An enzyme-linked immunosorbent assay for glial fibrillary acidic protein as an indicator of the presence of brain or spinal cord in meat.

The current methods to detect central nervous system (CNS) tissue in blood, lungs, or meat are cumbersome, time consuming, and costly. The objective of this study was to use glial fibrillary acidic protein (GFAP), which is restricted to the CNS, in an enzyme-linked immunosorbent assay (ELISA) for the detection of CNS tissue in blood and muscle from beef cattle. Bovine brain, cerebral cortex, spinal cord, sciatic nerve, diaphragm, blood clots, and other skeletal muscle were obtained from three animals at slaughter. The limit for detection of GFAP was approximately 1.0 ng and the standard curve was linear up to 40 ng. Tissue samples gave responses parallel to the GFAP standard, suggesting that standard and unknown samples were immunoreactively identical. No GFAP was detected in skeletal muscle (ground beef, shoulder clod, and diaphragm) and blood clots. Trace amounts (13.5 to 51 ng/mg) were present in sciatic nerve. In contrast, high levels of GFAP (55 to 220 microg/ mg) were present in spinal cord, cerebral cortex (17 microg/mg), and whole brain (9 to 55 microg/mg). In a storage study using two animals in two separate studies, immunoreactive GFAP was detectable for up to 8 days at 4 degrees C in all tissues containing neural elements. Thus, mixtures of muscle with spinal cord or brain retained almost 80% of their immunoreactivity after 8 days at 4 degrees C, while brain and spinal cord alone retained approximately 50% and 25%, respectively, of their initial activities. In a repeat experiment, 80 to 100% of the initial activity was retained in these tissues after 8 days at 4 degrees C. The results of the current study demonstrate that the GFAP ELISA provides a valid and repeatable method to detect CNS tissue contamination in meat.     

Comparison of commercially available antibodies for the detection of central nervous system tissue in meat products by enzyme-linked immunosorbent assay

An indirect competitive enzyme-linked immunosorbent assay was used to compare four commercially available monoclonal and polyclonal antibodies prepared against the markers of central nervous system tissue: glial fibrillary acidic protein, neurofilaments, and myelin basic protein. These antibodies were investigated for their usefulness at the detection of prohibited central nervous system tissue in meat products to prevent spread of bovine spongiform encephalopathy and its human form, a new variant of Creutzfeldt-Jakob disease. The detection limit was 2.5 to 10%. The specificity was tested using extracts of tissue samples of bovine and porcine brain, spinal cord, lymphatic node, diaphragm, skeletal muscle, and peripheral nerves. All antibodies reacted with both bovine and porcine tissue extracts.     

Comparison of immunochemical (enzyme-linked immunosorbent assay) and immunohistochemical methods for the detection of central nervous system tissue in meat products

Three methods are widely used in the United States to detect the presence of central nervous system (CNS) tissue in meat products: the fluorescent enzyme-linked immunosorbent assay (F-ELISA), developed in this laboratory, the colorimetric Ridascreen Risk Material 10/5 ELISA (R-ELISA), and the U.S. Department of Agriculture, Food Safety and Inspection Service immunohistochemical (IHC) procedure. These assays are based on the immunological detection of glial fibrillary acidic protein (GFAP), a neural antigen largely restricted to the CNS. The objective of the current study was to compare the sensitivity and repeatability of these tests for detecting the presence of neural tissue in meat. Ground beef spiked with 0.05 to 0.5% of bovine brain, spinal cord (SC), or dorsal root ganglia, as well as advanced meat recovery samples, were evaluated by each of the three GFAP detection procedures. Interassay coefficients of variation for the F-ELISA GFAP standards were 7 to 25%, and intra-assay variation due to sampling and extraction of spiked ground beef was 7 to 13% for SC and 8 to 14% for brain (n = 10). The F-ELISA was the most sensitive of the methods tested, capable of detecting 0.3 ng GFAP standard per well and the presence of 0.05% brain and SC in meat. The R-ELISA standards produced highly variable results (up to 36% variation) and, as a result, none of these standards were different from zero (n = 26). The R-ELISA resulted in high sample variation in SC-spiked ground beef samples (coefficients of variation were 23 to 50%) and did not detect the presence of brain contamination. After modification of the R-ELISA sampling and extraction methods, SC-spiked sample variation was reduced to 16 to 20%, and sensitivity was improved from 0.3 to 0.2% SC, although brain tissue was still not detected. The IHC analysis of CNS-adulterated ground beef had a sensitivity of 0.2% SC and 0.05% brain, with false-negative rates of 10 to 20% at and above the stated sensitivities. None of the methods examined detected dorsal root ganglia contamination. The F-ELISA detected the presence of CNS contamination in 20% of the advanced meat recovery samples, compared to 3.5 to 5% for the R-ELISA and 2% for IHC. This study suggests that the F-ELISA is much more sensitive and repeatable than either the R-ELISA or the IHC procedure method for the detection of CNS tissue in meat products.     

Verification of different glial fibrillary acidic protein (GFAP) analyses as accurate detectors of central nervous system tissue in advanced meat recovery (AMR) products

A glial fibrillary acidic protein (GFAP) fluorescent enzyme linked immunosorbant assay (ELISA) was compared with an ELISA test kit for GFAP to determine the level of central nervous system (CNS) tissue in advanced meat recovery (AMR) products. The test kit results were highly correlated (r=0.975) with the fluorescent ELISA. Meat cuts and AMR were analyzed on site at 14 meat plants utilizing the test kits. In seven of the plants all AMR samples had less than 1 ng GFAP. Seven of the plants had greater than 1 ng GFAP in AMR samples. Development of proper process controls to eliminate inclusion of spinal cord in AMR materials should bring all values to less than 1 ng GFAP, a level slightly above background.     

Glial fibrillary acidic protein and myelin basic protein as markers for the immunochemical detection of bovine central nervous tissue in heat-treated meat products

Because bovine central nervous tissue (CNT) is the main risk material in transmission of the infective agent of bovine spongiform encephalopathy, a suitable test is needed to enforce the ban on CNT in human foodstuffs in the United States and the European Union and to ensure that meat products are free of CNT To detect bovine CNT in heat-treated meat products, we used immunohistochemistry and Western blots with antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). Both antigens were resistant to processing methods used for meat products. The anti-GFAP antibody showed a high degree of tissue specificity, whereas the anti-MBP antibody had high species specificity, clearly differentiating between porcine and bovine CNT Therefore, immunochemistry performed with both proteins provides an effective means for detecting bovine CNT in meat products.     

Immunological one‐step determination of the central nervous system indicator proteins,neuron‐specific enolase and glial fibrillary acidic protein,in meat products

The determination of specific marker proteins is important in the prevention of infections and transmission of disease. Several diagnostic assays have been developed but these are mostly restricted to the detection of single antigens. Thus there is a need for multiplex detection assays for the simultaneous analysis of several disease indicators. Consumer protection against the transmission of prion diseases is ensured by the removal of specified risk material from cattle meat during slaughtering and this is regulated by law. The investigation reported here describes a one‐step determination on immunoblots of the simultaneous detection of two indicator proteins, neuron‐specific enolase and glial fibrillary acidic protein, in tissues of the central nervous system. Although the two proteins run in polyacrylamide gels with similar molecular masses the indicators are differentiated by immunological reactions followed by visualizing the different coloured specific protein bands which develop. The enolase exhibits a brown band, whereas the glial fibrillary acidic protein is red. Immunoblotting has proved to be a suitable assay for multiplex analysis of marker proteins possessing similar molecular weights and is therefore a suitable tool for application in food, veterinary and medical facilities.     

Detection of central nervous system tissue in meat and meat products with a newly developed immunoassay selective for Myelin proteolipid protein

Bovine spongiform encephalopathy (BSE) is most likely transmitted by the consumption of central nervous system (CNS) tissue of infected animals. In this study, an immunochemical assay for CNS in meat and meat products was developed using an antibody against Myelin proteolipid protein (PLP), which is very specifically expressed in the CNS. Solvent extraction of CNS-contaminated meat yielded a highly enriched PLP fraction. Subsequent Western blot analysis specifically detected the PLP band at 29 kDa. The detection limit for unprocessed CNS in raw meat was less than 0.025% and the quantification limit was calculated to be 0.049%. The PLP epitope was relatively stable during storage at 5 °C or –21 °C and during heating at 75 °C and 95 °C. Amounts of 0.1% CNS could be reliably detected in cooked bologna type sausage, cooked liver sausage and fermented sausage. Thus, the new assay allows highly specific and sensitive determination of CNS contaminations in meat and meat products.     

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.     

Assessment and modification of a Western blot assay for detection of central nervous system tissue in meat products in the United States

Health hazards associated with meat contaminated by the bovine spongiform encephalopathy agent have led to the development of tests for the presence of this agent. The objective of this study was to optimize a neuron-specific enolase Western blot assay for use in the United States. We compared the original test with a modified protocol to evaluate the detection limit for the presence of central nervous system (CNS) tissue in experimentally inoculated samples and compared and evaluated the utility of these tests for detecting CNS tissue in retail sausages. Sensitivity and specificity of the original and modified protocols were evaluated using the kappa statistic to assess agreement between the results of the two protocols. The original protocol resulted in 100% specificity and 92% sensitivity for raw samples and 92% specificity and 72% sensitivity for cooked samples. The modified protocol resulted in 92% specificity and 89% sensitivity for raw samples and 83% specificity and 75% sensitivity for cooked samples. The kappa statistic for protocol comparison was 0.94 for raw samples and 0.74 for cooked samples. Both protocols correctly identified CNS tissue in positive controls for each replicate. Although the Western blot technique should be considered for screening for the presence of bovine CNS tissue in meat samples, the techniques should be further optimized to address problems of low sensitivity. A test with higher sensitivity is needed to protect consumers from food safety threats associated with bovine CNS tissue.