ELISA to Measure
Cytochrome P450
Protein Concentration
by: William Collins
Objectives
• To develop an ELISA procedure
to measure Cytochrome P450
protein.
What Is An ELISA?
•
•
•
•
•
E- Enzyme
L- Linked
I- Immuno
S- Sorbent
A- Assay
• This technique is
designed to provide
an ultra-sensitive
process with
dependable results.
• It uses a 96-well
plate to measure a
protein or
substance based
on an
antigen/antibody
reaction.
Steps Involved in an ELISA
96 Well Plate
• Bind the protein or antigen
to the plate.
• Then you block the plate to
get rid of any non-specific
binding sites.
• Incubate with the primary
antibody which is specific
for the antigen.
• Secondary antibody that is
linked with an Enzyme is
allowed to bind with the
primary antibody.
• Use a Substrate for the
enzyme which will cause
color to be released.
Sulphan Blue Results
individual std
1.8
1.6
1.4
y = 0.9207x + 0.1012
1.2
2
R = 0.9506
Series1
1
abs
Series2
Linear (Series2)
0.8
Linear (Series1)
y = 1.073x + 0.0159
2
0.6
R = 0.9931
0.4
0.2
0
0
0.2
0.4
0.6
0.8
conc
1
1.2
1.4
1.6
Cytochrome P450
• Cytochrome P450 is a large
group of enzymes that are found
in the liver of mammals. They
are the main step in the
elimination and transformation
of foreign substances.
Abbreviations
•
•
•
•
•
uL- microliters
FBS- Fetal Bovine Serum
PBS- Phosphate Buffered Saline
TBS- Tris Buffered Saline
nm- nanometers
Microsomes
• We removed the liver from a
normal rat and from a
Phenobarbital treated rat.
• Use a Potter-Eljeham
homogenizer at 1000 RPM to
create a homogenate.
• Centrifuge the homogenate at
600g for 10 minutes to produce
a crude homogenate.
• Centrifuge the remaining
supernatant at 15,000g for 1
hour to separate out the
mitochondrial pellet.
• Centrifuge the remaining
supernatant at 100,000g for 1
hour to yield the microsomal
pellet.
ELISA Procedure
1.
2.
Add 100 uL protein to plate wells in triplicate.
Add 100 uL of 2x Carbonate-Bicarbonate buffer to
each well. Cover and store overnight at 4°C.
3. Add 200 uL of 50% FBS in PBS to each well. Mix
for 1 hour. This is the blocking solution.
4. Wash plate out with TBS-Tween 3 times
5. Add 200uL Primary Antibody Solution to each
well. Mix for 1hour at 37ºC
6. Wash plate out with TBS-Tween 3 times.
7. Add 200ul Secondary Antibody Solution to each
well. Mix for 1 hour at 37ºC.
8. Wash plate out with TBS-Tween 3 times.
9. Add 200 uL of alkaline phosphatase substrate. Mix
for 30 minutes at 25ºC.
10. Read the absorbance in a 96-well plate reader at
405 nm.
Experiment 1
• Antigen– CYP450 2B1 Varied from 1000 to 1
femtomoles per well.
– Microsomes from normal rat 10 to 1
ug/mL.
• 1º Antibody– Anti-rat CYP450 2B1 1:5000 dilution.
• 2º Antibody conjugated to Alkaline
Phosphatase
– 1:30,000 dilution.
• Resulted in no activity detected.
Experiment 2
• Antigen– CYP450 2B1 Varied from 1000 to 1
femtomoles per well.
– Microsomes from normal rat 10 to 1
ug/mL.
• 1º Antibody– Anti-rat CYP450 2B1 1:1000 or 1:2000
dilutions.
• 2º Antibody conjugated to Alkaline
Phosphatase
– 1:10,000 dilution.
• Resulted in variable and low activity.
ELISA Graph of Trial 2
0.350
Day 2 ELISA
0.300
y = 0.0001x + 0.188
R2 = 0.772
Absorbance
0.250
0.200
0.150
y = 9E-05x + 0.0833
R2 = 0.8931
0.100
1:1000AVE
1:2000AVE
Linear (1:1000AVE)
Linear (1:2000AVE)
0.050
0.000
0
200
400
600
800
Femtomoles of Cytochrome P450 2B1
1000
1200
Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mg
of rat microsomes
Experiment 3
• Antigen– CYP450 2B1 Varied from 1000 to 10
femtomoles per well.
– Microsomes from normal rat 10 to 2.5 ug/mL.
– Microsomes from Phenobarbital treated rat
10 to 2.5 ug/mL.
• 1º Antibody– Anti-rat CYP450 2B1 1:1000 or 1:500 dilution.
• 2º Antibody conjugated to Alkaline
Phosphatase
– 1:5,000 dilution.
Trial 3 Graph
0.900
0.800
y = 0.0004x + 0.4619
R2 = 0.782
Day 3 ELISA
0.700
Absorbance
0.600
0.500
y
0.400
= 0.0002x + 0.3103
R2 = 0.7103
0.300
Series1
Series2
Linear (Series1)
Linear (Series2)
0.200
0.100
0.000
0
200
400
600
800
1000
Femtomoles of Cytochrome P450
• Using 1:1000, found 874 picomoles of cytochrome P450 2B1 per
mg of normal rat microsomes and 4574 picomoles of cytochrome
P450 2B1 per mg of phenobarbital rat microsomes
1200
Comparison of 2º Antibody
Concentrations From Trial 2 and 3.
0.600
Comparison of Day 2 and Day 3 1:1000 Data
0.500
Day 3
y = 0.0002x + 0.3103
2
R = 0.7103
Absorbance
0.400
0.300
y = 0.0001x + 0.188
2
R = 0.772
0.200
Day 2
Series1
Series2
Linear (Series1)
Linear (Series2)
0.100
0.000
0
200
400
600
800
Femtomoles of Cytochrome P450
1000
1200
Experiment 4
• Antigen– CYP450 2B1 Varied from 1000 to 10
femtomoles per well.
– Crude extract of tissue culture from
H4IIE, an immortalized cell line of rat
hepatocytes, 10 to 2.5 ug/mL.
– Microsomes from cell extract of H4IIE 10
to 2.5 ug/mL.
• 1º Antibody– Anti-rat CYP450 2B1 1:500 dilution.
• 2º Antibody conjugated to Alkaline
Phosphatase
– 1:5,000 dilution.
Trial 4 Graph
Day 4 ELISA
0.700
0.600
y = 0.0004x + 0.2065
R2 = 0.9886
Absorbance
0.500
0.400
Series1
Linear (Series1)
0.300
0.200
0.100
0.000
0
200
400
600
800
femtomoles of Cytochrome P450
No activity was detected in either the
crude or microsomal cell extract.
1000
1200
Conclusions
• Successfully developed an ELISA assay to
measure Cytochrome P450 2B1 protein.
– Optimized antigen, 1º and 2º antibody concentrations.
• Measured Cytochrome P450 2B1 from normal
and phenobarbital treated rats.
– There was increased levels of P450 2B1 in the
phenobarbital treated animals.
• Unable to detect Cytochrome P450 2B1 in tissue
cultures of H4IIE rat hepatocytes.