Indirect ELISA for detection of goats' milk in ewes' milk and cheese

An indirect ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of goats' milk (1–25%) in ewes' milk and cheese. The assay uses polyclonal antibodies raised in rabbits against goats' caseins (GC). The anti-GC antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized goats' caseins. The anti-GC antibodies were biotinylated and rendered goats' milk specific by mixing them with lyophilized bovine and ovine caseins. The assay was developed in a non-competitive ELISA format and it comprised coating plates with extracts from samples. ExtrAvidin-peroxidase was used to detect the biotinylated anti-GC antibodies bound to goats' caseins immobilized on 96-well plates. The colour developed by the subsequent enzymic conversion of the substrate resulted in discernible differences in optical densities when assaying mixtures of ewes' milk and cheese containing variable amounts of goats' milk.     

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

An indirect enzyme-linked immunosorbent assay was successfully developed for the detection of defined amounts of cows' milk (1-50%) in sheeps' milk and cheese. The assay used polyclonal antibodies raised in rabbits against bovine caseins (BC). The anti-BC antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing BC. The antibodies were biotinylated and rendered cows' milk specific by mixing them with lyophilized ovine and caprine caseins. ExtrAvidin-peroxidase was used to detect the biotinylated anti-BC antibodies bound to BC immobilized on 96-well plates. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of sheeps' milk and cheese containing variable amounts of cows' milk.     

Detection and quantification of goat's cheese in ewe's cheese using a monoclonal antibody and two ELISA formats

A stable hybridoma cell line (B2B) has been produced secreting a monoclonal antibody (MAb) specific for the s α_s2‐casein of goats. The MAb B2B was used in two enzyme‐linked immunosorbent assays (ELISA) formats for the detection and quantification of the presence of goat's cheese in ewe's cheese samples. In the indirect ELISA format the limit of detection was 1–25% (w/w) substitution of ewe's cheese samples by goat's cheese. Afterwards, a competitive indirect ELISA was successfully developed for the detection of 0.5 to 25% (w/w) of goat's cheese in ewe's cheese samples. This competitive indirect ELISA is a very sensitive assay, can be performed in less than 5 h and is not influenced by the ripening process in cheese. © 1999 Society of Chemical Industry     

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.     

酶联免疫吸附法测定小麦中脱氧雪腐镰刀菌烯醇

目的 建立小麦中脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)含量测定的酶联免疫吸附(ELISA)分析方法。方法 选取山东省4地区小麦样品80份, 用不同溶剂对小麦中的DON进行提取后,构建小麦中DON的ELISA检测方法, 根据国家标准对检测结果进行评价。结果 选用84%的乙腈水溶液作为提取溶剂, 其平均回收率可达100.2%, 较为理想。该ELISA方法检测小麦中DON, 线性范围是5~5000 ng/g, 回归方程Y= 0.0419X 0.0222, 相关系数r=0.9868, 样品中DON含量为5.27~3639.38 ng/g , 中位数为79.25 ng/g, 在所检测的80份样品中, 有4份超出了国家规定的上限标准(1000 ng/g), 76份符合国家规定, 合格率为95％。结论 该方法灵敏、快捷, 适用于小麦中脱氧雪腐镰刀菌烯醇的检测。     

Ochratoxin a in pig kidney determined by enzyme-linked immunosorbent assay (elisa)

An indirect, double antibody enzyme-linked immunosorbent assay (ELISA) has been validated for application to pig kidney. The specificity of the anti-ochratoxin A antiserum employed is such that minimal sample preparation is required prior to assay, with consequent beneficial effects on sample throughput. Kidneys (303) obtained in the UK as being unsuitable for human consumption were examined for ochratoxin A content by ELISA. Of these, 191 (63%) contained no detectable toxin (the detection limit was 0.5 ng g^?1). Only eight samples (2.6%) contained more than 5 ng g^?1 ochratoxin A, and only two were above 10 ng g^?1 (at 11.5 and 12.4 ng g^?1). The significance of these findings is discussed.     

酶联免疫吸附法测定植物油中黄曲霉毒素B1

目的对酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)快速检测植物油中黄曲霉毒素B_1进行方法验证,考查植物油中黄曲霉毒素B_1污染情况。方法对酶联免疫吸附法检测植物油中黄曲霉毒素B_1进行了准确性与回收率、重复性、复现性等方法学实验。用验证后的试剂盒检测156份植物油中黄曲霉毒素B_1含量。结果酶联免疫吸附法的回收率在102.8%~113.8%,相对标准偏差为2.0%~6.1%,重复性相对标准偏差为4.3%,复现性相对标准偏差为7.1%和7.6%。156份植物油中黄曲霉毒素B_1检出率为60.90%,其中山茶油的检出率为78.38%,高于平均水平。结论试剂盒检测植物油稳定性较好,定量准确。156份植物油中黄曲霉毒素B_1含量均符合国家标准,花生油中黄曲霉毒素B_1检出浓度最高,其次是玉米油。茶油的加工生产过程可能存在污染黄曲霉毒素B_1的情况,应注意加工过程中黄曲霉毒素B_1的污染。     

Screening survey of deoxynivalenol in beer from the European market by an enzyme-linked immunosorbent assay

Deoxynivalenol (DON) was analysed in 313 beer samples collected from the European retail market using a commercially available immunoassay kit (enzyme-linked immunosorbent assay, ELISA). The incidence rate was about 87%, while most samples (73%) had contamination levels lower than 20 ng ml^-1. The contamination ranged between 4.0 and 56.7 ng ml^-1, with an average of 13.5 ng ml^-1. A statistically significant correlation between alcohol levels and DON contamination was found, as well as a significant difference between bottom, top and spontaneous fermenting beers. Twenty-seven beer samples were compared using a second ELISA kit and a good correlation was obtained between the two kits (r = 0.93). Although when compared with gas chromatography-mass spectrometry the ELISA tended to overestimate the results, a good correlation (r = 0.94) between the two methods was observed. Monitoring of DON in beer is important considering that DON production is dependent on the weather and that it can contribute significantly to the tolerable daily intake of DON, especially for frequent beer consumers.     

酶联免疫分析方法测定动物性食品中西马特罗残留

目的 建立检测西马特罗药物残留的酶联免疫分析方法(enzyme-linked immunosorbent assay,ELISA).方法 对主要影响因素,包括包被原稀释倍数、抗体稀释倍数、竞争时间、标准品稀释液pH等参数进行优化,对样品进行前处理,经提取净化后进行检测,并对ELISA检测结果与仪器方法进行对比.结果 西...     

Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

酶联免疫吸附法快速检测婴儿配方奶粉中乳铁蛋白含量

目的采用乳铁蛋白快速检测试剂盒,通过酶联免疫吸附法定量测定婴儿配方奶粉中的乳铁蛋白含量,并验证该方法的准确性、稳定性和可靠性。方法按照试剂盒操作说明制作标准曲线,选取乳铁蛋白阴性样品进行添加回收实验,验证方法的准确性;选取一个乳铁蛋白阳性样品,重复检测6次,计算结果偏差,验证方法的稳定性;选取12个乳铁蛋白阳性样品,分别采用试剂盒方法和高效液相色谱法进行检测,对比2种方法的检测结果,验证试剂盒方法的可靠性。结果试剂盒检出限为2 mg/100 g;试剂盒检测方法回收率在101.6%~109%;相对标准偏差(relative standard deviation,RSD)为5.34%;试剂盒方法检测结果与标签值的偏差为-6.90%~6.45%,和高效液相色谱法检测结果的偏差为-6.45%~6.9%,说明试剂盒方法具有很好的可靠性。结论乳铁蛋白试剂盒检测方法灵敏度高、操作简单,能快速准确地测定婴儿配方奶粉中的乳铁蛋白含量。     

酶联免疫吸附技术在食品检测分析中的研究进展

酶联免疫吸附技术(ELISA)是酶免疫测定技术中应用最广泛的技术,是将已知的抗原或抗体吸附在固相载体表面,使酶标记的抗原抗体反应在固相表面进行,通过酶与底物产生颜色反应,用洗涤法将液相中的游离成分除去后再进行定量测定。主要分为夹心法、间接法和竞争法。本文简要介绍了几种常用方法及其基本作用机制,详述了ELISA检测技术在食品检测中具体应用及发展历史与现状,对其在食品中农药残留、病原微生物、生物毒素、转基因食品、重金属残留、过敏性残留物及违规添加成分检测等方面的应用情况进行系统总结,并对其发展前景进行了分析。     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.     

酶联免疫法测定肌肉中氟喹诺酮类药物效果探讨

目的探讨酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)快速测定肌肉中氟喹诺酮类药物的效果。方法样品经过前处理之后,在肌肉中添加10、50、100、200和400 ng/g的氟喹诺酮类药物,利用ELISA法在20~25℃下测定。结果氟喹诺酮类药物酶联免疫试剂盒的相关系数r为0.9976,50%抑制浓度(IC50)介于0.306~0.351 ng/g之间;在0~400 ng/g范围内,肌肉中氟喹诺酮类药物的回收率在81.8%~100.3%之间,精密度在0.4%~12.0%之间,最低检测限为4.0 ng/g。结论酶联免疫吸附法检测肌肉中氟喹诺酮类药物的结果相对稳定、可靠,能够满足初检筛选要求,值得在基层检测部门推广使用。     

酶联免疫检测试剂盒检测水产品中孔雀石绿的应用研究

利用酶联免疫试剂盒检测水产品中孔雀石绿残留量,研究了国产试剂盒检测性能,并与高效液相色谱法进行了比较。结果表明：孔雀石绿含量在0．5μg／kg,8μg／kg范围时,试剂盒具有良好线性,最低检出限为0．16μg／kg;试剂盒测得孔雀石绿总量的加标回收率在7l％-120％,批内变异系数介于5．61％-18．6％;检测鳜鱼实际样品时,试剂盒和高效液相色谱法定性判定结果一致,定量结果有所差异,试荆盒测得鳜鱼样品中孔雀石绿残留量为37．75μg／kg,高效液相色谱法测得结果为46．29μg／kg,但准确度偏差范围尚符合国家质量监督检验检疫总局《残留分析质量控制》的要求。综上所述,国产酶联免疫检测试剂盒操作方便快速,灵敏度较高,重现性较好,适用于大量样品的快速检测。     

Free amino acid profile during ripening of ewe's milk cheese from the Croatian island Krk

The aim of this study was to determine the content of free amino acids (FAA) and their ratio in ewe's milk cheese from the island Krk during its ripening. FAA content was determined by reversed phase HPLC (RP‐HPLC) of cheese aqueous/ethanol extracts after FAA were transformed into their 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatives. Their concentration increased during ripening, reaching the value of 5% in cheese dry matter. The dominant FAA were glutamic acid>leucine>valine>aspartic acid>phenylalanine>serine>proline, and higher content of nonessential vs essential FAA was revealed. Krk cheese has, in relation to other cheeses, higher values for glutamic acid/leucine, glutamic acid/phenylalanine, glutamic acid/proline and smaller values for leucine/aspartic acid, valine/aspartic acid, phenylalanine/aspartic acid ratios, while other ratios are comparable to those of other hard ovine cheeses.     

Determination of the glycoalkaloid content of potato tubers by three methods including enzyme-linked immunosorbent assay

Potato tuber samples of diverse genetic backgrounds were analysed independently for total glycoalkaloid content by three different procedures. Two of the methods used were conventional chemical assays, the third an enzyme-linked immunosorbent assay (ELISA). Agreement between all three was extremely good, though the ELISA employed much simpler sample preparation, was technically easier to carry out, and could assay more rapidly large numbers of samples. For these and other reasons, the ELISA will find wider application in future.     

An assessment of commercially available reagents for an enzyme-linked immunosorbent assay (ELISA) of soya protein in meat products

An enzyme-linked immunosorbent assay (ELISA) procedure based on a limited supply of specially prepared experimental immunoreagents and designed to measure levels of soya protein in raw or processed mixed meat products has already been published. This report describes a modified method with commercially available immunoreagents suitable for routine use; its response to a range of commercial soya ingredients has been checked using a particular soya protein isolate (Unisol, Unimills BV) as arbitrary standard. Individual soya components and possible cross-reacting food materials have also been investigated. Both the original and modified methods have been applied to a set of model beefburgers containing known levels of Unisol. There was good agreement between observed and calculated levels in raw and heat-set beefburgers; sterilised samples gave a decreased but linear response. The procedure is recommended for the examination of meat products in non-specialised laboratories.     

Improved species identification of raw meat by double sandwich enzyme-linked immunosorbent assay

An improved enzyme-linked immunosorbent assay (ELISA) has been developed to enable differentiation of unprocessed beef, sheep, horse, kangaroo, pig, camel, buffalo and goat meats to less than 1% level of detection. This double sandwich system utilises species-specific capture antibodies raised in sheep or cattle and coated on to microtitre plates. Antibody coated plates are then used to immunoextract soluble protein from prepared meat samples. Bound meat proteins are detected by the addition of species-specific rabbit antisera followed by staphyloccocal Protein A conjugated to horse-radish peroxidase (HRPO), or by antisera directly conjugated to HRPO. Adoption of a capture antibody provides a number of advantages over previously described ELISA systems. Sample preparation is not critical because colour production is approximately constant between sample dilutions of 1000 and 10 g litre^?1. Increased sensitivity and selectivity allows the bulking of samples for screening purposes. In addition, the assay is faster; testing may be carried out in less than 2 h.     

采用GC-MS和ELISA试剂盒法对比分析白酒中的塑化剂

使用GC-MS和ELISA试剂盒法对白酒中的塑化剂进行测定,考察了GC-MS的精确度及回收率,ELISA试剂盒法的标准曲线,并比较了两种方法的不同前处理对测定结果的影响。结果表明:GC-MS的精密度标准偏差在-6.0%~6.3%,回收率在87%~113%,ELISA试剂盒法线性良好,两种方法对样品的测定结果接近,表明两种方法都可以对白酒中的塑化剂进行定性定量分析。ELISA试剂盒法前处理比GC-MS简便,但有一定的损失,可以进行快速的定性定量分析,如需更准确的测定塑化剂含量,可以配合GC-MS法进行验证检测。