Which plant is transgenic?
Determination using an
enzyme-linked immunosorbent assay
transgenic plants / GMOs
• A transgenic plant has incorporated gene(s)
from another species.
• Transgenics are useful for engineering a
specific phenotype or studying a gene’s
function.
transgenic selection: NPTII
• Selection of plants that incorporated the transgene
can be performed by simultaneously introducing a
drug resistance gene into the plant (just like we do
with E. coli).
• nptII (most common) codes for the enzyme
aminoglycoside 3'-phosphotransferase (NPTII)
that phosphorylates (inactivates) aminoglycoside
antibiotics including kanamycin, neomycin
geneticin (G418) and paromomycin.
transgenic selection:
kanamycin resistance due to NPTII
kanamycin
WT
kanamycin
Transgenic
NPTII
Which plant is transgenic?
Which plant is transgenic?
WT
WT
WT
transgenic
• Need both WT and transgenic plants for
experiment (today) so kanamycin selection is out.
• Perform test for transgene or transgene protein?
• ELISA is a fast way to test for the presence and
amount of a protein such as NPTII.
ELISA
understanding the acronym
• EL
Enzyme-linked
– An enzyme’s activity is used as a “reporter”
to test for the presence/amount of a protein
of interest.
– The enzymatic reaction will produce a
colored species.
ELISA
understanding the acronym
• EL
Enzyme-linked
• IS
Immunosorbent
– An antibody or antigen (“immuno”) is
adsorbed (“sorbent”) onto the polystyrene
wells in which we conduct the test.
– One antibody is already adsorbed added to
the wells and a second antibody with our
enzyme will be added in the type of ELISA
we will perform today.
ELISA
understanding the acronym
• EL
Enzyme-linked
• IS
Immunosorbent
• A
Assay
– We will could determine the amount of
NPTII (quantitative assay).
– We will use NPTII concentration standards
to construct a standard curve.
ELISA: uses antibodies
• What is an antibody?
• What types of antibodies exist?
• What kinds of antibodies are used in our lab
today?
•
What
is
an
antibody?
Protein secreted by B-cells that
specifically bind a foreign
substance (antigen)
• Immunoglobulin domains
• Complementarity-determining
Regions (CDRs)
• Fab= Fragment antigen binding
• Hinge
• Fc= Fragment crystalline
• F(ab)’2= Protease digestion still
useful to bind antigen
producing polyclonal antibodies
producing monoclonal antibodies
1
2
3
Antibody-based assays
Types of immunodetection systems
2. Indirect immunodetection
1. Direct immunodetection
Secondary antibody conjugated with
enzyme system
Primary antibody conjugated with
enzyme system
HRP
HRP
HRP
HRP
Ag
Ag
antigen
Ag
Ag
Ag
horseradish
HRP peroxidase
3. Sandwich indirect
immunodetection
streptavidin
Antigen applied in soluble form
HRP
HRP
HRP
4. Indirect immunodetection
with biotin linkers
Biotinylated primary antibodies
HRP
HRP
HRP
HRP
HRP
HRP
Ag
Ag
Ag
Ag
Streptavidin
Today’s assay
Sandwich indirect
Antigen appliedimmunodetection
in soluble form
Substrate
Substrate
HRP
HRP
HRP
Substrate
HRP
Substrate
Sandwich ELISA protocol
1. Coat primary antibody
onto microplate.
1a. Allow antibody adsorption
and block unoccupied sites
with neutral protein (BSA).
2. Add antigen sample to be detected
into each well. Incubate 30 min at 370 C.
3. Add second primary antibody
against antigen and HRP-conjugated
secondary antibody (antibody mix)
into each well. Incubate 30 min at 370 C.
4. Develop colorimetric reaction
with appropriate substrate. Incubate
15 min at room temperature.
5. Stop reaction with 3M H2SO4. Read
absorbance in ELISA spectrophotometer
and quantitate relative antigen levels.
What you will do today (1):
– Collect and weigh tissue sample plant A, B and C.
– Repeat for second and third plants.
– Add 400 μL PEBX1 buffer to each microcentrifuge
tube and grind plant tissue using pestle.
– Add 100 μL of PEBX1 to wells A and B.
– Add 100 μL of one sample to wells C and D.
– Add 100 μL of second sample to wells E and F.
– Add 100 μL of third sample to wells G and H.
– HANDLE THE WELLS LIKE A CUVETTE (read at
bottom)
A
H
B
C
D
E
F
G
•
What
you
will
do
today
(2):
Incubate 30 minutes at 37 °C.
– Place wells in a zip-close bag to prevent them from
drying out in the oven.
• Wash wells 3 times with PBST
• Add antibody-enzyme conjugate (MRS-2 Ab)
– Note the dilution factor for the conjugate
• Incubate 30 minutes at 37 °C.
– Place wells in a zip-close bag to prevent them from
drying out in the oven.
• Wash 4 times with PBST.
What
you
will
do
today
(3):
Add substrate and allow blue color to develop.
•
• Stop the enzymatic reaction with 3M H2SO4.
• Presence of NPTII will result in color change from
blue to yellow.
How we will detect:
read absorbance at 450 nm
Data
1
W
E
L
L
A
B
C
D
E
F
G
H
2
3
4
5
Absorbance 450 nm
6
7
8
9
10
11
NPTII Std
Data analysis (due 4/23/12)
0.8
y = 0.3631x - 0.0025 R² = 0.995
absorbance at 450 nm
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
W
E
L
L
A Blank
B Blank
C Plant __
D Plant __
E Plant __
F Plant __
G Plant __
H Plant __
Total
amount of
plant tissue
(mg)
Your
Sample
N/A
N/A
A450
Your
Sample
0.5
1
NPTII standard (ng/mL)
1.5
2
NPTII
NPTII amount
concentration (ng protein
(ng/mL)
/mg tissue)
Transgenic?
A 450
NPTII
concentration
(ng/mL)
Your Sample
0
0
Your Sample
N/A
N/A
Your sample
N/A
N/A
Standard
Standard
Average
Average
Yes/No
Average
Average
Yes/No
Average
Average
Yes/No
2
1
0.5
0.25
0.125
0.0625
0.03125
0.015625