Monoclonal antibodies against troponin I for the detection of rendered muscle tissues in animal feedstuffs

Regulatory controls to prevent the spread of BSE have prohibited the use of certain animal proteins in feed in several countries. Accurate analytical methods for detecting prohibited material in feedstuffs are needed to ensure compliance with the new regulations. Six IgG class monoclonal antibodies (MAbs) against troponin I (TnI), a thermostable marker protein, have been developed for the detection and differentiation of rendered muscle tissue in animal feed. MAbs 1F9, 2G3 and 7F7 reacted to TnI of all species, including mammalian, poultry and fish, while MAbs 7A12 and 8A12 recognized only mammalian TnI (porcine, bovine, ovine, equine, and deer). MAb 2A8 was able to differentiate TnI of ruminant origin (bovine, ovine and deer) from other species. Three indirect enzyme-linked immunosorbent assays (ELISAs) employing these MAbs were developed for the determination of animal muscle, mammalian muscle or ruminant muscle in animal feeds.     

Determination of processed animal proteins in feed: The performance characteristics of classical microscopy and immunoassay methods

Species-specific detection and detection of groups of species such as ruminants is required according to European legislation dealing with the safe use of animal by-products in animal nutrition. Various methods are applied to the analysis of feed samples for the presence of banned processed animal proteins (PAPs) including meat and bone meal (MBM). Classical microscopy as described in the Commission Directive EC/2003/126 is the only official method to detect the presence of constituents of animal origin in feed, nevertheless some deviating protocols allowed under the old Directive (EC/88/1988) claim to gain comparable results. An inherent limitation of the microscopic method is the lack of species specificity. Immunoassays showed the most promising potential in research projects or intercomparison studies being able to detect ruminant PAPs at a concentration level of 0.5%. The aim of this paper is to present the results of the intercomparison study conducted on behalf of European Commission's Directorate General for Health and Consumer Protection (SANCO) in 2004 to establish whether the two-solvent method would gain comparable results to the current European Method and to evaluate the current capability of immunoassays of determining the species in PAPs present in feed.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

New approach for the quantification of processed animal proteins in feed using light microscopy

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.     

Immunological detection of osteocalcin in meat and bone meal: a novel heat stable marker for the investigation of illegal feed adulteration

A sandwich ELISA was developed for the detection of bovine meat and bone meal (BMBM) in feed, based on polyclonal rabbit antibodies raised against the synthetic N-terminal amino acid sequence 1–9 (YLDHWLGAP) of bovine osteocalcin. To set up a sandwich ELISA pair, a commercial mouse monoclonal capture antibody binding to a highly conserved epitope in the mid-fragment of the peptide was employed. It is shown that the bone marker osteocalcin is immunologically well detectable in BMBM extracts obtained by a simple EDTA-based procedure even in a sample heated up to 145°C. Furthermore, a genus-specific restriction of the major specificity to cattle and horse was possible. The observed bi-specificity is consistent with theoretical predictions. The assay sensitivity with bovine osteocalcin of 1?ng was sufficient to enable the detection of 0.1% BMBM in compound plant feed or fish meal, for which no cross reaction was observed. In general the quantification of osteocalcin in extracts is possible using a standard curve procedure with pure bovine osteocalcin.     

胶体金免疫层析法快速检测动物源性食品中土霉素残留

目的 建立用于快速检测动物源性食品中土霉素残留的胶体金免疫层析试纸条.方法 采用柠檬酸三钠还原法制备胶体金,胶体金粒径选用20 nm,将经鉴定制备成功的胶体金溶液与土霉素多克隆抗体结合得到金标抗体.优化金标抗体的制备条件,将包被抗原(1.0 mg/mL)和羊抗鼠二抗(0.75 mg/mL)分别作为检测线和质控线包被在硝...     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

食品安全快速检测方法与参比方法一致性的评价方法探讨

食品安全快速检测方法是我国食品安全保障的重要手段,对食品安全快速检测方法（产品）进行规范化评价,是保障食品安全快速检测质量的有效手段。目前,国家相关部门制定的食品安全快速检测方法的评价规范及《SN/T 2775—2011商品化食品检测试剂盒评价方法》,对食品安全快速检测方法与参比方法一致性分析都采用了卡方检验。本文对现有一致性分析方法-卡方检验进行了探讨,指出其局限性（在阳性检测率不变的情况下会随着实验样品数的增加得出相反的结论）,并提出采用Kappa检验替代卡方检验,并推导简便的计算公式。Kappa检验能够更准确的评价食品安全快速检测方法与参比方法的一致性。     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

Application of immunological methods for the detection of species adulteration in dairy products

A number of enzyme‐linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.     

Application of a polymerase chain reaction (PCR) method as a complementary tool to microscopic analysis for the detection of bones and other animal tissues in home‐made animal meals

A polymerase chain reaction (PCR) method was compared with a variation of the official microscopic technique (Directive 98/88/EC) for the detection in animal meals of cereals (wheat and corn) and animal parts (bone, feathers, meat, liver, fat and blood). Microscopy successfully detected animal bones in raw feeds with a sensitivity of 1 g kg^?1, while the sensitivity of the PCR method was in the range of 5–10 g kg^?1. Microscopy also allowed the detection of animal bones and feathers in feeds processed at 115 and 133 °C but failed to detect other animal materials. The PCR method successfully detected cereals (wheat and corn) as well as meat, bone, liver, fat and feathers after processing at 115 °C for 20 min. Heating at 133 °C under overpressure (autoclave) conditions resulted in more intense DNA fragmentation and lower DNA extractability. Nevertheless, bone and liver, as well as wheat and corn in home‐made animal meals, were successfully detected even after heating at 133 °C for 20 min. Copyright © 2004 Society of Chemical Industry     

食品中糖皮质激素残留检测方法的研究进展

糖皮质激素具有抗炎、抗过敏等疗效, 被广泛应用于动物养殖业, 若使用不当会造成动物源性食品中药物残留; 也可能被非法添加到保健食品中, 威胁人体健康。本文介绍了国内外对食品中糖皮质激素的限量标准, 综述了国内外关于糖皮质激素的检测方法, 主要有高效液相色谱法、液相色谱/质谱联用法、液相色谱-串联质谱法、高效毛细管电泳法和免疫分析方法, 讨论了各类方法的优缺点及用于糖皮质激素残留检测的适用性, 展望了食品中糖皮质激素残留检测的发展趋势, 以期为食品中糖皮质激素残留检测相关研究和安全监管提供参考。     

Development of a lateral flow immunoassay strip for screening of sulfamonomethoxine residues

A rapid lateral flow immunoassay screening method has been developed for the determination of sulfamonomethoxine (SMM) residues in swine urine. For this purpose, a specific monoclonal antibody (mAb), SMM4B9 for SMM, was generated and characterized. The mAb showed low cross-reactivity (not larger than 0.3%) to other sulfonamides and other potentially occurring analytes. Based on the competitive immunoassay principle, the strip was developed with the mAb SMM4B9 and applied to the screening of SMM residues. The test strip is made up of a sample pad, a gold-conjugate SMM4B9 reagent pad, a blotted test membrane containing a test line, a control line (a nitrocellulose membrane spotted with SMM-BSA and goat anti-mouse antibody, respectively), and an absorbent pad. The test could be accomplished within 8-10 min. It was shown that the sensitivity of the test strip was as low as 5 ng ml^-1 of SMM and the half of maximal inhibition concentration (IC₅₀) was calculated to be 10.78 ± 0.22 ng ml^-1 by relative optical density. In unaided visual assessment the detection limit of the strip was 15 ng ml^-1. For samples spiked at 20 and 30 ng ml^-1 the coefficient of variation (CV (%)) was between 2.3 and 7.1%. When the test strip was compared with high-performance liquid chromatography (HPLC) analysis for naturally contaminated swine urine samples, the difference in results was less than 6.1%. The data suggest that the method has advantages of high sensitivity, specificity, simplicity and speed of performance, as well as the characteristics of repeatability, reproducibility or accuracy and assurance. Therefore, the test strip is suitable to determine SMM residues in swine urine rapidly and reliably by quantitative, semi-quantitative or qualitative detection.     

Development of a lateral flow immunoassay strip for screening of sulfamonomethoxine residues

A rapid lateral flow immunoassay screening method has been developed for the determination of sulfamonomethoxine (SMM) residues in swine urine. For this purpose, a specific monoclonal antibody (mAb), SMM4B9 for SMM, was generated and characterized. The mAb showed low cross-reactivity (not larger than 0.3%) to other sulfonamides and other potentially occurring analytes. Based on the competitive immunoassay principle, the strip was developed with the mAb SMM4B9 and applied to the screening of SMM residues. The test strip is made up of a sample pad, a gold-conjugate SMM4B9 reagent pad, a blotted test membrane containing a test line, a control line (a nitrocellulose membrane spotted with SMM-BSA and goat anti-mouse antibody, respectively), and an absorbent pad. The test could be accomplished within 8–10?min. It was shown that the sensitivity of the test strip was as low as 5?ng?ml^?1 of SMM and the half of maximal inhibition concentration (IC₅₀) was calculated to be 10.78?±?0.22?ng?ml^?1 by relative optical density. In unaided visual assessment the detection limit of the strip was 15?ng?ml^?1. For samples spiked at 20 and 30?ng?ml^?1 the coefficient of variation (CV (%)) was between 2.3 and 7.1%. When the test strip was compared with high-performance liquid chromatography (HPLC) analysis for naturally contaminated swine urine samples, the difference in results was less than 6.1%. The data suggest that the method has advantages of high sensitivity, specificity, simplicity and speed of performance, as well as the characteristics of repeatability, reproducibility or accuracy and assurance. Therefore, the test strip is suitable to determine SMM residues in swine urine rapidly and reliably by quantitative, semi-quantitative or qualitative detection.     

食品中大肠杆菌的快速检测方法

大肠杆菌是人及各种动物肠道中的正常寄居菌,食物或水中大肠杆菌的检出意味着直接或间接的近期粪便污染。大肠杆菌作为饮水、食品等的粪源性污染卫生细菌学指标;而且他在外界存活时间与一些主要肠道病原菌相近,它的出现也可能预示某些肠道病原菌（如沙门氏菌、志贺氏菌）的存在。大肠杆菌是国际上公认的卫生监测指示菌,因此大肠杆菌的检测技术显得十分重要,相应出现了大量的大肠杆菌的各种检测方法。     

Impedance detection of Salmonella in processed animal protein and meat.

The impedance technique showed a detection rate (95%) equal to that of conventional enrichment for raw meat contaminated with Salmonella. For processed animal protein samples impedance was less sensitive. A commercially available Easter and Gibson impedance medium used for the selective enrichment of salmonellae proved superior to the laboratory prepared equivalent for the detection of Salmonella in processed animal protein. The rate of false-positive results with the impedance technique was high.     

European interlaboratory trial regarding the official microscopic method for the detection of the presence of animal constituents in feedstuffs

The bovine spongiform encephalopathy epidemic is thought to have occurred as a consequence of feeding prion-infected material to cattle. To avoid the risk of bovine spongiform encephalopathy diffusion, the European Commission (Directive 2003/126/EC) established an official method to detect the presence of animal-derived constituents in feedstuffs, using microscopic examination. This method allows easy identification of bone fragments among other animal constituents. The analysis is based on morphological conformation of the fragments and their characterization (mainly of the shape of lacunae) to discriminate among mammalian, poultry, and fish tissues. The aim of this study was to assess the performances of nine European laboratories through a ring trial of the official microscopic method, and to calculate accuracy and reproducibility of the method. In general the reproducibility of the microscopic method was very good (kappa overall = 0.83), with a high sensitivity for all laboratories. Concerning the analysis on the different animal-derived constituents, the results show values of sensitivity with large variability between fish and poultry or mammal. It was generally more difficult to discriminate between mammalian and poultry tissues than fish tissue.     

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of prohibited ruminant proteins in feedstuffs

Regulations aimed to control the epidemic of bovine spongiform encephalopathy have banned the use of certain animal products, i.e., ruminant meat and bone meals, in ruminant animal feeds. A sensitive enzyme-linked immunosorbent assay has been developed to detect prohibited bovine and ovine muscles in feedstuffs. The assay utilizes a pair of monoclonal antibodies (MAbs) against skeletal troponin I (TnI). MAb 5G9, specific to bovine and ovine TnI, was used as the capture antibody and the biotin-conjugated MAb 2G3, reacting to all heterologous TnI, was used as the detection antibody. Quantitative procedures were applied to samples containing 5, 0.5, and 0.05% (wt/wt) of heat-treated (132 degrees C/2 bar, 2 h) bovine and ovine meat meals in three different feeds, coexisting with porcine, chicken, or turkey meat meal. The presence of these nonprohibited species did not affect the detection of bovine and ovine meat meals in the feed samples (P > 0.05). Quantitative determinations of extractable bovine and ovine TnI, with a detection limit of 5.0 and 4.0 ng/ml, respectively, were achieved when the matching feed matrixes were used in the calibration curves. This new assay provides a rapid and reliable way to detect animal protein products containing a trace amount of bovine or ovine muscle tissue in feedstuffs.     

食品中单增李斯特菌的检测新技术

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是一种人畜共患食源性致病菌,可使人畜患脑膜炎、心肌炎、败血症、死婴、早产等疾病,危害较大;有效控制食品中的LM,是食品安全的重要课题之一。对以免疫学、分子生物学为基础建立的一些方法,如酶联免疫吸收分析法(ELISA)、酶联荧光分析法(ELFA)、DNA探针、PCR、DNA微矩阵法,作一简单叙述,为深入研究提供参考。最后指出预防手段非常重要,从源头上杜绝LM污染是关键。     

海洋生物毒素检测的动物试验替代方法及标准化

海洋生物毒素结构多样、种类繁多、作用机制复杂,给人类健康带来潜在的风险。使用动物模型检测贝类组织中的海洋毒素是许多国家监控计划推荐的方法。近年来,新的基于毒性作用机制和明确化学结构的检测方法不断被开发,如细胞检测法、免疫学方法、化学分析法和生物传感器方法等。有的方法已进入标准化和验证程序,逐渐被认可用于监控和检测目的 。