This document describes the development of an ELISA procedure to measure Cytochrome P450 protein concentration. It details the optimization process, which included varying antigen, primary antibody, and secondary antibody concentrations over multiple experiments. The optimized ELISA was able to detect Cytochrome P450 2B1 levels in liver microsomes from normal and phenobarbital-treated rats, finding higher levels in treated rats. However, the ELISA was unable to detect Cytochrome P450 2B1 in tissue culture extracts of rat hepatocytes.

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.