LC–MS were comparable.

	CONCLUSIONS

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The results show that ELISA tests are inexpensive (approximately $5 per sample for tylosin and $15 per sample for tetracycline) and quick monitoring tools for the presence of antibiotics and transformed by-products in various water quality samples. Moreover, these tests require a small sample volume (<100 µL), are field portable, and work at very low but environmentally significant concentrations (>0.10 µg L^–1). These above-reported ELISA procedures can provide a low-cost screening tool for the presence of tylosin, tetracycline, and chlortetracycline in surface and ground waters, runoff samples, and manure. Comparison of ELISA with LC–MS technique showed that although ELISA is a reliable technique to screen a large number of samples, some samples should be run through LC–MS to confirm the presence of a given antibiotic or its transformation product.

	ACKNOWLEDGMENTS

 
This research was supported with funds from the U.S. Geological Survey through the National Institute of Water Resources; USDA Cooperative State Research, Education, and Extension Service through the National Center for Manure and Animal Waste Management; Minnesota Pork Producers Association; and the graduate school of the University of Minnesota. The authors are grateful to Professor Sagar Goyal for his comments on the manuscript. Constructive criticisms from Associate Editor Dr. Jorge S. Domingo and two anonymous reviewers have helped us improve this manuscript.

	REFERENCES

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This Article Abstract Figures Only Full Text (PDF) Free An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (25) Citing Articles via Google Scholar Google Scholar Articles by Kumar, K. Articles by Gupta, S. C. Search for Related Content PubMed PubMed Citation Articles by Kumar, K. Articles by Gupta, S. C. Agricola Articles by Kumar, K. Articles by Gupta, S. C. Related Collections Ground Water Quality Surface Water Quality Pharmaceuticals Organic Compounds