Quantitative determination of dexamethasone in bovine milk by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2ng/g for liver, 0.75ng/g for muscle and kidney tissues, and 0.3ng/ml for milk, while its use as growth-promoter is completely banned. The purpose of this study was to develop and validate a simple and reliable method to determine DXM residues in bovine milk. Milk proteins were removed by the addition of concentrated trichloroacetic acid and paper filtration. Solid-phase extraction clean-up on a C18 reversed phase column was performed to obtain an extract suitable for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation of DXM and the internal standard desoximetasone, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.1% (v/v) acetic acid in water (mobile phase A) and acetonitrile (mobile phase B) as the mobile phases. They were identified using the MS/MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at 0.15, 0.30 and 0.60ng/ml (being half the MRL, the MRL and double the MRL levels fixed by the EC). Calibration graphs were prepared in the 0.15-5ng/ml range and good linearity was achieved (r>or=0.99 and goodness of fit 相似文献   

Monoclonal antibody based enzyme-linked immunosorbent assay for aminoglycoside antibiotic kanamycin in foodstuff

Monoclonal antibodies to aminoglycoside antibiotic kanamycin (KM) were raised as a result of mice complex immunization with glutaraldehyde conjugates BSA with KM, tobramycin (TM) and gentamicin. Using antibodies an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows to determine antibiotic up to 1.2 ng/ml in water solutions, milk and eggs and up to 2.5 ng/ml in honey. The recovery rate from these products spiked with KM was 83, 84 and 96% respectively. The assay of KM based on homologous and heterologous solid-phase conjugates were estimated. The cross-reactivity with TM could vary from 7 to 54%. The same indexes for of amikacin were more constant and reached 7-8%. The other aminoglycosides showed no inhibitory activity.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

An enzyme-linked immunosorbent assay (ELISA) to measure growth hormone level in serum and milk of buffaloes (Bubalus bubalis)

An indirect Sandwich ELISA to measure growth hormone level in serum and milk of buffaloes was developed. The assay was based on purified anti rbST IgG raised in rabbits and chicken and rabbit anti chicken IgG horseradish peroxidase. The assay was validated in terms of sensitivity, specificity, precision and recovery. Parallelism was demonstrated between the standard curve and serially diluted serum, milk and pituitary derived growth hormone. Sensitivity of the assay was 0.1 ng/ml. Recovery of exogenous bovine somatotropin from serum and milk ranged from 90 to 102% and 96 to 108% respectively. The intra and inter assay variations to measure growth hormone in serum and milk were 3.36 to 8.81% and 6.01 to 12.31% respectively. Statistical analysis for parallelism and cross-reactivity of rbST with serum of other species confirmed the reproducibility of the assay.     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

Enzyme immunoassay for the determination of the glycopeptide antibiotic eremomycin

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using rabbit polyclonal antibodies against the eremomycin-glucose oxidase conjugated antigen. This technique allows the glycopeptide antibiotic eremomycin to be determined both in aqueous solutions (with a sensitivity as high as 0.1 ng/ml) and in blood plasma. The cross-reactivity of the antibodies with vancomycin was 0.4% of that for eremomycin, while teicoplanin was almost not recognized. Experiments with blood plasma samples diluted 1: 10 showed that the assay was linear over the concentration range 1–30 ng/ml and that the variation coefficient did not exceed 10%. The high sensitivity and selectivity of this test make it suitable for pharmacokinetic studies and drug monitoring analysis.     

Analysis of Neurotrophin/Receptor Interactions with a gD-Flag-Modified Quantitative Kinase Receptor Activation (gD.KIRA) Enzyme-Linked Immunosorbent Assay

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a “kinase receptor activation” or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC₅₀of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for the aminoglycoside antibiotic kanamycin in foodstuffs

Monoclonal antibodies to the aminoglycoside antibiotic kanamycin (KM) were obtained as a result of the complex immunization of mice with glutaraldehyde conjugates of BSA with KM, tobramycin (TM), and gentamicin. With the use of these antibodies, an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows for the determination of up to 1.2 ng/ml of an antibiotic in water solutions, milk, and eggs, and up to 2.5 ng/ml in honey. The recovery rate in these products spiked with KM was 83, 84, and 96%, respectively. The assays of KM based on homologous and heterologous solid-phase conjugates were estimated. Under these conditions the cross-reactivity with TM could vary from 7 to 54%. The same indexes for amikacin were more constant and reached 7–8%. The other aminoglycosides showed no inhibitory activity.     

Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.     

Milk composition in free-ranging polar bears (Ursus maritimus) as a model for captive rearing milk formula

The goals of this study were to have an improved understanding of milk composition and to help create a suitable milk formula for cubs raised in captivity. Milk samples were evaluated for fat, fatty acids, carbohydrate, vitamin D(3), 25(OH)D(3), vitamin A (retinol), vitamin E (α-tocopherol), protein, and amino acids. Total lipids in milk did not differ for cubs (mean ± SEM = 26.60 ± 1.88 g/100 ml vs. yearlings 27.80 ± 2.20 g/100 ml). Milk lipids were of 23.6% saturated fatty acid for cubs and 22.4% for yearlings. Milk consumed by cubs and yearlings contained 43.8 and 42.0% mono-unsaturated fatty acids and 23.4 and 21.9% polyunsaturated fatty acids, respectively. Carbohydrate content was higher in milk for cubs (4.60 ± 0.64 g/100 ml) than for yearlings (2.60 ± 0.40 g/100 ml). Vitamin D(3) concentration of milk was 18.40 ± 5.00 ng/ml in early lactation compared with 7.60 ± 2.00 ng/ml for mid-lactation. 25(OH)D(3) was lower in milk consumed by cubs (162.00 ± 6.70 pg/ml) than in milk consumed by yearlings (205.00 ± 45.70 pg/ml). Vitamin A concentrations were 0.06 ± 0.01 and 0.03 ± 0.01 μg/ml for cubs and yearlings, respectively. Vitamin E was higher in milk consumed by cubs (20.16 ± 4.46 μg/ml) than by yearlings (7.30 ± 1.50 μg/ml). Protein content did not differ in milk available to cubs (11.40 ± 0.80 g/100 ml compared with milk for yearlings 11.80 ± 0.40 g/100 ml). Taurine was the most abundant free amino acid at 3,165.90 ± 192.90 nmol/ml (0.04% as fed basis).     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

A Long Term Study on the Health Status and Performance of Sows on Different Feed Allowances during Late Pregnancy

The main objective of this study was to determine the total cell content, TCC, and the percentage of polymorphonuclear leucocytes, PMNLs, in colostrum and milk collected from sows during the first 22 days of lactation. The pH-values during the same sampling period were also determined. It should be emphasized that all the values obtained emanate from bacteriologically negative colostrum and milk. The potential influence of different levels of late gestation feeding regimes was also evaluated. The TCC-values obtained from milk samples during the first 3 weeks of lactation and exceeding the designated threshold of 10×106 cells/ml varied between 4% and 21%. Within the TCC-limitation of 10-19.99×l06 cells/ml neither the preceding nor the succeeding cell counts exceeded the threshold in 26.8%. TCC-values above 19.99×l06 cells/ml were preceded and succeeded by cell counts below the threshold in 58.8% and 58.8%, respectively. The TCC-levels below the threshold of 10×106 cells/ml, expressed as geometric least square means, increased significantly from day 1 to day 3 (1.23×l06 cells/ml versus 1.86×106 cells/ml) and decreased thereafter gradually to day 22 (1.38×106 cells/ml). When all values were included, the TCC-values increased in a similar pattern from day 1 to day 3 (1.38×106 cells/ml versus 3.18×106 cells/ml). The value on day 22 of lactation was still on a significantly elevated level compared with that of day 1 (2.10×106 cells/ml versus 1.38×106 cells/ml). The 2 different feeding regimes were not found to influence the TCC-values during the first 22 days of lactation. In the whole material the PMNL-values, expressed as percentages of the TCC, declined from approximately 60% on day 1 of lactation to between 40% and 50% for the remaining sampling period. This decline was comparable with the one seen in the cell class below the threshold of 10×106 cells/ml. In the 2 cell classes above 9.99×106 cells/ml, 78.0% and 88.8% of PMNLs on day 1 declined to about 40% on day 22. This might indicate an inflammatory response on day 1 but without any detectable bacteriological growth. The increase in lactation number, if lactation 1 was compared with the following lactations, revealed a significant rise (p<0.05) in TCC-level and percentage level of PMNLs. A stepwise and significant increase in pH-level occurred between days 1, 3 and 8 (6.18, 6.56, 7.03) followed by a significant decrease to day 22 (6.91) when pH-values from milk of all cell classes were included.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Use of cow-side progesterone tests to improve reproductive performance of high-producing dairy cows

This study evaluated the effectiveness of the cow-side ELISA milk progesterone test in improving postpartum reproductive performance in the Dordt College dairy herd. Cows that produced more than 18,500 lb of milk per lactation were assigned to the high production group (40 cows), while cows that produced less than 18,500 lb of milk (42 cows) were assigned to the low production group. Twenty-one cows in the high production group and 19 cows in the low production group received no ELISA testing (untreated controls), while the remaining cows in each group were evaluated by ELISA test every 7 d beginning on Day 27 post partum (treated cows). A sequence of 2 high progesterone tests and 1 low test indicated the cows were cycling normally. Cows that had low milk progesterone levels (<5 ng/ml) for 3 consecutive tests were assumed to have follicular cysts and were treated with 2 ml GnRH (Cystorelin, 50 mug/ml). Cows that had 3 consecutive high tests (>5 ng/ml) were assumed to have persistent corpora lutea (CL) and were treated with 5 ml PGF(2)alpha (Lutalyse, 5 mg/ml). In both the high and low production groups, treated cows had higher (P < 0.08) pregnancy rates by Day 210 than the untreated controls (63.2 vs 38.1% and 56.5 vs 42.1%, respectively). The days open were reduced (P < 0.05) for the treated animals by 41.6 d compared with the controls. The treated cows produced a net savings of $70.42 (US) per cow assuming a $3.00 savings/day open.     

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.     

Production and characterization of antibodies against microcystins

Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively.     

Production and characterization of antibodies against microcystins.

Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively.     

Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3-63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 microg/l) by several orders of magnitude.     

Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3–63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 μg/l) by several orders of magnitude.