Evaluation of a commercial lateral flow feed test for rapid detection of beef and sheep content in raw and cooked meats.

Meat species adulteration is a common problem in the retail market. This study investigated the feasibility of a commercial lateral flow immunoassay designed to detect ruminant muscle tissue in feedstuffs, such as "meat-and-bone meal" (MBM) for detection of beef and/or sheep flesh in meat mixtures, and developed a simple method for meat sample extraction. Laboratory adulterated samples including raw, cooked (100°C, 30min), and sterilized (121°C, 15min) beef-in-chicken, beef-in-turkey, and lamb-in-pork at 0 to 1.00% (w/w) adulteration levels were extracted by different solvents (tap water, NaCl, and PB-NaCl with and without EDTA; and a kit-provided "Extraction Solvent") using three mixing methods. The test rapidly (20min) detected 0.50% (w/w) bovine or ovine meat; Extraction Solvent was the most efficient extractant tested; EDTA coupled with heating (100°C, 10min) improved the assay sensitivity; and all the mixing methods achieved the same results. This immunoassay can be conveniently applied to detect low levels of beef/sheep meat in a wide range of meat products.

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.

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.

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.

Porcine troponin I: a thermostable species marker protein.

In this study, we confirmed our previous hypothesis that the 24 kD thermostable skeletal muscle protein (TSMP) recognized by a panel of porcine-specific monoclonal antibodies (MAbs) is skeletal troponin I (sTnI). The TSMP and sTnI purified from porcine muscle have identical electrophoretic mobilities, isoelectric characteristics, and specific antigenicities. The heterogeneity of sTnI between porcine and other species, and between porcine sTnI and other troponin subunits or cardiac isoforms can be immunologically differentiated by the MAbs. Heat treatment of sTnI up to 126 °C for 120 min did not diminish its solubility and antigenicity. The antigenic specificity and thermal stability of sTnI indicate its potential as a thermostable species marker for the identification of the origin of meats in severely heated products.