L. van Raamsdonk, M. Bremer, W. Hekman, A. Kemmers-Vonken, J. Vliege
RIKILT Institute of food safety, Wageningen, the Netherlands.
For the monitoring of the presence of a prohibited animal proteins several methods can be applied. The strengths of the microscopic detection method are, among others, the low level of detection as low as 0.02% and the precise indication of the detected materials. The different detection and identification methods such as PCR (DNA detection), immunoassays (protein detection) and microscopy provide the possibility to identify detected animal proteins in terms of avian or mammalian origin, or ruminant vs. non-ruminant. This information, additional to the mere presence or absence, is vital for support of the species-to-species ban. Combination of methods can be achieved in order to combine the strengths of several methods, whereas the disadvantages can be minimalised. In situ detection or hybridisation is known for years as a powerful method for detection and identification of small quantities and small particles. The combination of PCR or immunoassay analysis and microscopy adds the possibility of identifying individual particles to the low level of detection and the specificity of the microscopic method.
For the development of a method combining microscopy with an identification technique (in situ identification), muscles were chosen, because this is a well recognisable type of particles in animal proteins, and a primary target for the examination of sieve fractions. Muscle material shows the combination of high abundance and the presence of an identification mark with high specificity (DNA, protein). Both rt-PCR and immunoassay are suitable for application to muscle material. Immunoassay techniques were chosen for the identification, since antibodies are available for troponin I as well as other muscle proteins, whereas a sandwich system with a staining enzyme connected to a second antibody is available as well.
The design of a combination method comprises several steps. The chosen target, present at low frequencies if any is found, has to be concentrated and selected from the feed sample. A solvent is required with a relatively low density, which allows to get a flotation with the muscle fibres, and a sediment with the majority of the other particles. A second step is to immobilise the particles from the concentrate on a microscopic slide. The dried flotation is sprinkled on a slide which is coated with Norland Resin 81, and hardened with UV light in order to immobilise the particles. Optimal circumstances have to be established for hybridisation of the first and second antibody, and for the staining procedure. Therefore, slides are at first blocked, and washed at several points in the procedure with a TRIS-buffer. Optimal discrimination between the targeted and other muscle fibres has to be established. It appears that several enzyme-substrate combinations connected to a goat-anti-mouse antibody can be use effectively, either with alkaline phosphatise (blue staining) or with horse-radish peroxidise (red staining). These systems can be used simultaneously, allowing a discrimination system for muscle fibres from different target animal species. The sensitivity and specificity of the several available antibodies at the micro level appears to be a problem. Some commercially available antibodies react well with the muscle, but do not show a homogeneous reaction along an entire muscle fibre. Others are specific to a certain extent, but do not seem to react strongly with the muscle.