Elisa Based Monitoring and Analysis of Tetracycline Residues in Cattle Milk in Various Districts of Punjab

Introduction in the literature, mainly due to difficulties related to differences in physico-chemical properties between Antibiotic residues in foods of animal origin are families of compounds [6-12]. However, there are one of the sources of concern among the public and several methods to detect tetracyclines but chromato-medical health professionals [1]. Antibiotics are used graphy including high performance liquid chromato-to treat infections in sick and injured animals and as graphy and liquid chromatography [13, 14] are the prophylactics and growth promoters in lower efficient techniques. This efficiency can be gathered in concentrations than those used for treatment that is a multi-detection methods based on liquid chromatography potentially dangerous practice since it can encourage coupled with tandem mass spectrometry [15-18]. the production of antibiotic resistant strains of bacteria Microbiological and bioassay techniques are still used [2, 3]. Side effects from tetracyclines are not common, for antibiotic qualitative screening purposes [19-23] but of particular note is phototoxicity. Therefore, to mainly because of their low cost and simplicity; protect consumers, regulatory agencies in the European however, they lack sensitivity and specificity. In this Union published several of? cial documents regulating study, competitive enzyme immunoassay technique the control of veterinary drugs in food products from was used to determine tetracycline residues in milk. animal origin. Tolerance levels, as described by This technique has the advantage that it does not European Commission Regulation 470/2009/EC [4], include cleanup and extraction procedure of sample as were set for compounds that can be used for therapeutic is needed in chromatography techniques. In addition, purposes. The MRLs set for the tetracyclines by this technique is time saving as it provides optimal limit European Union is 100 µg/kg. Regulation 37/2010 [5] of detection (LOD) and reproducibility in a complex also lists pharmacologically active substances and their milk matrix in a very short span of time. maximum residue level (MRL) in foodstuffs of animal This study was undertaken with the aim of origin. monitoring of tetracycline residues in milk samples For the simultaneous analysis of compounds of from Punjab by employing ELISA technique, as it is different antibiotic classes in a multi-class residue the most commonly used antibiotic on dairy farms. analysis, only a restricted number of methods are reported Materials and Methods Collection of milk samples: In total 133 individual animal milk samples were collected from 5 districts of Punjab viz. Abstract Background: Tetracycline antibiotics are widely used for prevention and …


Introduction
in the literature, mainly due to difficulties related to differences in physico-chemical properties between Antibiotic residues in foods of animal origin are families of compounds [6][7][8][9][10][11][12].However, there are one of the sources of concern among the public and several methods to detect tetracyclines but chromatomedical health professionals [1].Antibiotics are used graphy including high performance liquid chromatoto treat infections in sick and injured animals and as graphy and liquid chromatography [13,14] are the prophylactics and growth promoters in lower efficient techniques.This efficiency can be gathered in concentrations than those used for treatment that is a multi-detection methods based on liquid chromatography potentially dangerous practice since it can encourage coupled with tandem mass spectrometry [15][16][17][18].the production of antibiotic resistant strains of bacteria Microbiological and bioassay techniques are still used [2,3] include cleanup and extraction procedure of sample as were set for compounds that can be used for therapeutic is needed in chromatography techniques.In addition, purposes.The MRLs set for the tetracyclines by this technique is time saving as it provides optimal limit European Union is 100 µg/kg.Regulation 37/2010 [5] of detection (LOD) and reproducibility in a complex also lists pharmacologically active substances and their milk matrix in a very short span of time.maximum residue level (MRL) in foodstuffs of animal This study was undertaken with the aim of origin.
monitoring of tetracycline residues in milk samples For the simultaneous analysis of compounds of from Punjab by employing ELISA technique, as it is different antibiotic classes in a multi-class residue the most commonly used antibiotic on dairy farms.analysis, only a restricted number of methods are reported

Materials and Methods
Collection of milk samples: In total 133 individual animal milk samples were collected from 5 districts of Punjab viz., Ludhiana, Muktsar, Ferozpur, Amritsar and Patiala.These collected samples were transported to stop the reaction and absorbance was recorded at 450 o o nm within 30 minute.to the laboratory at 4 C and were preserved at -20 C, to The results were calculated by obtaining the O.D. conduct further analysis.These samples were tested and analyzed for tetracycline residues by using values and calculating the percentage (%) absorbance.RIDASCREEN competitive enzyme immunoassay % absorbance = Absorbance standard per sample/ kits (Biopharm AG, Darmstadt, Germany).These milk Absorbance zero standard x 100.samples were initially thawed and then centrifuged at Finally a calibration curve was plotted between 3000 g for 10 min for quanti-tative estimation of the standard concentration and O.D. (Figure -1).tetracycline.The upper cream layer was removed from

Results and Discussion
the centrifuged milk samples and the skimmed milk The objective of this study was to monitor and samples were diluted in a ratio of 1:10 with sample analyze the residual levels of tetracyclines in raw milk dilution buffer (50 µl milk +450 µl buffer) provided in samples from Punjab.A total of 133 cattle milk samples kits.A 50 µl aliquot from this dilution was used per well were obtained from dairies of 5 districts of Punjab of ELISA plate in the test.
state, India in May, 2013 and assessed their Antibiotic assay: RIDASCREEN (Biopharm AG, contamination with tetracycline antibiotic, using the Darmstadt, Germany) tetracycline kit was used for the ELISA method to assess the exact residue levels in the monitoring of the tetracycline residues from milk.This dairy milk in the targeted area.The determined levels kit is a competitive enzyme immunoassay for the of tetracyclines residue in milk samples were then quantitative analysis of tetracycline, minocycline, analyzed to compare the prevalent residue levels with rolitetracycline, chlortetracycline and oxytetracycline recommended maximum permissible residue limits to in milk, honey and meat.All the reagents required for find whether the prevalent residue levels are within the the enzyme immunoassay including standards were prescribed maximum limits in terms of consumer contained in the test kit.Each kit contained sufficient health protection.It was found that out of all the milk materials for 96 measurements.Each test kit contained samples analyzed, 18 samples were found to contain 12 strips with 8 removable wells each, standard tetracycline residues.The concentrations of concentrations (0, 0.15, 0.45, 1.35, 4.05 ppb in aqueous tetracycline residues in cattle milk samples were in the solutions), conjugate (peroxidase conjugated secondary range 16-134.5 ppb (Table-1).Three samples exceeded antibodies), anti-tetracycline antibody, substrate the maximum residue levels (MRLs) for tetracycline (containing urea peroxide), chromogen (containing antibiotic, according to the regulations set by the tetramethylbenzidine), stop solution (1N sulfuric acid) European Union and the Codex Alimentarius and buffer (sample and standard dilution buffer).
Commission.Experimental methodology: The RIDASCREEN competitive enzyme immunoassay kit (Biopharm AG, Darmstadt, Germany) which is commercially available including LOD with 1.5 ppb (µg/L) was used according to the manufacturer's instructions for antibiotic determination in milk samples.A volume of 50 µl of each standard solution or prepared sample was added to each well followed by 50 µl of anti-tetracycline antibody solution.Plate was incubated at room temperature for 1 hr.Thereafter, three times washing was given by adding 250 µl of washing buffer to each well and then 100 µl of enzyme conjugate was added to each well.Plate was again incubated at room temperature for 15  tetracycline were found in these samples.However, the Union [29]. Of

Fig. 1
Fig. 1 Calibration curve for estimation of tetracycline by using ELISA min.Again washing was given three times by adding A study was conducted in which the screening of 250 µl of washing buffer.Then, 50 µl of substrate and locally produced milk samples in Czech Republic 50 µl of chromogen were added to each well and the indicated the presence of tetracyclines in 50.6 % of the plate was incubated in dark for 15 min at room analyzed samples with concentrations under the MRL temperature.Finally, 100 µl of stop solution was added regulation tetracycline concentrations found were below the (EU) No. 37/2010 of 22 December 2009: on pharmacolomaximum residue limits permitted by the European gically active substances and their classi?cation regarding maximum residue limits in foodstuffs of animal origin.

Table - 1
. District wise samples detected positive with their mean, maximum and MRL values parliament and of the council.Of?cial Journal of the 60 milk samples analyzed.The high incidences of European Union., L152 ; 11-22.