One of the WP2 objectives is to “Develop a more appropriate sample extraction method for use with Neogen Lateral Flow Devise (LFD) and in house validation of the improved method”. Two protein extraction techniques were evaluated under the VLA responsibilities of this aspect of the project. The first method involved an anhydrous ammonium sulphate(AAS) salting out procedure. In which a selective ammonium sulphate precipitation was carried out and a concentrations which precipitates specific antigen appeared to be
primary and secondary precipitations at 75%/ 95% or 35%/65%
AAS. A combination of precipitation and dialysis has allowed the detection in 0.5% MBM spiked samples using this method. The second approach used was aimed at complete breakdown of bone fragments into their mineral and protein constituents. It is known that the proteins are covalently bonded to mineral structures and the mineral structure of the bone could potentially protect the protein molecules during the rendering process. Therefore, to recover these proteins a new extraction procedure focused on bone particles by decalcifying the bones with EDTA free acid was developed by VLA. The main advantages of this extraction procedure is that the extract can be used for different analytical methods. The results from our laboratory show that the feed contaminated with ruminant material is detected at 0.2% level. The method is not suitable on samples containing Pap-in-Pap
(processed animal protein). The bone decalcification method is currently being validated through
interlaboratory
studies .
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M. Karanam
Veterinary Laboratory Agency , United Kingdom |
