ELISA to Measure ELISA to Measure Cytochrome P450 Protein Cytochrome P450 Protein ConcentrationConcentration

by: William Collinsby: William Collins

ObjectivesObjectives

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

What Is An ELISA?What Is An ELISA?

• E- EnzymeE- Enzyme• L- LinkedL- Linked• I- ImmunoI- Immuno• S- SorbentS- Sorbent• A- AssayA- Assay

• This technique is This technique is designed to provide designed to provide an ultra-sensitive an ultra-sensitive process with process with dependable results.dependable results.

• It uses a 96-well It uses a 96-well plate to measure a plate to measure a protein or substance protein or substance based on an based on an antigen/antibody antigen/antibody reaction.reaction.

Steps Involved in an Steps Involved in an ELISAELISA

• Bind the protein or antigen to Bind the protein or antigen to the plate.the plate.

• Then you block the plate to get Then you block the plate to get rid of any non-specific binding rid of any non-specific binding sites.sites.

• Incubate with the primary Incubate with the primary antibody which is specific for antibody which is specific for the antigen.the antigen.

• Secondary antibody that is Secondary antibody that is linked with an Enzyme is linked with an Enzyme is allowed to bind with the allowed to bind with the primary antibody.primary antibody.

• Use a Substrate for the enzyme Use a Substrate for the enzyme which will cause color to be which will cause color to be released.released.

96 Well Plate

Sulphan Blue ResultsSulphan Blue Results

individual std

y = 1.073x + 0.0159

R 2 = 0.9931

y = 0.9207x + 0.1012

R 2 = 0.9506

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conc

abs

Series1

Series2

Linear (Series2)

Linear (Series1)

Cytochrome P450Cytochrome P450

• Cytochrome P450 is a large group Cytochrome P450 is a large group of enzymes that are found in the of enzymes that are found in the liver of mammals. They are the liver of mammals. They are the main step in the elimination and main step in the elimination and transformation of foreign transformation of foreign substances.substances.

AbbreviationsAbbreviations

• uL- microlitersuL- microliters• FBS- Fetal Bovine SerumFBS- Fetal Bovine Serum• PBS- Phosphate Buffered Saline PBS- Phosphate Buffered Saline • TBS- Tris Buffered SalineTBS- Tris Buffered Saline• nm- nanometersnm- nanometers

MicrosomesMicrosomes• We removed the liver from a We removed the liver from a

normal rat and from a normal rat and from a Phenobarbital treated rat.Phenobarbital treated rat.

• Use a Potter-Eljeham homogenizer Use a Potter-Eljeham homogenizer at 1000 RPM to create a at 1000 RPM to create a homogenate.homogenate.

• Centrifuge the homogenate at Centrifuge the homogenate at 600g for 10 minutes to produce a 600g for 10 minutes to produce a crude homogenate.crude homogenate.

• Centrifuge the remaining Centrifuge the remaining supernatant at 15,000g for 1 hour supernatant at 15,000g for 1 hour to separate out the mitochondrial to separate out the mitochondrial pellet.pellet.

• Centrifuge the remaining Centrifuge the remaining supernatant at 100,000g for 1 supernatant at 100,000g for 1 hour to yield the microsomal hour to yield the microsomal pellet.pellet.

ELISA ProcedureELISA Procedure

1.1. Add 100 uL protein to plate wells in triplicate.Add 100 uL protein to plate wells in triplicate.2.2. Add 100 uL of 2x Carbonate-Bicarbonate buffer to each Add 100 uL of 2x Carbonate-Bicarbonate buffer to each

well. Cover and store overnight at 4well. Cover and store overnight at 4°C.°C.3.3. Add 200 uL of 50% FBS in PBS to each well. Mix for 1 Add 200 uL of 50% FBS in PBS to each well. Mix for 1

hour. This is the blocking solution.hour. This is the blocking solution.4.4. Wash plate out with TBS-Tween 3 timesWash plate out with TBS-Tween 3 times5.5. Add 200uL Primary Antibody Solution to each well. Mix Add 200uL Primary Antibody Solution to each well. Mix

for 1hour at 37ºCfor 1hour at 37ºC6.6. Wash plate out with TBS-Tween 3 times.Wash plate out with TBS-Tween 3 times.7.7. Add 200ul Secondary Antibody Solution to each well. Add 200ul Secondary Antibody Solution to each well.

Mix for 1 hour at 37ºC.Mix for 1 hour at 37ºC.8.8. Wash plate out with TBS-Tween 3 times.Wash plate out with TBS-Tween 3 times.9.9. Add 200 uL of alkaline phosphatase substrate. Mix for Add 200 uL of alkaline phosphatase substrate. Mix for

30 minutes at 25ºC.30 minutes at 25ºC.10.10. Read the absorbance in a 96-well plate reader at 405 Read the absorbance in a 96-well plate reader at 405

nm.nm.

Experiment 1Experiment 1

• Antigen- Antigen- – CYP450 2B1 Varied from 1000 to 1 CYP450 2B1 Varied from 1000 to 1

femtomoles per well.femtomoles per well.– Microsomes from normal rat 10 to 1 ug/mL.Microsomes from normal rat 10 to 1 ug/mL.

• 1º Antibody-1º Antibody-– Anti-rat CYP450 2B1 1:5000 dilution.Anti-rat CYP450 2B1 1:5000 dilution.

• 2º Antibody conjugated to Alkaline 2º Antibody conjugated to Alkaline PhosphatasePhosphatase– 1:30,000 dilution. 1:30,000 dilution.

• Resulted in no activity detected.Resulted in no activity detected.

Experiment 2Experiment 2

• Antigen- Antigen- – CYP450 2B1 Varied from 1000 to 1 femtomoles CYP450 2B1 Varied from 1000 to 1 femtomoles

per well.per well.– Microsomes from normal rat 10 to 1 ug/mL.Microsomes from normal rat 10 to 1 ug/mL.

• 1º Antibody-1º Antibody-– Anti-rat CYP450 2B1 1:1000 or 1:2000 dilutions.Anti-rat CYP450 2B1 1:1000 or 1:2000 dilutions.

• 2º Antibody conjugated to Alkaline 2º Antibody conjugated to Alkaline PhosphatasePhosphatase– 1:10,000 dilution. 1:10,000 dilution.

• Resulted in variable and low activity.Resulted in variable and low activity.

ELISA Graph of Trial 2ELISA Graph of Trial 2

Day 2 ELISA

y = 0.0001x + 0.188

R2 = 0.772

y = 9E-05x + 0.0833

R2 = 0.8931

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Femtomoles of Cytochrome P450 2B1

Absorbance

1:1000AVE1:2000AVELinear (1:1000AVE)Linear (1:2000AVE)

Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mg Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mg of rat microsomesof rat microsomes

Experiment 3Experiment 3

• Antigen- Antigen- – CYP450 2B1 Varied from 1000 to 10 femtomoles per CYP450 2B1 Varied from 1000 to 10 femtomoles per

well.well.– Microsomes from normal rat 10 to 2.5 ug/mL.Microsomes from normal rat 10 to 2.5 ug/mL.– Microsomes from Phenobarbital treated rat 10 to 2.5 Microsomes from Phenobarbital treated rat 10 to 2.5

ug/mL.ug/mL.

• 1º Antibody-1º Antibody-– Anti-rat CYP450 2B1 1:1000 or 1:500 dilution.Anti-rat CYP450 2B1 1:1000 or 1:500 dilution.

• 2º Antibody conjugated to Alkaline 2º Antibody conjugated to Alkaline PhosphatasePhosphatase– 1:5,000 dilution. 1:5,000 dilution.

Trial 3 GraphTrial 3 Graph

Day 3 ELISA y = 0.0004x + 0.4619

R2 = 0.782

y = 0.0002x + 0.3103

R2 = 0.7103

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Femtomoles of Cytochrome P450

Absorbance

Series1

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Linear (Series1)

Linear (Series2)

• Using 1:1000, found 874 picomoles of cytochrome P450 2B1 per Using 1:1000, found 874 picomoles of cytochrome P450 2B1 per mg of normal rat microsomes and 4574 picomoles of cytochrome mg of normal rat microsomes and 4574 picomoles of cytochrome P450 2B1 per mg of phenobarbital rat microsomes P450 2B1 per mg of phenobarbital rat microsomes

Comparison of Day 2 and Day 3 1:1000 Data

y = 0.0002x + 0.3103

R2 = 0.7103

y = 0.0001x + 0.188

R2 = 0.772

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0 200 400 600 800 1000 1200Femtomoles of Cytochrome P450

Absorbance

Series1

Series2

Linear (Series1)

Linear (Series2)

Comparison of 2º Antibody Comparison of 2º Antibody Concentrations From Trial 2 and 3.Concentrations From Trial 2 and 3.

Day 3

Day 2

Experiment 4Experiment 4• Antigen- Antigen-

– CYP450 2B1 Varied from 1000 to 10 femtomoles per CYP450 2B1 Varied from 1000 to 10 femtomoles per well.well.

– Crude extract of tissue culture from H4IIE, an Crude extract of tissue culture from H4IIE, an immortalized cell line of rat hepatocytes, 10 to 2.5 immortalized cell line of rat hepatocytes, 10 to 2.5 ug/mL.ug/mL.

– Microsomes from cell extract of H4IIE 10 to 2.5 Microsomes from cell extract of H4IIE 10 to 2.5 ug/mL.ug/mL.

• 1º Antibody-1º Antibody-– Anti-rat CYP450 2B1 1:500 dilution.Anti-rat CYP450 2B1 1:500 dilution.

• 2º Antibody conjugated to Alkaline Phosphatase2º Antibody conjugated to Alkaline Phosphatase– 1:5,000 dilution.1:5,000 dilution.

Trial 4 GraphTrial 4 Graph

Day 4 ELISA

y = 0.0004x + 0.2065

R2 = 0.9886

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femtomoles of Cytochrome P450

AbsorbanceSeries1

Linear (Series1)

No activity was detected in either the crude or microsomal cell extract.

ConclusionsConclusions

• Successfully developed an ELISA assay to Successfully developed an ELISA assay to measure Cytochrome P450 2B1 protein.measure Cytochrome P450 2B1 protein. – Optimized antigen, 1º and 2º antibody concentrationsOptimized antigen, 1º and 2º antibody concentrations..

• Measured Cytochrome P450 2B1 from normal Measured Cytochrome P450 2B1 from normal and phenobarbital treated rats. and phenobarbital treated rats. – There was increased levels of P450 2B1 in the There was increased levels of P450 2B1 in the

phenobarbital treated animals.phenobarbital treated animals.• Unable to detect Cytochrome P450 2B1 in tissue Unable to detect Cytochrome P450 2B1 in tissue

cultures of H4IIE rat hepatocytes.cultures of H4IIE rat hepatocytes.