重金属汞酶联免疫检测方法的建立

建立了重金属汞离子的间接竞争酶联免疫(IC-ELISA)分析方法。由于汞离子不具有免疫原性,因此以异硫氰酸苄基乙二胺四乙酸(ITCBE)为双功能螯合剂连接汞离子和钥孔血蓝蛋白(KLH)作为免疫原,免疫新西兰大白兔获得特异性抗体。用获得的抗体建立汞的ELISA分析方法。检测限为0.45μg/L,灵敏度为4.10μg/L。特异性实验表明,抗体对镍和铜的交叉反应率分别为10.8%和13.5%,与其他金属均无明显交叉反应。本实验以白芷、胡萝卜和皮皮虾为基质,添加回收实验表明,白芷、胡萝卜、皮皮虾回收率均在70%～120%。使用电感耦合等离子体质谱(ICP-MS)验证方法的准确性,仪器检测结果和建立的ELISA分析方法具有很好的相关性(相关系数为0.988),表明建立的方法具有很好的准确性。     

牛乳铁蛋白的酶联免疫吸附分析(ELISA)方法的建立

用牛乳铁蛋白(Bovine Laetoferrin，BLf)、纯品免疫新西兰白兔制得兔抗BLf的多克隆抗体，琼扩效价最高为l：64。经ELlSA方法检测，该抗体与牛乳中的主要蛋白质：α—乳清蛋白、β—乳球蛋白、血清白蛋白以及酪蛋白之间不存在免疫交叉性，说明抗体特异性良好。以BLf纯品为包被抗原，自制抗体为一抗，辣根过氧化物酶(HRP)标记的羊抗兔IgG为二抗，邻苯二胺(OPD)为底物，建立了BLf的竞争抑制性ELISA方法。包被抗原及抗体的浓度均由方阵稀释实验确定。所建立的ELlSA方法孔间误差1．6％，板间误差4．3％，灵敏度25μg／L，检测范围25～800μg／L。标缝曲线线性方程（Logit）Y=-1.488IgC 3.8116，相关系数R=0.990。     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

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.     

Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of mustard in foods

ABSTRACT:  Undeclared mustard residues in food products could trigger allergic reactions in mustard-allergic consumers. Our objective was to develop and validate a sandwich-type ELISA for the detection of mustard residues in foods. A mixture of yellow, brown, and oriental mustard seeds was used to immunize 3 rabbits and 1 sheep. Two mustard ELISAs were developed by utilizing the reciprocal combination of rabbit and sheep polyclonal antimustard sera as the capture and detector reagents. Binding was visualized by addition of rabbit antisheep or goat antirabbit IgG antibody labeled with alkaline phosphatase and subsequent addition of substrate. The optimized ELISAs have limits of quantification (LOQ) of 1 and 3 ppm (μg of ground, whole mustard seeds/mL) for the sheep capture and rabbit capture formats, respectively. Only rapeseed cross-reacted in the rabbit and sheep capture mustard ELISAs at a level equivalent to 12300 and 16900 ppm of mustard. The mean percent recovery for cooked frankfurters spiked with 0 to 1000 ppm mustard flour was 95.3%± 10.7%. A limited retail survey of 29 foods revealed that, of 15 samples having mustard declared on the ingredient list, 2 baked bean products contained no detectable mustard, possibly owing to a decrease in extractability and detectability of mustard proteins after subjecting to thermal processing. For the remaining 14 samples without mustard declared on the label, 3 samples contained detectable mustard, presumably due to the labeling of mustard as "spice" or inadvertent cross-contamination. This sandwich-type ELISA can serve as a powerful tool for food manufacturers and regulatory agencies to detect and quantify mustard residues in processed foods.     

Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods

The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements.     

Development of a chemiluminescent enzyme‐linked immunosorbent assay for five sulfonamide residues in chicken muscle and pig muscle

BACKGROUND: An enzyme‐linked immunosorbent assay (ELISA) based on polyclonal antibodies with enhanced chemiluminescent (ECL) detection of sulfonamides in food samples has been optimised and characterised. The specificity of the assay was assessed by determining cross‐reactivities with a set of 16 sulfonamides. The aim of this study was to develop a method for determining sulfonamides with high sensitivity. RESULTS: The sensitivity of the developed ECL‐ELISA was higher than that of colorimetric ELISA. The sensitivities of five of the sulfonamides (sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine) ranged from 0.73 to 2.92 µg L^?1, with limits of detection of 0.10–0.43 µg L^?1. The coefficients of variation of intra‐assay and inter‐assay studies carried out over 5 days were mostly less than 10%. Recovery studies of chicken muscle and pig muscle were performed with simple and rapid extraction. Good recoveries (62.1–110.3%) were achieved and the results correlated well with those obtained using high‐performance liquid chromatography analysis. CONCLUSION: This study has provided an effective analytical technique for the rapid and reliable determination of residues of sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine in food samples with high sensitivity. To the authors' knowledge, this is the first report on chemiluminescent ELISA for sulfonamide analysis. Copyright © 2008 Society of Chemical Industry     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

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.     

Development of an enzyme-linked immunosorbent assay for diniconazole in agricultural samples

Diniconazole hapten was synthesized and conjugated to bovine serum albumin (BSA) by the carbodiimide method to produce an immunogen and to ovalbumin (OVA) by mixed anhydride method to produce a coating antigen. Polyclonal antibody against diniconazole was generated by immunizing New Zealand rabbits with the immunogen. Under optimised conditions (20% methanol, 0.4 mol/L Na⁺, pH 7.5), an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for detecting diniconazole. The 50% inhibitory concentration (IC₅₀) was 0.071 ± 0.013 mg/L and the limit of detection (LOD) was 1.28 ± 0.80 μg/L. Triazole fungicide analogues of diniconazole were tested and did not obviously cross-react, except for uniconazole (2.25%). The recoveries obtained after addition of standard diniconazole to agricultural samples, including water, soil, pear, grape, tomato and wheat flour, ranged from 70% to 120%. The ic-ELISA developed could successfully be applied to analysis of diniconazole residues in agriculture samples.     

Development of an enzyme-linked immunoassay for the detection of gentamicin in swine tissues

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.     

Detection of cows' milk in ewes' milk and cheese by a sandwich enzyme-linked immunosorbent assay (ELISA)

A sandwich enzyme-linked immunosorbent assay (ELISA) has been successfully developed for the detection of defined amounts of cows' milk in ewes' milk and cheese. Polyclonal antibodies were raised in goats against bovine caseins (BC). The resultant antibodies were recovered from the crude antiserum by ammonium sulphate precipitation and further purified by immunoatisorption of the cross-reacting antibodies onto columns containing immobilised ovine, caprine and bovine caseins, followed by elution of the bovine caseins specific antibodies (anti-BC) from the column containing the bovine caseins. The anti-BC bound to the wells of a microtitre plate were used to capture the BC from milk and cheese mixtures. Further immunorecognition of the captured proteins was attained with the same specific antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of ewes' milk and cheese containing variable amounts of cows' milk.     

Validation of an enzyme-linked immunosorbent assay for qualitative screening of neomycin in muscle, liver, kidney, eggs and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was used for the qualitative screening analysis of neomycin in food of animal origin (muscle, liver, kidney, eggs and milk) at levels corresponding to the European Union maximum residue limit (MRL) set for this substance. The method validation was performed according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, detection limit (LOD), quantification limit (LOQ), recovery, precision, linearity and ruggedness. LODs ranged from 5.7 microg kg(-1) in kidney to 29.3 microg kg(-1) in milk; LOQs ranged from 11.4 microg kg(-1) in kidney to 59.7 microkg(-1) in eggs. The recoveries from spiked samples at the MRL, half the MRL and double the MRL levels ranged from 65.8% to 122.8%, with a coefficient of variation (CV) between 5.9% and 28.6%. The CCβ value was less than the MRL for all examined matrices. Moderate variations of some critical factors in the sample pretreatment for muscle, milk and eggs were deliberately introduced for ruggedness evaluation and had a slight but not statistically significant effect on method performance. The proposed method is suitable for qualitative screening analysis of neomycin in the above-mentioned food in conformity with current European Union performance requirements.     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15?±?0.02 µg l^?1 and an IC₅₀ value of 1.13?±?0.16 µg l^?1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg^?1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r ²?=?0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

酶联免疫法与乳及乳制品中抗生素残留的检测

对酶联免疫检测技术(ELISA)做了介绍,并对该技术在乳及乳制品抗生素残留检测的应用进行了评述.阐述了该项技术在乳及乳制品抗生素残留检测中的应用前景.     

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC₅₀ values were determined to be 0.20?ng/ml for metronidazole, 4.0?ng/ml for tinidazole, 0.17?ng/ml for dimetridazole and 0.24?ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0–90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.     

Development of a sandwich enzyme-linked immunosorbent assay for the detection of egg residues in processed foodst.

Chicken eggs are used extensively as an excellent source of dietary proteins. These proteins have many functional properties, making them valuable food ingredients. However, eggs are a frequent cause of food hypersensitivity, especially in children. Of major concern to food processors is the inadvertent cross-contact of food products with allergenic residues, which could result in potentially life-threatening reactions in those with a food allergy. The aim of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of undeclared egg residues in foods. Commercially purified ovalbumin (OVA) and dehydrated egg white solids were used as antigens to induce antibodies in rabbits and goats. Reference pasta standards and various food samples were extracted, then clarified by centrifugation. Goat anti-egg white antibodies were used as the capture reagent, nonspecific sites were blocked with gelatin, then standard and sample extracts were added. Rabbit anti-OVA antibodies were used as detector antibodies, followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. Twenty brands of egg-free pasta (two lots each) were analyzed using the ELISA. Fourteen common pasta ingredients were also evaluated for cross-reactivity problems in the method. The detection limit of the assay was 1 ppm spray-dried whole egg. Fifty-five percent (22 samples) of the egg-free pasta samples tested positive for the presence of undeclared egg residues, with values ranging from 1 to >100,000 ppm. Minimal cross-reactivity was encountered in general, but portobello mushrooms and basil caused some minor matrix effects. This sandwich-type ELISA method can be used to detect undeclared egg residues in processed foods and to evaluate industrial clean-up operations.     

A sensitive and selective direct competitive enzyme-linked immunosorbent assay for fast detection of Sudan I in food samples

BACKGROUND: Sudan I, a synthetic azo dye, is considered to be a genotoxic carcinogen and is prohibited in foodstuffs for any purpose at any level worldwide. In this study, a sensitive and specific direct competitive enzyme‐linked immunosorbent assay (dc‐ELISA) for fast detection of Sudan I in food samples was developed for the first time. The monoclonal antibody against Sudan I was used as capture protein, while horseradish peroxidase labeled Sudan I conjugate prepared by the periodate method via ovalbumin (OVA) as a bridge was used as enzyme tracer. RESULTS: The standard curve of dc‐ELISA for Sudan I was constructed in the range 0.1–100 ng mL^?1 and the assay time was within 80 min. Sensitivity was 2.6 ng mL^?1 and the limit of detection was 0.08 ng mL^?1. Cross‐reactivity values of the assay with Sudan II, III and IV were 5.78%, 1.72% and 0.64%; no cross‐reactivity was found with six other edible colorants. The assay was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀. Recoveries of spiked Sudan I in five different samples including chilli powder, tomato sauce, hotpot seasoning and chilli sauce I and II were within 88.4–113.2% and the intra‐assay relative standard deviation was less than 14%. The dc‐ELISA was confirmed by conventional high‐performance liquid chromatography and the correlation coefficient of the two methods was 0.9902. CONCLUSION: The proposed dc‐ELISA method provides an alternative method for sensitive, specific and fast determination of Sudan I in food samples. Copyright © 2011 Society of Chemical Industry     

Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples

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.     

酶联免疫吸附技术及其在食品安全检测中的应用研究进展

本文主要综述了酶联免疫吸附技术原理、分类及其在食品安全检测中的应用,并根据当前研究现状及存在问题进行了研究展望和提出几点发展建议.     

Evaluation of enzyme-linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin in foods

Two ELISA kits were employed to detect staphylococcal enterotoxin A, B, C and D in foods to which enterotoxin had been added or which had been artificially contaminated with enterotoxin-producing strains of Staphylococcus aureus. The sensitivity was satisfactory, and enterotoxins were detected by both ELISA kits in all positive samples. Weak positive reactions with one ELISA kit caused some difficulties in evaluation of samples. Enterotoxins A, B and C detected in small amounts (2-22 ng/ml) in minced fried beef were not detectable after heating at 80 degrees C, whereas enterotoxin D detected in considerably greater amounts (783 ng/ml) was reduced to 0.4% of this amount.