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

Abstract

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.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures

“…This result shows high specifity of HRP inactivation originating from the first conjugated antibody although the marker proteins were used in concentrations revealing clear positive signals. The limit of detection was approximately at 0.5 μl brain homogenate in a 10 μl food suspension aliquot (Kuczius et al, 2010).…”

“…the relative expression levels of proteins which overlay in dot blots or may exhibit similar or identical molecular masses in Western blot analysis. Several methods have been described for simultaneous or serial determination of different antigens on one immunoblot which allow protein discrimination by using various chromogenic, fluorescence and chemiluminescence reactions (Hsu and Soban, 1982;Lee et al, 1988;Poor et al, 1988;Schilling and Aletsee-Ufrecht, 1989;Kondoh et al, 1998;Martin et al, 2003;Kuczius et al, 2010). However, the techniques are limited when antigenic determinants overlapping in molecular masses.…”