|
Prions
can be detected on the basis of the inherent physical and biochemical
properties.
Properties
Glycoprotein
Resistance to protease digestion
Insolubility in detergents
Difference in conformation
between normal & abnormal forms of the prion protein
USE THIS TO ATTACK THE PROBLEM!!
SOURCES
· Brain
· Peripheral
Tissue
· Blood
· Urine
· Other
Biological samples
Common Tests Employed
1.
ELISA
s
2.
IMMUNO
FLUORESCENCE
3.
WESTERN
BLOTS
4.
PK
digestion followed by Western Blots
More Radical
Approaches
· PMCA – Protein Misfolding
Cyclic Amplification Procedure
Normal prion
protein is subjected to repeated cycles of incubation with the abnormal
isoform and then sonicated. Aggregates are formed at the end of each
incubation step that contain the abnormal protein. Sonication serves to
break the aggregates and increase the surface area for binding and eventual
transformation of the protein.
(Nature/Vol 411/14June2001/)
· Affinity Chromataography methods
The Antibody
to the prion protein can be bound to a column through which the sample is
passed through. After elution of the unwanted materials the prion protein
can be isolated and then subjected to detergent treatment and /or
Proteinase digestion to isolate the abnormal Isoform that may then be
detected by a western blot. It is possible to develop this system into an
automated kit – like method.
· RNA Aptamer Technology
Patents to
this method have currently been filed by V.I. Technologies (Watertown
M.A.). This method has been proposed to disinfect blood that might be
contaminated.
Two RNA
aptamers highly specific to the cellular Prion protein are used that bind
to it at the NH2 terminus.
· Can Lectins be used?
Lectins are
Glycoproteins that can specifically recognize sugar groups on molecules and
bind them. Using this approach it might be possible to easily purify the
prion protein from biological samples. This possible might save the cost
factors involved in generating monoclonal antibodies to the prion protein.
Lectin based affinity beads can be used to purify the protein. It might
also be possible to increase the isolation of the prion protein from a
sample by using Lectin – Antibody combination in which one arm of the
antibody has been replaced by the Lectin binding domain and the other arm
is specific to the prion. This will help to increase the specificity of
protein binding.
Unfortunately
this approach will be of limited use in distinguishing between the normal
and abnormal form of the protein.
· Flow Cytometry
As the prion
protein is membrane bound it is possible to use flow cytometry using
labeled Antibodies.
· RT PCR
Using probes
it may be possible to determine if there is altered gene expression as seen
in the case of the human prion diseases that involve point mutations.
· Prion interacting proteins
As more
information is gathered on the role and function of the prion protein in
cells, information on the interacting proteins will subsequently become
available. Human plasminogen, fibrinogen, antithrombin III & factor IX
has been shown to specifically bind to the abnormal prion protein and not
the normal cellular form. Magnetic beads with bound plasminogen could be
used in assays to detect prions. Techniques like FRET maybe helpful in assessment of the interaction.
(Prion
Conformation and Diagnostic tests: The Prion Protein – Bennion &
Daggett – Clin. Chemistry – 48:12 ; 2105 – 2114; 2002)
|
