Optimizing Fecal Sample Preparation to Determine Presence of Download

Transcript
Aya Murakami
 Background
 Transmissible Spongiform Encephalopathies and Prion
 Other for TSEs
 CWD Distribution in the US
 Symptoms and Diagnosis
 Biomarkers
 Research Goals
 Results
 Conclusion
 Transmissible spongiform encephalopathy (TSE) is
a group of progressive diseases that affect the
nervous systems and brains of many mammals,
including human.
 Caused by accumulation of a misfolded isoform of
the protein called prion protein (PrP)
 Prion is an infectious agent which forms
aggregates in nervous tissues.
 Two protein conformations exist…
 PrPsen= native conformation of PrP present in all
mammals and is sensitive to digestion with protease.
It is a membrane associated protein but its function
is unknown.
 PrPres= misfolded conformation and is resistant to
digestion with protease
 Mad Cow Disease (Bovine
Spongiform Encephalophathy; BSE)
 Scrapie
 Chronic Wasting Disease (CWD)
 Creutzfeldt- Jakob Disease (CJD)
Cows and
other
mammals
Sheep and goats
Deer and elk
humans
 Symptoms
 Weight loss
 Behavioral changes:
 Decreased interactions with other animals
 Listlessness
 Lowering of the head
 Blank facial expression
 Repetitive walking in set patterns
 A smell like meat starting to rot
 Diagnosis
 Post-mortem: detection of PrPres and vacuolization in
nervous tissues.
 Ante-mortem: RAMALT (recto-anal mucosal associated
lymphoid tissue) to detect PrPres.
 Biomarkers= indicators of diseases/ conditions
 Pregnancy
 Albumin in urine to measure the kidney functions
 ALT/AST level to evaluate the liver functions
 many other…
 Easy, fast, non-invasive testing: use of urine/ feces/
blood
Biomarkers
Possible Functions
Dynamin 1
GTPase responsible for endocytosis
Calpain
Calcium dependent protease
TCP-1 α
A subunit of chaperonin involved in tubulin and actin folding
And others…
The Mission:
To detect these biomarkers in feces using
Western Blotting
Why feces?
-Easy to collect in the field.
-Non- invasive
For method development, we used feces samples from captive
white tail deer that were inoculated with chronic wasting disease.
Western blot is a common technique to detect a protein of interest in a sample using
antibodies specific to the protein.
Protein
Separate
proteins on
SDS-PAGE
Transfer
proteins to
nitrocellulose
membrane
Membrane
is exposed
to specific
antibodies
Protein bands
detected by
specific
antibodies
250
150
100
75
50
Banshee
Banshee
Dundee
Dundee
Kool aid
Kool Aid
Red
Red
Niblet
Niblet
Loki
Indy
Swift
Marker
kD
1 21 2 1 2 1 21 2
This is very DIRTY!!
We would like to see nice clear bands…
Our Goals:
37
To make this better looking…
By altering
20
15
extraction buffer
 dilution of the primary and secondary
antibodies
 Extraction time/ methods

antibody: decided 1:300
dilution was the best
1 21 2 1 2 1 21 2
kD
 Alteration of secondary
antibody: decided
1:10,000 was the best
 This is a blot with 1:300
of 1°antbody and 1:
10,000 of 2°antibody
250
150
100
75
kD
Banshee
Banshee
Dundee
Dundee
Kool aid
Kool Aid
Red
Red
Niblet
Niblet
Loki
Indy
Swift
Marker
Banshee
Banshee
Dundee
Dundee
Kool aid
Kool Aid
Red
Red
Niblet
Niblet
Loki
Indy
Swift
Marker
 Alteration of primary
1 21 2 1 2 1 21 2
250
150
100
75
50
37
50
37
20
15
20
15
There is not much
improvement.
incubated the
samples for 15 hrs to
extract the mucus
layers.
 In this experiment, 1,
3 and 5 hr extraction
was performed.
1 hr extraction
seemed to work
the best.
Dundee hr
Banshee
Dundee hr
Banshee
Dundee
Dundee hr
Red
hr
Dundee
Kool
Redaid hr
Kool Aid hr
Red
RedRed
(cont’l)
IndyRed hr
Niblet
Indy hr
Niblethr
Indy
Swift Loki
hr
Swift Indy
hr
Swift
Swift hr
Marker
Swift (cont’l)
Marker
 We previously
1 3 5 1 3 5
kD
250
150
100
250
150
100
75
75
50
50
37
37
20
20
15
10
15
1 3
5 1 3 5
1 21 2 1 2 1 21 2
1 hr extraction on the rotary shaker
10
15
10
Kool Aid
15
20
Red
20
50
37
Loki
50
37
250
150
100
75
CWD +
CWD -
250
150
100
75
kD
Indy
extracted the feces
1 2
using tumbler shaker
1
2
1 2 1 2
1 2
kD (360°rotation).
 In this experiment, we
used a rotary shaker
instead.
Marker
Kool Aid
Kool Aid
Niblet
Niblet
Red
Red
Banshee
Banshee
Dundee
Dundee
Indy
Swift
Loki
Marker
extraction on the tumbler shaker
1 hr
We
previously
20 45 90 20 45 90 20 45 90 20 45 90
 Tried our methods on
kD
250
150
100
75
50
37
20
15
10
CWD+
CWD –
Marker
feces from deer of
unknown CWD status.
 Blot was a little ugly.
 Decided to alter the
buffer to have less salt
and detergent.
1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B
 Alter the salt and
NaCl: 130 mM
detergent
Detergent:concentration.
0.5%
1A 1B 2A 2B 3A0.5%
3B 4A 4B→0.05%
5A 5B 6A 6B
 Detergent:
kD
250Addition of phenyl
150
100
75
50
37
20
15
10
methyl sulfonyl fluoride
(protease inhibitor)
kD
250
150
100
75
50
37
20
15
10
CWD+
CWD –
Marker
CWD+
CWD –
Marker
 NaCl: 130 mM→10 mM
NaCl: 10 mM
Detergent: 0.05%
1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B
 Improved the method for protein extraction from deer
feces for biomarker detection.
 Furthermore, these methods seemed to work for deer
feces with unknown CWD status.
 This method has potential to serve as a non-invasive
screen for CWD in the field.
 I would like to thank all the faculty members in Dr.
Lewis’ Lab, especially Ted John for walking through
the experiments with me and being a great mentor.