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1.
An indirect competitive immunoassay for the insecticide parathion has been optimized and characterized. This assay is based on a monoclonal antibody (2H9) produced from an immunogen, a bovine thyroglobulin (BTG) conjugate wherein the reduced form of parathion was multiply bound to the carrier protein via diazo bonds. Assay was performed in the parathion-HSA coated (0.25 μg/ml) ELISA format in which antibody was diluted 1:2000. Several physicochemical factors (pH, ionic strength, BSA concentrations and organic solvent) that influence assay performance were studied and optimized. Finally, the assay was applied to the analysis of parathion in spiked vegetable samples. The sensitivity, estimated as the IC50 value, was 360 ng/ml, with a practical working range between 47 and 6000 ng/ml, a limit of detection of 26 ng/ml, and inter-assay and intra-assay variations less than 10%. The average recovery of parathion added to potato, celery and Chinese cabbage were 173 ± 34%, 108 ± 15% and 98 ± 6%, respectively.  相似文献   

2.
A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC50 value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r2 values 0.9909 for chicken liver and 0.9916 for chicken muscle.  相似文献   

3.
We developed an enzyme-linked immunoassay that provides rapid and sensitive detection of gentamicin in swine tissues. Rabbit was immunized with gentamicin-BSA conjugate and antiserum was collected after the fifth immunization. After optimizing the concentration of immunoreagents, competitive indirect ELISA (ciELISA) gave an IC50 value of 0.98 ng/ml, while competitive direct ELISA (cdELISA) exhibited lower IC50 value of 0.92 ng/ml, thus cdELISA was further optimized under various pH values and ionic strengths of assay buffer, different coating methods and incubation time. The optimized ELISA can be completed within 45 min and it showed negligible cross-reactivity with other aminoglycosides. The recoveries of gentamicin from spiked swine tissues at levels of 25–200 μg/kg ranged from 64.7% to 101.2% with CVs of 4.5–12.1%, and the detection limits were 6.2 μg/kg in muscle, 3.6 μg/kg in liver and 2.7 μg/kg in kidney, respectively.  相似文献   

4.
Chicory root (Cichorium intybus L. var. sativum) is an important foodstuff appreciated for its bitter taste, which is caused by sesquiterpene lactones. These compounds represent a quality parameter for monitoring the raw material. Using polyclonal antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify the bitter compound 11β,13-dihydrolactucin in chicory root. Assay linearity ranged from 4.6 to 300 ng/ml, with intra- and inter-assay variations of 4.9% and 7.2%, respectively. An IC50 of 2 ng/ml and a detection limit of 0.16 ng/ml were obtained. No or little cross-reactions with other sesquiterpene lactones occurred. Roots of three different chicory varieties were evaluated for their bitter taste and were investigated by the ELISA. Distinct concentrations of 11β,13-dihydrolactucin ranging from 485 to 1720 mg/kg dry matter were correlated with the bitterness degree (r = 0.9). The ELISA appeared sensitive, selective, accurate and may serve as screening tool in breeding of chicory roots for bitterness.  相似文献   

5.
Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I50 of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.  相似文献   

6.
A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC50) of 0.243 μg mL−1, workable range (IC30–IC70) of 0.050–12.8 μg mL−1 and limit of detection (LOD, IC20) of 0.021 μg mL−1. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320 μg mL−1 (R2 = 0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.  相似文献   

7.
A new heterologous ELISA method for detecting acetylgestagen multi-residues in animal fat was developed. An antibody generated has high cross-reactivity with relative progestogens (up to 50%) and no cross-reactivity with other steroids (<0.1%) in homologous ELISA. Three heterologous immunoassay were developed and one of them improved not only sensitivity but also the class-selectivity compared with the homologous assay against these progestogens. The IC50 for four acetylgestagens, chlormadinone acetate (CMA), 17α-hydroxyprogesterone acetate (HPA), megestrol acetate (MEGA) and medroxyprogesterone acetate (MPA) were, 4.5, 2.5, 2.9 and 1.8 μg/L, respectively. The cross-reactivity for CMA, HPA and MEGA were 40%, 72% and 62% when they were compared with MPA. Progestogens recoveries from spiked swine fat averaged between 61% and 78%. Results obtained from LC/MS/MS method showed the heterologous ELISA method developed was reliable and suitable for rapid screening the four progestogens residues in fat tissues.  相似文献   

8.
The influence of sarafloxacin (SAR) hapten orientation in immunogen on antibody specificity was examined. The spatial orientation of SAR linked through its carboxyl group to the carrier resulted to SAR-selective response (Anal Methods 2016, 8:5843–5850). To provide opposite orientation of SAR in immunogen, it was linked by its secondary amine to (1) succinic anhydride-modified BSA using carbodiimide-mediated conjugation and to (2) unmodified BSA using formaldehyde condensation method. Two versions of indirect competitive enzyme-linked immunosorbent assays (ELISA) based on generated antibodies were developed. A panel of homo- and heterologous conjugates was examined as potential coating antigens. No cross-reactions were registered in ELISA-1 and ELISA-2 except for fluorophenyl-containing fluoroquinolones difloxacin (DIF), SAR, and tosufloxacin as 138, 100 and 25% and 175, 100 and 8%, respectively. Among the recognized analytes, only DIF is authorized in meat products, so the developed tests were selective for DIF determination in bovine and porcine muscles and sera samples. The values of half-maximal inhibition concentration (IC50) for these assay versions were estimated to be 1.2 and 0.25 ng/ml. DIF detection limits (IC10) were, respectively, 0.15 and 0.015 ng/ml that allowed measuring of MRL and ten times lower level of DIF in the animal muscles. The developed ELISAs were also suitable for DIF determination in animal sera in the concentration range 1000–1 ng/ml.  相似文献   

9.
Three ELISA formats, antigen coated, antibody coated and the second antibody coated for the determination of carbofuran were investigated with conjugations including hapten–BSA, hapten–OVA, hapten–HRP and anticarbofuran IgG–HRP. Results showed that the second antibody-coated method of ELISA had a better performance in the establishment of standard curves and detection of carbofuran residue in vegetables samples. The sensitivity for detection, the I50 value was 36.1 ng/ml at a practical working concentration range from 3.44 to 380.1 ng/ml and the limit of detection for carbofuran was 3.44 ng/ml. The average recoveries of determination for carbofuran spiked in cabbage, lettuce, carrot, winter fragrant-flowered garlic, bamboo shoot and green soy bean were 85.24%, 101.8%, 103.6%, 90.52%, 106.9% and 94.08%, respectively. Additional analyses confirmed that the results given by the ELISA method was in agreement with those of the gas chromatography (GC) method.  相似文献   

10.
The purpose of the study was to determine polyphenols, anthocyanins and ascorbic acid in the extracts of black raspberry fruits and wine, along with their anti-oxidant, anti-proliferative and anti-inflammatory activities. Black raspberry fruits without or with seeds crushed were blended in 60% ethanol (FE and FES, respectively) or in water (FW and FWS, respectively). Black raspberry wine without or with seeds crushed (W and WS, respectively) were prepared. Polyphenol content was the highest in the FES (8.25 mg/g fruit). Generally the ethanol extracts with seeds crushed showed higher anti-oxidant activities with the lowest DPPH IC50 (130 μg/ml (freeze-dried extract/reaction solution)) for the FES and the lowest ABTS IC50 (198 μg/ml) for the WS. Cell viabilities were reduced by 13–70% when treated with 100 μg/ml (freeze-dried extract/medium) for HT-29 cells and 1000 μg/ml for LNCaP cells. The FES most actively suppressed nitric oxide production in LPS-stimulated RAW264.7 cells (p < 0.05). Superoxide dismutase and glutathione peroxidase activities treated with the extracts were higher than the control (p < 0.05).  相似文献   

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