Development of an Integrated Procedure for the Detection of Central Nervous Tissue in Meat Products Using Cholesterol and Neuron-Specific Enolase as Markers

Abstract

The emergence of a new variant of Creutzfeldt-Jakob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissue from the central nervous system (CNS) in food. So far, the banning of CNS tissue could not be effectively controlled because procedures for detection were missing. With regard to preventive health protection and labeling law enforcement, we have developed an integrated procedure for the detection of CNS tissue in meat products. Herein, we show that antigenic characteristics of neuron-specific enolase (NSE) quantitatively survive technological treatment including severe homogenization and pressure heating. Using both poly- and monoclonal antibodies against NSE in the Western blot, bovine and porcine brain could be detected in sausages, albeit with varying sensitivity (1 to 4%). Sensitivity was increased after reduction of fat content (30 to 40%) of the samples by means of a soxhlet extraction. This made possible the detection of brain addition as low as 0.25% when using monoclonal antibodies. Immunohistology showed distribution of CNS tissue in heat-treated meat products to be homogeneous. Immunoreaction was not found to be bound to morphologically intact histological or cytological structures; however, it proved to be highly specific. The quantification of cholesterol provides a low-cost screening method for the rapid identification of meat products, suspicious with regard to CNS tissue addition. Cholesterol content increased by 26 mg per 100 g of fresh substance for each percentage of brain added to internally produced reference material. Using three different approaches (internal reference material, raw material, and field samples), a provisional cutoff point of normal cholesterol content was calculated for emulsion-type cooked sausages to be 115 mg/100 g (P < 0.05).

Spread and Control of Prion Diseases in the Food and Feed Chains

“…PCR methods are also prone to contamination. [130][131][132] Immunochemical detection methods based on the specific immunoreactions between an antibody and its target antigen have been extensively employed for the detection of CNS contamination in meat (Table 1), generally in the form of an enzyme-linked immunosorbent assay (ELISA), [76,128,[133][134][135][136][137][138][139][140][141][142][143] immunoblotting [137][138][139][140][144][145][146][147][148][149] or immunohistochemistry. [145,146,[150][151][152] There are a number of advantages associated with using an immunoassay.…”

“…The immunological CNS specificity of γγ-enolase has been used in clinical research for a long time [159], and in 1999 Lucker et al were the first to adapt this clinical method to permit the use of NSE as a marker for the detection of CNS tissue in meat products using immunoblotting [148]. The detection limit of this assay was 1% (g/g) spiked brain in cooked (100 °C for 1 h) sausages of the emulsion type.…”

“…Hughson et al [140] compared the performance of two commercial kits, the RIDASCREEN GFAP kit and the Brainostic NSE kit (Table 3), for the detection of CNS tissue in meat products; these kits were based on the methods proposed by Schmidt et al [134] and Lucker et al [148], respectively. Hughson et al [140] reported that levels down to 0.1% (g/g) of ovine spinal cord tissues could be detected in raw lamb meat using the GFAP kit, while 1.0% (g/g) was detected by the NSE kit.…”

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products

“…The remaining two peptides (5,6) were synthesised by Severn Biotech Ltd (UK). All peptides were synthesised to 75% purity (Table 1).…”

“…brain and spinal column, and these methods include direct tissue examination, [2] microscopic examination [3], immunoassay [4] or gas chromatography detection of brain cholesterol or specific fatty acids [5,6]. The immunoassay procedures have been shown to be effective with only lightly cooked meat samples [2], and this is also likely to be the case with more recent methods based on tissue-specific mRNA [7] but less so with the methods based on methylation of tissue-specific DNA promoters [8].…”