ELISA to MeasureCytochrome P450Protein Concentration
Objectives• To develop an ELISA procedureto measure Cytochrome P450protein.
What Is An ELISA?• E- Enzyme• L- Linked• I- Immuno• S- Sorbent• A- Assay• This technique isdesigned to providean ultra-sensitiveprocess withdependable results.• It uses a 96-wellplate to measure aprotein orsubstance basedon anantigen/antibodyreaction.
Steps Involved in an ELISA• Bind the protein or antigento the plate.• Then you block the plate toget rid of any non-specificbinding sites.• Incubate with the primaryantibody which is specificfor the antigen.• Secondary antibody that islinked with an Enzyme isallowed to bind with theprimary antibody.• Use a Substrate for theenzyme which will causecolor to be released.96 Well Plate
Microsomes• We removed the liver from anormal rat and from aPhenobarbital treated rat.• Use a Potter-Eljehamhomogenizer at 1000 RPM tocreate a homogenate.• Centrifuge the homogenate at600g for 10 minutes to producea crude homogenate.• Centrifuge the remainingsupernatant at 15,000g for 1hour to separate out themitochondrial pellet.• Centrifuge the remainingsupernatant at 100,000g for 1hour to yield the microsomalpellet.
ELISA Procedure1. Add 100 uL protein to plate wells in triplicate.2. Add 100 uL of 2x Carbonate-Bicarbonate buffer toeach well. Cover and store overnight at 4°C.3. Add 200 uL of 50% FBS in PBS to each well. Mixfor 1 hour. This is the blocking solution.4. Wash plate out with TBS-Tween 3 times5. Add 200uL Primary Antibody Solution to each well.Mix for 1hour at 37ºC6. Wash plate out with TBS-Tween 3 times.7. Add 200ul Secondary Antibody Solution to eachwell. Mix for 1 hour at 37ºC.8. Wash plate out with TBS-Tween 3 times.9. Add 200 uL of alkaline phosphatase substrate. Mixfor 30 minutes at 25ºC.10. Read the absorbance in a 96-well plate reader at405 nm.
Experiment 1• Antigen-– CYP450 2B1 Varied from 1000 to 1femtomoles per well.– Microsomes from normal rat 10 to 1ug/mL.• 1º Antibody-– Anti-rat CYP450 2B1 1:5000 dilution.• 2º Antibody conjugated to AlkalinePhosphatase– 1:30,000 dilution.• Resulted in no activity detected.
Experiment 2• Antigen-– CYP450 2B1 Varied from 1000 to 1femtomoles per well.– Microsomes from normal rat 10 to 1ug/mL.• 1º Antibody-– Anti-rat CYP450 2B1 1:1000 or 1:2000dilutions.• 2º Antibody conjugated to AlkalinePhosphatase– 1:10,000 dilution.• Resulted in variable and low activity.
ELISA Graph of Trial 2Day 2 ELISAy = 0.0001x + 0.188R2= 0.772y = 9E-05x + 0.0833R2= 0.89310.0000.0500.1000.1500.2000.2500.3000.3500 200 400 600 800 1000 1200Femtomoles of Cytochrome P450 2B1Absorbance1:1000AVE1:2000AVELinear (1:1000AVE)Linear (1:2000AVE)Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mgof rat microsomes
Experiment 3• Antigen-– CYP450 2B1 Varied from 1000 to 10femtomoles per well.– Microsomes from normal rat 10 to 2.5 ug/mL.– Microsomes from Phenobarbital treated rat10 to 2.5 ug/mL.• 1º Antibody-– Anti-rat CYP450 2B1 1:1000 or 1:500 dilution.• 2º Antibody conjugated to AlkalinePhosphatase– 1:5,000 dilution.
Trial 3 GraphDay 3 ELISA y = 0.0004x + 0.4619R2= 0.782y = 0.0002x + 0.3103R2= 0.71030.0000.1000.2000.3000.4000.5000.6000.7000.8000.9000 200 400 600 800 1000 1200Femtomoles of Cytochrome P450AbsorbanceSeries1Series2Linear (Series1)Linear (Series2)• Using 1:1000, found 874 picomoles of cytochrome P450 2B1 permg of normal rat microsomes and 4574 picomoles of cytochromeP450 2B1 per mg of phenobarbital rat microsomes
Comparison of Day 2 and Day 3 1:1000 Datay = 0.0002x + 0.3103R2= 0.7103y = 0.0001x + 0.188R2= 0.7720.0000.1000.2000.3000.4000.5000.6000 200 400 600 800 1000 1200Femtomoles of Cytochrome P450AbsorbanceSeries1Series2Linear (Series1)Linear (Series2)Comparison of 2º AntibodyConcentrations From Trial 2 and 3.Day 3Day 2
Experiment 4• Antigen-– CYP450 2B1 Varied from 1000 to 10femtomoles per well.– Crude extract of tissue culture fromH4IIE, an immortalized cell line of rathepatocytes, 10 to 2.5 ug/mL.– Microsomes from cell extract of H4IIE 10to 2.5 ug/mL.• 1º Antibody-– Anti-rat CYP450 2B1 1:500 dilution.• 2º Antibody conjugated to AlkalinePhosphatase– 1:5,000 dilution.
Trial 4 GraphDay 4 ELISAy = 0.0004x + 0.2065R2= 0.98860.0000.1000.2000.3000.4000.5000.6000.7000 200 400 600 800 1000 1200femtomoles of Cytochrome P450AbsorbanceSeries1Linear (Series1)No activity was detected in either thecrude or microsomal cell extract.
Conclusions• Successfully developed an ELISA assay tomeasure Cytochrome P450 2B1 protein.– Optimized antigen, 1º and 2º antibody concentrations.• Measured Cytochrome P450 2B1 from normaland phenobarbital treated rats.– There was increased levels of P450 2B1 in thephenobarbital treated animals.• Unable to detect Cytochrome P450 2B1 in tissuecultures of H4IIE rat hepatocytes.
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