1. ELISA to Measure
Cytochrome P450
Protein Concentration

2. Objectives
• To develop an ELISA procedure
to measure Cytochrome P450
protein.

3. 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.

5. Steps Involved in an ELISA
• 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.
96 Well Plate

6. Sulphan Blue Results
individual std
y = 1.073x + 0.0159
R
2
= 0.9931
y = 0.9207x + 0.1012
R
2
= 0.9506
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
conc
abs
Series1
Series2
Linear (Series2)
Linear (Series1)

7. 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.

8. Abbreviations
• uL- microliters
• FBS- Fetal Bovine Serum
• PBS- Phosphate Buffered Saline
• TBS- Tris Buffered Saline
• nm- nanometers

9. 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.

10. ELISA Procedure
1. Add 100 uL protein to plate wells in triplicate.
2. 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.

11. 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.

12. 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.

13. ELISA Graph of Trial 2
Day 2 ELISA
y = 0.0001x + 0.188
R2
= 0.772
y = 9E-05x + 0.0833
R2
= 0.8931
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0 200 400 600 800 1000 1200
Femtomoles of Cytochrome P450 2B1
Absorbance
1:1000AVE
1:2000AVE
Linear (1:1000AVE)
Linear (1:2000AVE)
Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mg
of rat microsomes

14. 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.

15. Trial 3 Graph
Day 3 ELISA y = 0.0004x + 0.4619
R2
= 0.782
y = 0.0002x + 0.3103
R2
= 0.7103
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
0 200 400 600 800 1000 1200
Femtomoles of Cytochrome P450
Absorbance
Series1
Series2
Linear (Series1)
Linear (Series2)
• 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

16. Comparison of Day 2 and Day 3 1:1000 Data
y = 0.0002x + 0.3103
R
2
= 0.7103
y = 0.0001x + 0.188
R
2
= 0.772
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 200 400 600 800 1000 1200
Femtomoles of Cytochrome P450
Absorbance
Series1
Series2
Linear (Series1)
Linear (Series2)
Comparison of 2º Antibody
Concentrations From Trial 2 and 3.
Day 3
Day 2

17. 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.

18. Trial 4 Graph
Day 4 ELISA
y = 0.0004x + 0.2065
R2
= 0.9886
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0 200 400 600 800 1000 1200
femtomoles of Cytochrome P450
Absorbance
Series1
Linear (Series1)
No activity was detected in either the
crude or microsomal cell extract.

19. 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.