BAVARIAN RESEARCH ASSOCIATION PRIONS
Erl 3 High Sensitive Immunassay for Detection of Central Nervous Tissue in Food
Bovine spongiform encephalopathy (BSE) and the new variant of Creutzfeld-Jakob disease (vCJD) are most likely transmitted by the consumption of prion containing tissue of infected animals. Highest infectivity was hereby detected in brain and spinal cord. Tissue from the central nervous system (CNS) was, therefore, defined as risk material and banned from the food chain. In order to enforce the ban, analytical methods are required which detect CNS contaminations in food highly specifically and sensitively. The aim of the project was, therefore, to develop a reliable and sensitive immunochemical assay for the analysis of CNS tissue in meat products. A two-step assay design allows fast screening of a high number of samples in the first step and a highly reliable verification of positive results in the second step. For this purpose, myelin proteolipid protein (PLP) was identified for the first time as a highly specific and heat stable marker protein for CNS tissue. A highly selective polyclonal antibody was generated, which recognizes an epitope, present in the full length PLP protein, but not in the developmentally regulated splice variant DM-20, which is expressed in peripheral nerves. Due to the hydrophobicity of PLP, the marker protein could be selectively enriched by organic solvent extraction. In the next step, the PLP-antibody was used for the development of a two-step immunochemical assay. First, a Western blot test was designed for the reliable detection of CNS-tissue in meat products. Additionally, a dot blot assay was developed as a screening test. Compared to the Western blot, the dot blot assay is faster, easier to handle and does not rely on advanced technical equipment. Both assays showed an excellent specificity for CNS. Whereas spinal cord as well as cortex and cerebellum from the brain were reliably detected, tissue from other organs, which are regular ingredients of meat products, such as peripheral nerves, did not interfere with the test results. For antibody generation, a relatively heat stable epitope was chosen. Therefore, the assay was also able to record CNS contaminations in all tested types of sausages. The detection limit of the Western blot was 0.025 % CNS tissue in meat. Due to the very good linearity, the test could also be used for quantification. In several types of sausages, 0.1 % brain or spinal cord, which was added prior to preparation, was reliably detected. For the dot blot assay, the detection limit was even cut down to 0.01 % CNS tissue in meat allowing its application as a screening method. Furthermore, the dot blot test was combined with a swab test, so that CNS contaminations of meat and working surfaces, which occur during the slaughter process, were consistently detectable down to 0.5 mg. The newly developed CNS Western blot test was further applied in a survey of retail products analyzing 152 representative sausages, commercially available in Bavaria. In about 10 % of the samples, CNS was detectable. Both, the amount of the detected CNS, as well as the results of follow-up tests indicate that in some samples CNS, most likely brain, was added in considerable concentrations as an ingredient for sausage manufacturing. The newly developed, two-step test system thus allows a reliable and highly sensitive method for the analysis of CNS contaminations in meat products which can be applied in private and official food control.