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Chapter: Biotechnology Applying the Genetic Revolution: Viral and Prion Infections

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Detection of Pathogenic Prions

The emergence of mad cow disease (BSE) has created the need to screen cows and their products for the presence of the pathogenic form of the prion protein (PrP Sc ).

DETECTION OF PATHOGENIC PRIONS

The emergence of mad cow disease (BSE) has created the need to screen cows and their products for the presence of the pathogenic form of the prion protein (PrP Sc ). This is presently done by immunological detection. Unfortunately, even though the 3D folding of the normal (PrP c ) and pathogenic (PrP Sc ) forms is different, it has not yet proven possible to obtain antibodies specific to each form. Because the pathogenic form of the prion is protease resistant, samples are first treated with protease to destroy the normal (PrP c ) form and then subjected to immunological testing by Western blotting. The overall procedure is tedious and of only moderate sensitivity. In particular, it would be valuable to have a test that reveals prion disease well before symptoms develop to allow time for possible treatments.




The protein misfolding cyclic amplification (PMCA) procedure amplifies the levels of misfolded prion in a manner analogous to the use of PCR for amplifying DNA ( Fig. 22.14 ). This allows greatly increased sensitivity of detection of PrP Sc in clinical samples. Small samples suspected of containing PrP Sc are mixed with normal brain homogenate containing a surplus of the normal PrP c . The PrP c is converted to PrP Sc and incorporated into the growing PrP Sc aggregates. The sample is then sonicated to break up the aggregates. This procedure is repeated for several cycles. Increases of around 60-fold over five cycles are typical.


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