Chapter 6
Immunochemical Detection of Streptomycin in Honey Downloaded by SWINBURNE UNIV OF TECHNOLOGY on May 11, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch006
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Ewald Usleber , Richard Dietrich , Erwin Märtlbauer , and Wolfgang Unglaub 2
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Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, University of Munich, Schellingstrasse 10, 80799 Munich, Germany Staatliches Tierärztliches Untersuchungsamt, Löwenbreite 18/20, 88326 Aulendorf, Germany
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A sensitive method was developed for the detection of streptomycin residues in natural honey. Streptomycin was recovered from honey samples using a solid phase extraction (SPE) procedure, and determined by polyclonal antibody-based competitive direct enzyme immunoassay (EIA). SPE extracts were further purified using monoclonal antibody-based immunoaffinity (IA) chromatography columns, and these SPE/IA extracts were reanalyzed by EIA. The detection limits for streptomycin in honey using SPE and SPE/IA extracts were 32 and 11.5 ng/g, the recoveries at the 50 ng/g level were 100% and 93%, respectively. In a first series of analyses, streptomycin was detected in several honey samples obtained from retail shops in Southern Germany, maximum values found were in the range of 100 ng streptomycin per gram honey. The aminoglycoside antibiotic streptomycin (Figure 1) is intensively used in several countries to control fire blight, a devastating bacterial (Erwinia amylovora) disease affecting apple and pear trees. Recommended streptomycin concentrations for field applications are in the range of 100 /xg/mL (7). High streptomycin contamination levels of the orchard environment imply the possibility of an inadvertent cocontamination chain: pollen, nectar, honey bees, and finally "carry-over" of streptomycin into the honey. This paper is the first report of the detection of streptomycin in honey, and describes a simple and sensitive analytical approach, using a direct competitive EIA (2) to detect streptomycin after sample clean-up on reversed-phase ( C ) extraction cartridges. Further confirmation of the results obtained by this method was achieved employing a selective immunoaffinity chromatography procedure (3) for streptomycin. 18
0097-6156/96/0621-0074$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
6. USLEBER ET AL.
Immunochemical Detection of Streptomycin in Honey
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Materials Honey Sample Materials. Streptomycin-negative samples of natural honey were kindly supplied by F . Neumann, Tieràrztliches Untersuchungsamt Aulendorf, Germany. Commercial honey (n = 18) was purchased from retail stores in Southern Germany, mostly in the Munich area. Solid Phase Extraction (SPE). Throughout the study, Sep-Pak C cartridges (Waters-Millipore, Milford, M A , U.S.A.) were used for SPE. The honey extraction buffer (modified from the method described by Kurosawa et al. (4)) was 1heptanesulfonic acid (50 mM) and tribasic sodium phosphate ( N a P 0 · 12 H 0 ; 25 mM) in distilled water. The pH of this solution was adjusted to 2.0 with orthophosphoric acid. For elution of streptomycin from SPE cartridges, methanol (analytical grade) was used. The SPE extracts were diluted with phosphate buffered saline (PBS; 0.01 M phosphate buffer containing 0.1 M NaCl, p H 7.2-7.3), to give a methanol concentration of 10% for EIA analysis. Further dilutions of this extract were made with PBS containing 10% methanol.
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Immunoaffinity (IA) Chromatography. The IA columns consisted of monoclonal antibodies against streptomycin/dihydrostreptomycin (Strep II 4E2), coupled to CNBr-activated Sepharose 4B (3), with a gel volume of 200 /zL per minicolumn (Mobitec, Gôttingen, Germany). The streptomycin elution buffer for IA was glycine (0.1 M)/HC1 (0.2 M) buffer, pH 2.5. The neutralisation buffer for IA eluates was carbonate-bicarbonate buffer, 0.05 M , pH 9.6. Immunoreagents for E I A . Sheep anti-rabbit immunoglobulin G (Anti-rabbit IgG), purified by immunoaffinity chromatography, was used as described earlier (5). Rabbit antiserum against streptomycin, and streptomycin-horseradish peroxidase (HRP) conjugate were used in direct competitive EIA as reported previously (2). This EIA is specific for streptomycin and dihydrostreptomycin, having relative crossreactivities of 100% and 150%, respectively. Streptomycin sulfate was purchased from Sigma-Aldrich Vertriebs GmbH, Deisenhofen, Germany. Microtiter plates (Maxisorp) were from Nunc GmbH, Wiesbaden, Germany. Methods Sample Preparation. Approximately 1 g of honey was weighted into a 100 mL beaker. The extraction buffer was added to give a tenfold weight, thus 1 mL of buffer corresponded to 0.1 g of honey. The mixture was stirred for 10 min on a magnetic stirrer. Ten mL of this solution was passed through a SPE cartridge which had been conditioned with methanol (20 mL) and distilled water (20 mL). The cartridge was washed with distilled water (2 mL), and then dried by passing air (5 mL) through the cartridge with a syringe. Streptomycin was eluted with methanol (2 mL), the solvent was completely recovered from the cartridge with air (5 mL). The eluate was diluted with PBS (18 mL), and 300 /xL of this mixture was directly used for EIA analysis (SPE extract).
Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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IMMUNOASSAYS FOR RESIDUE ANALYSIS
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The remaining diluted extract (19.7 mL) was further purified by IA C . A n IA minicolumn was rinsed with glycine/HCl buffer (5 mL), followed by PBS (10 mL). Then the extract was passed through the column at a flow rate of 2—3 mL/min. The effluent waste was collected and assayed for presence of unretained streptomycin by EIA. The column was washed with PBS (5 mL), then streptomycin was eluted with glycine/HCl buffer (2 mL). The pH of the eluate was raised to near neutral with 850 μL carbonate-bicarbonate buffer prior to EIA analysis (IA extract). The IA minicolumn was rinsed with 10 mL PBS for immediate reuse, or rinsed with 10 mL of PBS containing 0.05% sodium azide for storage at 4 °C. Competitive Direct Enzyme Immunoassay. Microtiter plates were coated with anti-rabbit IgG (10 jxg/mL carbonate-bicarbonate buffer; 100 ^L/well) overnight in a humid chamber. Free protein binding sites of the plate were blocked with PBS containing sodium caseinate (Sigma-Aldrich; 20 g/L) for 30 min at room temperature, then the plate was washed three times with Tween 20 solution (0.25 m L / L of 0.15 M sodium chloride solution). To each well, 35 μL of streptomycin standard or sample extract solution was added. To analyse SPE extracts, standards were prepared in 10% methanol/PBS, for IA extracts the standards were performed in 100% PBS. Then, antiserum dilution (1:6000 in PBS; 35 /xL/well) and streptomycin-HRP solution (1:5000 in 1% sodium caseinate/PBS; 35 μL/well) were added and incubated for 2 h at room temperature. The plate was washed again, and enzyme substrate/chromogen (6) solution (1 mmol3,3\5,5'-tetramethylbenzidineand 3 mmol H 0 per liter potassium citrate buffer, pH 3.9) was added (100 ^L/well). After 15 min, the color development was stopped with 1 M H S 0 (100 ^L/well) and the absorbance was measured at 450 nm. The tests were evaluated using a competitive EIA calculation software (7). 2
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Evaluation of Test Parameters. A l l standard and sample solutions were analysed in quadruplicate throughout the study. Mean detection limits and 50% inhibition values were calculated from streptomycin standard curves (Figure 2) obtained over a period of 2 months (n=84). Repeated (n=38) analysis of streptomycin-free honey samples, using both SPE and SPE/IA purification, was performed to study sample extract variability and its influence on the EIA response. For SPE extracts as well as for effluent waste of IA columns, two independent dilutions of each honey extract were analyzed, corresponding to final sample dilution factors of 20 and 40, respectively. For IApurified extracts, three independent dilutions were assayed, corresponding to final sample dilution factors of 2.89, 5.78, and 11.56. For SPE extracts (absorbance values 70-120% B/B ), the detection limit was derived from the mean relative absorbance value (B/B xl00) minus three standard deviations obtained for streptomycin-negative sample extracts (Figure 3). For SPE/IA purified extracts (absorbance values 70-100% B / B ) , the detection limit was defined as the mean calculated concentration for these samples (ng/g) plus three standard deviations (Figure 4). To check recoveries obtained by either sample preparation procedure, honey was artificially contaminated with streptomycin at levels of 50 and 100 ng/g. Additionally, on each day of analysis (10-20 samples per day), a spiked honey 0
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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
6. USLEBER ET AL.
Immunochemical Detection of Streptomycin in Honey
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Downloaded by SWINBURNE UNIV OF TECHNOLOGY on May 11, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch006
NH-C-NH
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Figure 1. Structure of streptomycin (R: =0) and dihydrostreptomycin (R: -OH) 100
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Streptomycin (ng/mL) Figure 2. Comparison of typical standard curves of the EIA for the detection of streptomycin, using standards in PBS or in 10% methanol/PBS. The 50% inhibition value (3.5 ng/mL) and the detection limit (1.5 ng/mL) are indicated by arrows. Absolute B values were routinely 0.8-1.0 absorbance units. Intraassay coefficients of variation (n=4) were between 1.5 and 7.5%. 0
Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Downloaded by SWINBURNE UNIV OF TECHNOLOGY on May 11, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch006
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IMMUNOASSAYS FOR RESIDUE ANALYSIS
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Determination # ("blank" samples)
Figure 3. Evaluation of the detection limit of the streptomycin EIA after SPE, using repeated analysis of streptomycin negative honey sample extracts. Blank extracts gave % B / B values ("noise") ranging from 70% to 118%. The mean response was 97.3+9.8 % B / B . Subtracting three standard deviations from the mean value, the limit of detection was calculated to be at 67.9 % B / B , corresponding to a streptomycin concentration of 1.6 ng/mL. Considering a minimum sample dilution factor of 20, the detection limit for streptomycin in honey is 32 ng/g. 0
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Figure 4. Evaluation of the detection limit of the streptomycin EIA after combined SPE/IAC cleanup. A l l blank extracts gave % B / B values
