ELISA%20to%20Measure%20Cytochrome%20P450%20Protein%20Concentration

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ELISA to Measure Cytochrome P450 Protein
Concentration

• by William Collins

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Objectives

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

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What Is An ELISA?

• 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.

• E- Enzyme
• L- Linked
• I- Immuno
• S- Sorbent
• A- Assay

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Alkaline Phosphatase
Secondary Antibody
Primary Antibody
CYP450
Blocking Agent
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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
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Sulphan Blue Results
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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.

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Abbreviations

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

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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.

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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 4C.
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.

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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 15000 dilution.
• 2º Antibody conjugated to Alkaline Phosphatase
• 130,000 dilution.
• Resulted in no activity detected.

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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 11000 or 12000 dilutions.
• 2º Antibody conjugated to Alkaline Phosphatase
• 110,000 dilution.
• Resulted in variable and low activity.

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ELISA Graph of Trial 2
11000
12000

• Using 11000, found 705 picomoles of cytochrome
  P450 2B1 per mg of rat microsomes

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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 11000 or 1500 dilution.
• 2º Antibody conjugated to Alkaline Phosphatase
• 15,000 dilution.

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Trial 3 Graph
1500
11,000

• Using 11000, 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

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Comparison of 2º Antibody Concentrations From
Trial 2 and 3.
Day 3
15,000
110,000
Day 2
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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 1500 dilution.
• 2º Antibody conjugated to Alkaline Phosphatase
• 15,000 dilution.

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Trial 4 Graph
No activity was detected in either the crude or
microsomal cell extract.
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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.