Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%–102% and the limits of detection were in the range of 1.5–6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%–106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.     

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%–113% and the limits of detection were in the range of 0.8–6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%–94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.     

Production of the monoclonal antibody against tylosin for immunoassay of macrolide antibiotics in milk

The objective of the present study was to develop an immunoassay for multi-determination of the residues of four macrolide antibiotics in milk. Tylosin was derivatized with 6-aminohexanoic acid to synthesize a hapten that was used to produce the monoclonal antibody. The obtained monoclonal antibody simultaneously recognized tylosin, acetylisovaleryltylosin, tilmicosin and desmycosin with crossreactivities of 100%, 91%, 49% and 76%, respectively. Then an indirect competitive immunoassay was developed to determine the four analytes in milk simultaneously. The limits of detection for the four analytes were in the range of 5.5–11.7 ng/mL depending on the compound. The recoveries of the four analytes from fortified blank milk were in the range of 78%–96% with coefficients of variation lower than 11.4%. The developed immunoassay could be used as a practical tool for rapid screening the residues of the four anlytes in milk.     

Immunoassay of red dyes based on the monoclonal antibody of β-naphthol

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1–4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%–79% (with β-naphthol as 100%), and the limits of detection were in the range of 1.3–1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%–115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.     

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18–107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody–drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.     

Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%–98%. The limits of detection for the five drugs in feeds were in a range of 0.1–3.0 μg g^?1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5–0.8 ng g^?1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%–96.5% with coefficients of variation of 6.4%–15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.     

Screening of antibiotic residues in fresh milk of Kathmandu Valley,Nepal

The prevalence of two groups of antibiotics; namely penicillin and sulfonamides was studied in fresh milk available in Kathmandu Valley of Nepal. The milk samples (n = 140) were collected from three different sources; individual farmers, cottage dairies and organized dairies of Kathmandu valley. Qualitative and semi-quantitative analysis with rapid screening kits revealed that 23% samples were positive for antibiotic residues in the fresh milk for penicillin and sulfonamide groups (1–256 µg/kg). High performance liquid chromatography (HPLC) analyses detected 81% samples positive for amoxicillin (68–802 µg/kg), 41% for sulfadimethoxine (31–69 µg/kg), 27% for penicillin G (13–353 µg/kg), and 12% for ampicillin (0.5–92 µg/kg). Due to the precision and accuracy of liquid chromatography method, it detected more positive samples and consequently presented higher prevalence than the rapid screening kits. The antibiotic residues were found above the maximum residue limits that presented serious threat to consumer health and raised a serious concern regarding the implementation and monitoring of international regulations in developing countries.     

Analysis of organophosphorus pesticides in whole milk by solid phase microextraction gas chromatography method

Solid phase microextraction (SPME) was used for the extraction of residual coumaphos and dichlorvos in whole milk. The residues were analyzed by capillary gas chromatography equipped with nitrogen phosphorus detector (GC-NPD). A manual SPME holder with a 100-microm polyacrylate fiber was used. The optimized conditions for extraction by SPME method were: sample agitation, absorption temperature of 30 degrees C, absorption time of 40 min, desorption time of 10 min, and sample volume was 16.0 mL in the vial. Under these conditions, the calibration graphs were linear in the range of 0.17 microgL-1 to 1.75 microgL-1 for coumaphos and 0.69 microgL-1 to 6.90 microgL-1 for dichlorvos. Precision was good with RSD values of 13% for coumaphos and 14% for dichlorvos. The detection limits (LOD) were 0.060 microgL-1 for dichlorvos and 0.052 for coumaphos. The quantification limits (LOQ) were 0.086 microgL-1 for dichlorvos and 0.066 microgL-1 for coumaphos. The results obtained in this study suggest that SPME is a suitable technique for residual pesticide analysis of milk. The data demonstrate that particular OP pesticides used in dairy farming in the region of Minas Gerais were found to contaminate cow whole milk, and the residues are not removed by treating the milk by boiling.     

Development of in-house ELISA for detection of chloramphenicol in bovine milk with subsequent confirmatory analysis by LC-MS/MS

This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC₂₀ and IC₅₀, of 0.09 and 0.44 ng mL^?1, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 &; 1.5 ng mL^?1 and extracted in methanol. The mean recoveries were found to be 73–100% with coefficient of variance 7–11%. The decision limit (CCα) and detection capability (CCβ) were calculated as 0.10 and 0.12 ng mL^?1, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL^?1 and CCβ 0.12 ng mL^?1. As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL^?1 with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL^?1 and CCβ 0.10 ng mL^?1. Since CCα and CCβ are less than half of the MRPL (0.15 ng mL^?1), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL^?1, slightly above the MRPL.     

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I?? value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.     

Development of competitive immunoassays to hydroxyl containing fungicide metabolites

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 μg/L and a limit of detection (LOD) of 1.1 μg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 μg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 μg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.     

Development of competitive immunoassays to hydroxyl containing fungicide metabolites

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 μg/L and a limit of detection (LOD) of 1.1 μg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 μg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 μg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.     

Organochlorine compounds in bovine milk from the state of Mato Grosso do Sul – Brazil

Organochlorines are highly hydrophobic, synthetic organic pollutants that accumulate in the environment and in food webs. The primary route of human exposure to organochlorines is through food-mainly fat-rich food of animal origin such as meat, fish, and dairy products. Here we determined the presence and concentration of organochlorine residues in pasteurized milk from Mato Grosso do Sul, Brazil, to monitor consumer exposure to these contaminants. Organochlorine pesticides in milk samples were analyzed using solid phase extraction in octadecyl silica-prepacked columns and identified by gas chromatography using an electron capture detector. Of the 100 composite samples analyzed, more than 90% contained residues of organochlorine pesticides: aldrin was present in 44% of the samples, followed by ∑DDT (36%), mirex (34%), endosulfan (32%), chlordane (17%), dicofol (14%), heptachlor (11%) and dieldrin (11%). Compared to the values established by law, the concentration of the compounds in some samples was above the reference values. Given the importance that milk and its products have in the human diet, it is essential to know whether the levels of pesticide residues are kept well below the recommended levels to minimize the risk to human health.     

Analysis of Organophosphorus Pesticides in Whole Milk by Solid Phase Microextraction Gas Chromatography Method

Solid phase microextraction (SPME) was used for the extraction of residual coumaphos and dichlorvos in whole milk. The residues were analyzed by capillary gas chromatography equipped with nitrogen phosphorus detector (GC-NPD). A manual SPME holder with a 100-μm polyacrylate fiber was used. The optimized conditions for extraction by SPME method were: sample agitation, absorption temperature of 30°C, absorption time of 40 min, desorption time of 10 min, and sample volume was 16.0 mL in the vial. Under these conditions, the calibration graphs were linear in the range of 0.17 μgL^?1 to 1.75 μgL^?1 for coumaphos and 0.69 μgL^?1 to 6.90 μgL^?1 for dichlorvos. Precision was good with RSD values of 13% for coumaphos and 14% for dichlorvos. The detection limits (LOD) were 0.060 μgL^?1 for dichlorvos and 0.052 for coumaphos. The quantification limits (LOQ) were 0.086 μgL^?1 for dichlorvos and 0.066 μgL^?1 for coumaphos. The results obtained in this study suggest that SPME is a suitable technique for residual pesticide analysis of milk. The data demonstrate that particular OP pesticides used in dairy farming in the region of Minas Gerais were found to contaminate cow whole milk, and the residues are not removed by treating the milk by boiling.     

Characteristics of penicillin bacterial residue

IMPLICATIONS: During the production of penicillin, a mass of waste bacterial residue is generated. In the past, the bacterial residues have been used for food additives. Unfortunately, doubts of their suitability as a feedstock have been raised because of the small amount of antibiotics and the degradation products remaining in the bacterial residues. So, penicillin bacterial residue is one of the hazardous wastes. Therefore, penicillin bacterial residue should be managed in accordance with the hazardous waste. To get a right method, the penicillin bacterial residue was characterized.     

Determination of chloramphenicol, enrofloxacin and 29 pesticides residues in bovine milk by liquid chromatography-tandem mass spectrometry

A sensitive method for determination of chloramphenicol, enrofloxacin and 29 pesticides residues in bovine milk by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. Residues of the targets were extracted from milk with acetonitrile, cleaned up by C₁₈-SPE cartridge, and then determined by HPLC-MS/MS. The MS detection was operated in positive or negative ionization mode, depending on the compounds. For confirmation of each target compound, two precursor ion > product ion transitions were selected by multi-reaction monitoring mode (MRM). The method showed good linearity for all the tested compounds over the studied concentration range with correlation coefficient higher than 0.9910. Recoveries for the studied compounds at three spiked levels (0.05, 0.10, 0.19 mg kg⁻¹) in bovine milk were in the range of 71-107% with RSDs not larger than 13.7%, except that recoveries of trifluralin ranged between 62% and 70% at the spiked levels. Limits of quantitation for the analytes were estimated to range between 0.03 × 10⁻³ and 14.5 × 10⁻³ mg kg⁻¹. The proposed method was applied for the determination of the analytes residues in real samples. The found levels of the analytes in milk samples were lower than maximum residues levels (MRL).     

Comparative study of methodologies for the analysis of PCDDs and PDCFs in powdered full-fat milk. PCB, PCDD and PCDF levels in commercial samples from Spain

Feasibility of two different extraction methods involving solid-liquid extraction and Soxhlet extraction, previously used for the determination of lipid contents and PCB levels on powdered full-fat milk, are now examined for simultaneous PCDD/F analysis. The results of this study are consistent with those found for PCBs. The solid-liquid procedure provides the most efficient extraction of both labelled spiked and endogenous PCDD/Fs with the lowest variability. The average recoveries were 101% (R.S.D. = 6.9%) for 13C12-2,3,7,8-TCDD and 95% (R.S.D. = 11%) for 13C12-1,2,3,4,7,8-HxCDD. The R.S.D.s for endogenous 2,3,7,8-PCDD/Fs were in the 9.3-25% range. In addition, this methodology simplified the subsequent clean-up step as it allowed a semi-selective extraction of the apolar lipids from the milk. The proposed method was applied to simultaneous PCB and PCDD/F analyses in different brands of powdered full-fat milks commercially available in Spain. In all cases, very similar PCB and PCDD/F levels were found. Differences among batches from the same manufacturer were even lower. The total i-TEQ average for PCDDs and PCDFs in the 22 powdered full-fat milk samples analysed was 1.87 pg/g fat basis. This value was similar to those found in cow's milk from other European countries.     

Metabolic fate and elimination in milk, urine and bile of deoxynivalenol following administration to lactating sheep

Using a combination of radioisotopic counting and chromatographic detection techniques, the kinetics and metabolic fate of deoxynivalenol (DON) in plasma, urine and bile were studied in lactating sheep, as was the transmission of residues to milk. Following intravenous administration, the plasma clearance of 14C-DON-derived radioactivity was rapid and followed a tri-phasic decay curve comprised of a bi-exponential decrease in DON (rapid distribution phase, t1/2 alpha = 16.2 min; slower elimination phase t1/2 beta = 66.5 min) and the formation and elimination (t1/2 beta = 188.0 min) of its major plasma metabolite, DON-glucuronide conjugate, which accounted for 13% of the plasma radioactivity levels. DON was rapidly cleared from the body by metabolism to 7 possible metabolites, which were excreted essentially in the urine (91%) and to a lesser extent in the bile (6%). Most (67%) of the recovered radioactivity was in the form of the glucuronide conjugates of DON (54%) and the de-epoxide metabolite, DOM-1 (13%). Excretion of unmetabolized DON accounted for 11%. The remaining recovered dose (18%) comprised of minor amounts of DOM-1 (6%), DON-sulfate conjugate (2%) and 3 unidentified radioactive components (10%). Studies on the presence of DON-derived residues in milk indicated that, relative to the dose, only trace amounts were transmitted following either oral or iv administration of the toxin.     

Development,validation of QuEChERS-based method for simultaneous determination of multiclass pesticide residue in milk,and evaluation of the matrix effect

Extraction and quantification of pesticide residue from the milk matrix at or below the established maximum residue limit (MRL) is a challenging task for both analytical chemists and the regulatory institutions to take corrective actions for the human health and safety. The main aim of the study is to develop a simple rapid and less expensive QuEChERS extraction and cleanup method for simultaneous analysis of 41 multiclass pesticide residue in milk by gas chromatography-electron capture detector (GC-ECD), followed by confirmation of the residues with gas chromatography-mass spectrometer (GC-MS). Effect of sorbent type, temperature, spiking concentration, matrix effect (ME), measurement uncertainty (MU), inter- and intra-assay repeatability, reproducibility of recovery, and trueness of the results were investigated to validate the effectiveness of the method. Limit of determination (LOD) and limit of quantitation (LOQ) for all the analytes ranged within 0.001–0.02 and 0.002–0.05 µg mL^?1, respectively. The % recovery of all the pesticides ranged between 91.38 and 117.56% with relative standard deviation (RSD) below 2.79%. The MU for all the analytes was ≤29% of respective LOQs, and except for few pesticides, the ME was largely negative. The method fulfilled all the SANTE guidelines and thus can be extended for routine analysis of multiclass pesticide residue in milk.     

A survey of PCB congeners in U.K. cows'' milk

Samples of unpasteurised bulked milk, taken directly from ten herds of dairy cattle on rural and urban farms in the north west of England on five separate sampling occasions, were analysed for a range of PCB congeners. ΣPCB concentrations (sum of 37 congeners) ranged from 3.4–16.4 ng/g milk fat with a mean ΣPCB concentration of 8.4 ng/g milk fat. The dominating congeners were 118, 153, 138 and 180, which contributed 15%, 20%, 17% and 9% of the ΣPCB load respectively. The chlorine pattern of the congeners which made moderate or major contributions to the ΣPCB concentration were typically substituted at both para positions (4, 4′), while the PCB congeners not detected in the milk had at least one ring that was not 4-substituted. These results indicate the 4,4′ substitution pattern as being the key to PCB persistence in cows. It is estimated that consumption of typical daily intakes of milk with the PCB concentrations measured in this study would contribute 11 % of the average daily ΣPCB intake for individuals in the UK. This contribution would increase to 30% when exposure through the consumption of dairy products prepared from such milk (e.g. cheese, butter) is taken into account. It is estimated that the inclusion of the TEF assigned PCBs would typically increase the TEQ rating of cows' milk by approximately 40% over that attributed to PCDD/Fs alone.