Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of horse meat (1-50%) in unheated meat mixtures. The assay uses horse-specific antibodies obtained by immunoadsorption of the crude horse antisera onto immobilised sarcoplasmic extracts from chicken, beef and pig to remove cross-reacting antibodies. The purified antibodies bound to a solid support sequester horse muscle soluble proteins from meat mixtures. Further immunorecognition was made with the same antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of minced beef and pig containing variable amounts of horse meat.     

Detection of chicken meat in raw meat mixtures by a sandwich enzyme immunoassay

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of low levels of chicken meat (1–30%) in unheated meat mixtures. The assay uses chicken-specific antibodies, obtained by immunoadsorption of the crude chicken antisera onto immobilized sarcoplasmic extracts from beef, pig and horse, to remove cross-reacting antibodies. The purified antibodies, bound to the wells of a microtitre plate, sequester chicken muscle soluble proteins from saline extracts of meat mixtures. Immuno-recognition is made with similar purified antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gives clear optical density differences, when assaying minced beef and pig containing variable amounts of chicken meat.     

Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork

A sandwich ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of chicken meat (1-100%) in beef and pork meat mixtures. The assay uses a monoclonal antibody (BC9) specific to a chicken muscle soluble protein to capture this protein from complex meat mixtures. Further immunorecognition of the captured protein was attained with rabbit polyclonal antibodies against chicken muscle proteins (anti-CHSP). A commercial goat anti-rabbit immunoglobulin conjugated to peroxidase was used to detect the anti-CHSP antibodies bound to the chicken protein. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of beef and pork meats containing variable amounts of chicken meat.     

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.     

Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products

A monoclonal antibody‐based sandwich enzyme‐linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat‐processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin‐conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory‐adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy‐based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy‐based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety.     

Quantitative Detection of Poultry in Cooked Meat Products

ABSTRACT: There is a growing awareness of perceived harm from meat species adulteration, both intentional and accidental. The present study developed a monoclonal antibody (Mab)-based enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chicken and turkey meat adulterated in cooked (100 °C, 15 min) mammalian meat. The specificity of Mab 5D2 to different species (pork, beef, lamb, deer, horse, duck, chicken, and turkey) and tissues (serum, gizzard, heart, and liver) was studied by noncompetitive ELISA. The detection of cooked chicken in beef, and turkey in pork was accomplished by competitive and noncompetitive ELISAs. Both ELISAs were optimized to quantify cooked poultry in red meats. The new Mab-based ELISAs enabled the detection of cooked poultry in red meats at levels as low as 1% (v/v) or better. The correlation ( r > 0.994) between chicken or turkey concentrations and ELISA signals permitted the quantification of poultry adulterants in cooked non-poultry meats.     

Identification of novel peptides for horse meat speciation in highly processed foodstuffs

There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species.     

Monoclonal Antibody-based ELISA for Assessment of Endpoint Heating Temperature of Ground Pork and Beef

ABSTRACT: :
An indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb) 2F8 specific to a mammalian muscle protein was developed to evaluate the endpoint heating temperature (EPT) in ground pork and beef. The ELISA response of MAb 2F8 with meat extracts increased rapidly as the EPT increased from 60 to 80 °C and leveled off above 80 °C. Results of immunoblot revealed that the 12 kD antigenic thermo-stable muscle protein, which is common in both pork and beef, can be used as an EPT indicator for heat-processed pork and beef based on changes in the ratio of this protein to total soluble proteins in the meat extract which occurs at the EPT.     

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.     

Competitive enzyme-linked immunosorbent assay for quantitative detection of bovine blood in heat-processed meat and feed

Animal blood is widely used as a functional additive in food and as a protein supplement in animal feed. Effective analytical methods are therefore needed to enforce labeling regulations for product quality control, as well as to address safety concerns. This study developed a monoclonal antibody (MAb)-based competitive enzyme-linked immunosorbent assay (ELISA) for quantitative detection of ruminant (bovine and ovine) blood in heat-processed meat and feedstuff. MAb BblH9 immunoglobulin G1, which recognizes a 12-kDa, thermostable, ruminant blood protein, was used as the detecting agent in the competitive ELISA. The immunoreactivity of MAb Bb1H9 was confirmed in both an indirect noncompetitive ELISA and a competitive ELISA, in which antigens are presented in immobilized form and free form, respectively. The competitive ELISA was optimized, and three spiked samples adulterated at 0 to 10% were tested to determine the detection limits of the optimized assay. Results showed that MAb Bb1H9 is specific to ruminant blood protein, with no cross-reaction with nonblood samples tested. The optimized competitive ELISA quantitatively detected heat-processed bovine blood over the tested range. The detection limit of the assay for bovine blood in beef (P < 0.001), bovine blood in porcine blood (P < 0.01), and bovine blood meal in soybean meal (P < 0.01) was found to be 0.5% in all cases. This MAb-based competitive ELISA is thus suitable for the sensitive and quantitative detection of ruminant blood proteins in heat processed meat and feed products.     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

Identification of cattle, llama and horse meat by near infrared reflectance or transflectance spectroscopy

Visible and near infrared reflectance spectroscopy (VIS-NIRS) was used to discriminate meat and meat juices from three livestock species. In a first trial, samples of Longissimus lumborum muscle, corresponding to beef (31) llamas (21) and horses (27), were homogenised and their spectra collected in reflectance (NIRSystems 6500 scanning monochromator, in the range of 400-2500 nm). In the second trial, samples of meat juice (same muscle) from the same species (20 beef, 19 llama and 19 horse) were scanned in folded transmission (transflectance). Discriminating models (PLS regression) were developed against “dummy” variables, testing different mathematical treatments of the spectra. Best models indentified the species of almost all samples by their meat (reflectance) or meat juice (transflectance) spectra. A few (three of beef and one of llama, for meat samples; one of beef and one of horse, for juice samples) were classified as uncertain. It is concluded that NIRS is an effective tool to recognise meat and meat juice from beef, llama and horses.     

Capillary electrophoresis for bovine and ostrich meat characterisation

The objective of this study was to characterise, compare and quantify the water soluble protein (WSP) and salt soluble protein (SSP) fractions from bovine and ostrich muscle by using sodium dodecyl sulphate polymer-filled capillary gel electrophoresis (CE-SDS). Samples were raw ostrich leg and eye round beef collected 24 and 48 h, respectively, after sacrifice from local slaughter houses. WSP were extracted with cold double distilled deionized water and SSP with 0.6 M NaCl/0.01 M phosphate buffer pH 6 with 0.5% polyphosphates. Separation of WSP and SSP extracts was achieved by CE-SDS. Quantitative data for individual proteins was generated by constructing a calibration curve using bovine serum albumin (BSA) as a standard. The WSP profiles showed differences for bovine and ostrich meat, both qualitatively and quantitatively and could be employed for species differentiation. Quantitative data derived for WSP and SSP from bovine and ostrich muscle showed significant differences among individual proteins. A comparison of protein profiles form ostrich and bovine meat should be useful for meat species differentiation and muscle characterisation for establishing relations to meat quality.     

Identification of the species origin of fresh meat using an enzyme-linked immunosorbent assay procedure

A modification of indirect enzyme-linked immunosorbent assay (ELISA) has been successfully applied to the detection of horse meat and beef. This technically simple assay requiring species specific antibody, conjugated enzyme anti-IgG and a polystyrene protein-binding solid phase, can be adapted for the identification of meat species in circumstances where laboratory facilities are minimal.     

The Use of a Poultry-Specific Murine Monoclonal Antibody Directed to the Insoluble Muscle Protein Desmin in Meat Speciation

Monoclonal antibodies were produced using chicken smooth muscle desmin as the immunogen and one antibody, 4B4/B2, was found to be reactive with desmin from chicken, turkey, pheasant and duck but not from lamb, beef and pork. This antibody reacts with poultry desmin from both smooth muscle and skeletal muscle, allowing its use for the detection of poultry meat in other meats. In addition to reacting with intact forms of this cytoskeletal protein (apparent subunit molecular size 54 kDa), 4B4/B2 also reacts with degradation products of desmin (30–40 kDa), thus enhancing its use for speciation purposes. When the antibody is used in an indirect ELISA involving antigen-coated plates, it can detect <0·04 μg ml⁻¹ poultry desmin. As desmin is one of the most insoluble proteins in cells, it is an ideal marker for speciation. Desmin-enriched samples can be produced from chicken meat and chicken/red meat mixtures by differential extraction, culminating with the solubilisation of desmin in 8M urea. These extracts can be analysed using the ELISA system, routinely detecting chicken at the 100 g kg⁻¹ mixed meats level. Alternatively, Western blots of the samples, resolved by 100 g kg⁻¹ SDS PAGE, can be probed with 4B4/B2 allow-ing detection at the 20 g kg⁻¹ mixed meats level. This monoclonal antibody rep-resents a standard reagent of unlimited supply which can, for the first time, detect a defined insoluble protein in poultry speciation assays. Preliminary applications indicate that it is superior to other reagents and demonstrate that desmin has the potential to be a valuable marker for speciation.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

基于ROAV法分析木瓜蛋白酶对马肉挥发性风味物质的影响

为探究木瓜蛋白酶对马肉挥发性风味物质的影响,采用顶空固相微萃取（HS-SPME）结合气相质谱联用（GC-MS）法对马肉的挥发性风味物质进行鉴定,通过计算风味物质的相对气味活度值（ROAV）确定其关键风味成分。结果表明,空白组检出30种风味物质,木瓜蛋白酶处理组检出25种风味物质,表明马肉经过木瓜蛋白酶处理后,挥发性风味物质种类减少,但是醛类风味物质的相对含量（68.56%）明显提高。空白组和木瓜蛋白酶处理组的共有关键性风味物质种类的差异不大,醛类物质是马肉典型的风味物质,辛醛、壬醛、癸醛和戊醛对马肉的风味均有较大贡献,3-甲基丁醛对木瓜蛋白酶处理组的风味贡献很大,ROAV为93.03。     

A modified indirect ELISA procedure for raw meat speciation using crude anti-species antisera and stabilised immunoreagents

Indirect enzyme-linked immunosorbent assay (ELISA) for species identification of unheated meat materials has been modified to facilitate the use of non-specific (unpurified) anti-species antisera by integral assay inhibition of heterologous cross-reactivity. Effective ELISA treatments of three antisera (identifying beef, horse and pig) and four commercial products (identifying beef, horse, pig and sheep/goat) were determined using chequer-board microtitration assays against pure species antigens. Subsequent tests with aqueous meat extracts gave consistent absorbance differences between the homologous (high colour) and heterologous (low colour) responses and provided reliable, accurate species identification without the extensive prior purification of antisera (i.e. by affinity chromatography) previously required. This modified ELISA enabled efficient use of stabilised anti-species sera discs, prepared initially for application in a simplified agar-gel immunodiffusion (AGID) test; i.e. one disc (20 μl freeze-dried antiserum) for testing two meat samples by the prescribed AGID made sufficient treated solution to test 50 meat samples on microtitre plates. Authentic species meat extracts are also stabilised on discs for convenience (reference responses and routine controls). ELISA in this form is a practical alternative to AGID screening tests or can be used in parallel back-up tests providing sensitive results in less than 3 h on a large or small scale.     

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.