2. ELISA to Measure Cytochrome P450 Protein Concentration by: William Collins

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

4. What Is An ELISA? E- EnzymeE- Enzyme L- LinkedL- Linked I- ImmunoI- Immuno S- SorbentS- Sorbent A- AssayA- 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.

6. 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

7. Sulphan Blue Results

8. 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.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.

9. Abbreviations uL- microlitersuL- microliters FBS- Fetal Bovine SerumFBS- Fetal Bovine Serum PBS- Phosphate Buffered SalinePBS- Phosphate Buffered Saline TBS- Tris Buffered SalineTBS- Tris Buffered Saline nm- nanometersnm- nanometers

10. 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.

11. 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.

12. Experiment 1 Antigen-Antigen- –CYP450 2B1 Varied from 1000 to 1 femtomoles per well. –Microsomes from normal rat 10 to 1 ug/mL. 1º Antibody-1º Antibody- –Anti-rat CYP450 2B1 1:5000 dilution. 2º Antibody conjugated to Alkaline Phosphatase2º Antibody conjugated to Alkaline Phosphatase –1:30,000 dilution. Resulted in no activity detected.Resulted in no activity detected.

13. Experiment 2 Antigen-Antigen- –CYP450 2B1 Varied from 1000 to 1 femtomoles per well. –Microsomes from normal rat 10 to 1 ug/mL. 1º Antibody-1º Antibody- –Anti-rat CYP450 2B1 1:1000 or 1:2000 dilutions. 2º Antibody conjugated to Alkaline Phosphatase2º Antibody conjugated to Alkaline Phosphatase –1:10,000 dilution. Resulted in variable and low activity.Resulted in variable and low activity.

14. ELISA Graph of Trial 2 Using 1:1000, found 705 picomoles of cytochrome P450 2B1 per mg of rat microsomes

15. Experiment 3 Antigen-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-1º Antibody- –Anti-rat CYP450 2B1 1:1000 or 1:500 dilution. 2º Antibody conjugated to Alkaline Phosphatase2º Antibody conjugated to Alkaline Phosphatase –1:5,000 dilution.

16. Trial 3 Graph 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 microsomesUsing 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

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

18. Experiment 4 Antigen-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-1º Antibody- –Anti-rat CYP450 2B1 1:500 dilution. 2º Antibody conjugated to Alkaline Phosphatase2º Antibody conjugated to Alkaline Phosphatase –1:5,000 dilution.

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

20. Conclusions Successfully developed an ELISA assay to measure Cytochrome P450 2B1 protein.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.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.Unable to detect Cytochrome P450 2B1 in tissue cultures of H4IIE rat hepatocytes.