Characterization of a 12 kDa thermal-stable antigenic protein in bovine blood

We have previously developed an immunoassay based on monoclonal antibody (MAb) Bb1H9 for quantitative detection of ruminant blood in processed food and feedstuffs. The purpose of this study was to characterize the unknown 12 kDa thermal-stable ruminant-specific antigenic protein recognized by MAb Bb1H9 in order to better define the application scope of the developed assay. Extracts obtained from raw and heat-treated bovine blood-derived products were analyzed with indirect ELISA and Western blot. Target proteins resolved by 2D electrophoresis were subjected to N-terminal sequencing. Results indicated that the 12 kDa protein is a monomer of the tetrameric hemoglobin molecule (64.5 kDa) and that the heme group is not required for its binding with MAb Bb1H9. This MAb can be utilized as a probe for red blood cell derived products of ruminant origin in raw or processed food and feedstuffs to enforce labeling regulations and to address consumer concerns. PRACTICAL APPLICATION: MAb Bb1H9 represents the first antibody with the capacity to recognize bovine hemoglobin both in the absence and presence of the heme group, regardless of the heat treatment. MAb Bb1H9 can therefore be utilized in immunoassays by manufacturers and regulators to detect any ingredients containing hemoglobin or globin (hemoglobin without the heme group) in both raw and processed food and feed materials for product quality control and labeling law enforcement.     

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.     

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.     

Development of primers for detection of meat and bone meal in ruminant feed and identification of the animal of origin

The safe use of cattle feed free from meat and bone meal is an important prerequisite to prevent further spread of bovine spongiform encephalopathy. We designed primers to detect very small amounts of meat and bone meal in ruminant feed. Mitochondrial subunit 8 of the ATP synthase gene was used as a target sequence. PCR-based assays revealed amplification of DNA from mammals, ruminants, and individual species using these primers. The method allowed detection of the presence of meat and bone meal in ruminant feed from 0.1 to 0.01%. Sensitivity and effectiveness of the method for detecting prohibited animal proteins in ruminant feed was evaluated.     

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.     

Development of immunoassay for detection of meat and bone meal in animal feed

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.     

Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.     

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.     

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.     

Development of a PCR assay for the detection of animal tissues in ruminant feeds

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.     

Detection of fish DNA in ruminant feed by PCR amplification

The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an official method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the official method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01-0.001%.     

Kinetics of tropomyosin denaturation as a predictive model for verifying thermal processing of beef products

An enzyme-linked immunosorbent assay (ELISA) was developed to study thermal denaturation of tropomyosin (Tm) using the time-temperature requirements for cooked beef. The ELISA employed a monoclonal antibody (MAb 2C9) raised against bovine Tm for quantifying residual Tm in muscle extracts. The specificity of MAb 2C9 to bovine Tm was demonstrated by Western blot and the analytical validity of ELISA was confirmed by dot blot. Thermal denaturation of Tm, in the temperature range between 54.4 and 70.0 degrees C, showed first-order dependency. Kinetic parameters of Tm denaturation were derived from isothermal heating of beef muscle extract at 54.4, 57.2, 60.0, and 62.8 degrees C. Temperature dependency of the rate constant (k) was demonstrated by Arrhenius plot; the activation energy (E(a)) of Tm denaturation was determined to be 484 kJ x mol(-1). A mathematic model describing the impact of the heating time-temperature on Tm denaturation was developed. Predicted Tm from the integrated time-temperature model agreed closely with the measured Tm in dynamically heat-processed beef samples. Percent errors between the measured and the predicted values ranged from -5.1 to 5.3%. The kinetic model provides an accurate and reproducible prediction of the impact of actual heating time-temperature on residual Tm in cooked beef. The MAb-based ELISA and kinetic model developed in this study have the potential to be adapted by the meat industry as a quality control tool.     

Evaluation of a rapid PCR-based method for the detection of animal material

A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.     

Characterization of a 60‐kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma‐Derived Food Ingredients

A sandwich enzyme‐linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60‐kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60‐kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma‐derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60‐kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2‐dimensional gel electrophoresis (2‐D GE) and amino acid sequencing revealed the 60‐kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons.     

Identification of a Biomarker for the Detection of Prohibited Meat and Bone Meal Residues in Animal Feed

ABSTRACT: A unique biomarker, h-caldesmon, was identified and purified from bovine intestine smooth muscle. It was used to develop monoclonal antibodies (MAbs) for use in immunochemical assays to detect prohibited meat and bone meal (MBM) in animal feed. This biomarker with a molecular weight of 150 kDa was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be present in MBM samples that were obtained from different manufacturers. The presence of this biomarker in MBM and smooth muscle was also demonstrated by immunostaining with MAb 8B4 in Western blot assay. h-Caldesmon in intestinal smooth muscle was demonstrated to be stable after autoclaving at 130 °C for up to 1 h. Because MAb 8B4 was sensitive in detecting MBM in animal feed at >0.05%, it can be used in immunoassays for MBM detection.     

A competitive enzyme-linked immunosorbent assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine beta-casein.

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine beta-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine beta-casein. The bovine caseins in milk or cheese samples compete with the bovine beta-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine beta-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.     

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.     

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.     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

酶联免疫吸附法快速测定不同样品 基质中三聚氰胺

目的 探求不同样品基质中三聚氰胺残留量的快速检测方法.为快速筛查不同种类食品中非法添加三聚氰胺提供技术保障.方法 采用酶联免疫吸附法在奶制品(奶粉、液态奶)、成品饲料(鸡饲料、猪饲料)、饲料原料(鱼粉、肉骨粉、豆粕、麸皮)、肉类(鸡肉、猪肉、内脏)等样品基质中添加一定浓度的三聚氰胺进行测定,并对检测结果进行分析.结果 酶联免疫试剂盒对奶制品和肉类检出限均能达到1.0 mg/kg;对成品饲料基质中的三聚氰胺的检测,检出限可达到2 mg/kg,而对饲料原料中的三聚氰胺的检测,检出限都不能达到2 mg/kg.结论 对奶制品中的三聚氰胺检测完全符合我国的临时限量标准;对成品饲料中的三聚氰胺残留的检测同样符合其限量标准(2.5 mg/kg),而对饲料原料中的三聚氰胺的检测,由于不同基质中检出限不同,不能直接采用酶联免疫法进行快速筛查;酶联免疫法也适用于肉类中的三聚氰胺的检测.