ELISA determination of Aflatoxin M1 in milk and dairy products in Pakistan

A highly sensitive competitive ELISA was standardized to determine aflatoxin M₁ (AFM₁) in milk and dairy samples randomly collected from Faisalabad city, using Riedel-de-Haen, Aflatoxin M₁ ELISA Systems, (Art–No. 45169). Standards of 5, 10, 25, 50, and 100 ng L^?1 AFM₁ in sample/standard buffer were used in optimizing anti-AFM₁ antibodies and peroxidase–AFM₁ conjugate. Linear standard curve showed increase in concentration (log C) accompanied by decrease in maximal absorbance (%B/B₀) values (%B/B₀ = 124.3 ? 45.0 log C) with highly significant negative correlation (?0.973). The middle of the test (50% B/B₀) was at 30 ng L^?1 with 6.7 ng L^?1 the lowest detection limit (90% B/B₀). Duplicate analysis of 41 samples showed AFM₁ levels below EC permissible limits (50 ng L^?1) with maximums of 40, 13, and 7.4 ng L^?1 in milk, yogurt and butter, respectively. However, possibility of exceeding limits cannot be excluded, and demands regular monitoring.     

单克隆抗体间接竞争ELISA测定水体中氟虫腈

制备了氟虫腈单克隆抗体 ,在此基础上建立了水体中氟虫腈测定的间接竞争性酶联免疫吸附测定 (ELISA)法 .在优化条件下 ,氟虫腈测定的线性浓度范围为 1 0 -1 μg·L-1 ～ 1 0 3μg·L-1 ,最低检出浓度 (I1 0 )为 0 0 8μg·L-1 ,检测灵敏度 (I50 )为6 6 8μg·L-1 ;常见的几种与氟虫腈结构类似的农药的交叉反应率均低于 1 % ,不干扰氟虫腈的检测 ;2 0 %丙酮、5 %甲醇、2 %二甲基甲酰胺、2 %乙腈和 2 %乙酸乙酯基本不影响免疫测定 .利用建立的间接竞争性ELISA方法检测不同水样中氟虫腈 ,结果表明不同水样均可直接进行测定 ,方法的精密度和准确度均符合残留测定的要求     

Determnation of ochratoxin A in grain by monoclonal antibody-based enzyme-linked immunosorbent assay

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农药残留的酶联免疫检测技术研究进展

简要介绍了农药残留的酶联免疫分析的技术原理，关键技术环节，综述了国内外的酶联免疫测定农药残留的研究动态，并展望了此技术的发展及应用前景。     

Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria, which cause lots of accidents and threatens human health. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established and used to detect microcystin-LR (MC-LR) in drinking and surface waters. The concentration of coating antigen was 5 μ/mL, the dilution of monoclonal antibody MC10E7 was 1:3 000, the dilution of enzyme tracer (goat anti-mouse IgG-peroxidase) was 1:3 000, the standard concentration of MC-LR ranged from 0.001 μg/L to 30 μg/L, and o-phenylenediamine was used as substrate. The assay showed high relativity with high performance liquid chromatography (HPLC) with a correlation coefficient of more than 99%. The relative standard deviation was less than 10%, the detection limit was achieved down to 0.01 μg/L and up to 5.1 μg/L. The quantitative detection range was from 0.03 μg/L to 3 μg/L, and the antibody had high specificity for [4-arginine] microcystins. It performed well in spite of the influence of the real samples. Translated from Environmental Science, 2006, 27(6): 1166–1170 [译自: 环境科学]     

Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues

Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5^th booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL^?1 of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC₅₀ value of anti-Gent IgY was 2.69 ng mL^?1 and regression curve equation was y = ?16.27x + 56.97 (R² = 0.95, n = 3), confirming that, the detection limit (LOD, IC₁₀ value) was 0.01 ng mL^?1. Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.     

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.     

Sorptive interactions and binding of δ‐endotoxin protein from bacillus thuringiensis subsp. kurstaki inforest soils

Abstract

The adsorption, desorption and binding of the insecticidal protein from Bacillus thuringiensis subsp. kurstaki (Btk toxin) onto autoclaved sandy and clay loam forest soils were studied at 23°C in a buffer medium (pH 10.2) using the precipitated protein mixture (active + inactive) obtained from a commercial Btk formulation. The active protein in the buffer solution was quantified by ELISA technique. Maximum adsorption of the toxin onto the sandy (301 μg/g) and clay (474 μg/g) loam soils was found to occur after 3 and 4 hours of agitation, respectively. Adsorption of the toxin was higher in the clay loam soil than in sandy loam. Adsorption parameters were calculated using the Freundlich and linear isotherm equations. The K_F and 1/n values for the soils were 1.12 and 1.48 (sandy), and 20.42 and 0.874 (clay), respectively, indicating stronger affinity of the toxin for the clay compared to the sandy loam soil. The linear model showed deviations at higher concentrations, nevertheless using the best fit, K_D and K_OC values were computed for the two soils. For sandy loam, the K_D and K_OC values were 9.38 and 391, respectively; the corresponding values for clay loam were 13.19 and 425, confirming the higher sorption affinity of the toxin for clay loam. The adsorption data did not fit the Langmuir equation because of heterogeneity of the soil surface. Desorption studies showed that more than half of the adsorbed toxic protein remained firmly attached to sandy (162.6 μg/g or 54.5%) and clay (314.0 μg/g or 67.4%) loam soils after six 0.5‐h washes (total 3.0 h wash time). Although the toxin appears to be a non‐leacher, its lateral mobility, soil persistence and biological consequences, including bioavailability of the bound residues, are poorly understood and require further investigation.     

Comparison of in-house-developed ELISA with HPLC techniques for the analysis of atrazine residues

In-house developed ELISA was standardized to monitor atrazine residues in different environmental samples. The standard curve was linear, indicating an increase in log concentration with decrease in absorbance (%B/B₀ = 1.075–0.042 Log C; r = ?0.966). The middle of the test was at 75 ng/L and the lowest detection limit at 4 ng/L. ELISA significantly correlated with the high performance liquid chromatography (HPLC) (r = 0.990). Internal validation showed good accuracy and precision. Maximum atrazine residues were present in Jehlum River water/sediments and maize/sugarcane plant roots. Most of the food samples were found to be contaminated. ELISA required less clean-up steps than HPLC, but showed matrix effect in soil/colored extracts.     

A sensitive enzyme‐linked immunosorbent assay amplified by biotin–streptavidin system for detecting non‐steroidal anti‐inflammatory drug ketoprofen

A sensitive biotin–streptavidin‐amplified enzyme‐linked immunosorbent assay (BA‐ELISA) method was developed for detecting non‐steroidal anti‐inflammatory drug ketoprofen. Compared with traditional ELISA method, the sensitivity of proposed immunoassay was enhanced by the biotin–streptavidin system. Under the optimal condition, the median inhibitory concentration (IC₅₀) was 0.25 ng mL^?1, with minor cross‐reactivity to a number of structural analogs. This developed assay was successfully applied to detect the ketoprofen residues in different fish samples, and good recoveries (72.6–105.5%) were obtained. The results indicated that this immunoassay method could specifically detect trace ketoprofen residues and could be widely used for routine monitoring of food samples.