CETP Activity Assay Kit Protocol

Supplied by Roar Biomedical, Inc

Catalog Number MAK106
Storage Temperature 2–8 °C

Components

The kit is sufficient for 100 assays in 200 µL total assay volume.

Donor Particle, 260 nmoles/mL 0.4 mL
Catalog Number MAK106A

Acceptor Particle 0.4 mL
Catalog Number MAK106B

CETP Assay Buffer 20 mL
Catalog Number MAK106C

Reagents and Equipment Required but Not Provided.
• 96 well U-bottom black plates for fluorescence assays.
• 37 °C water bath incubator
• Fluorescence multiwell plate reader
• 2-propanol (isopropanol, Catalog Number 34863)
• Torcetrapib (Catalog Number PZ0170), for assay validation

 

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Briefly centrifuge vials before opening.

Storage/Stability

The kit is shipped on wet ice. Storage at 2–8 °C, protected from light, is recommended. DO NOT FREEZE.
Components are stable for 1 year, if stored properly.

Assay substrates are stable at high DMSO concentration (up to10% v/v).
Note: High DMSO concentration affects the activity of purified CETP.

Procedures

All samples and standards should be run in duplicate.

Standards for Fluorometric Detection

  1. Disperse 5 µL of the Donor Particle solution (Catalog Number MAK106A) into 2 mL of 2-propanol. For example, a 260 nmoles/mL Donor Particle solution prepares a 1.3 nmole/tube standard solution. Using the 1.3 nmole/tube standard solution, make four serial 2-fold dilutions (1 mL of previous standard solution and 1 mL of 2-propanol), generating five tubes with decreasing amounts of fluorescent donor substrate.

  2. Add 200 µL of each standard dilution to a separate well of the multiwell plate, generating standards of 130, 65, 32.5, 16.3, and 8.1 pmole/well. 200 µL of 2-propanol is used as the 0 (Blank) Standard. Measure the fluorescence intensity (λex = 465/λem = 535 nm) of each standard. The standard curve is used for calculating pmoles of substrate transferred from fluorescence intensity units.

Sample Preparation

Notes:
If the CETP sample is plasma, the transfer reaction will occur without the exogenous Acceptor Particles due to endogenous acceptor plasma lipoproteins.

The CETP sample should NOT be stored at 2–8 °C. Samples should be stored at –80 °C to maintain activity. Rabbit plasma or serum has 2 to 2.5-fold the CETP activity of normal human plasma and must be kept frozen. Different plasma samples will have different CETP activity levels.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

  1. Combine 4 µL of Donor Particle solution and 4 µL of Acceptor Particle solution with the desired CETP sample (0.2–0.8 µL of undiluted plasma or serum, fresh or frozen). Bring final volume in well to 200 µl with CETP Assay Buffer. Prepare a sample blank that contains 4 µL of Donor Particle solution and 4 µL of Acceptor Particle solution and 192 µL of CETP Assay Buffer.

    Notes: Donor and Acceptor Particle solutions may be mixed with buffer and pipetted as one step.
    Plasma should be diluted ten-fold with buffer and then pipetted at 10x the original volume.

Assay Reaction

  1. Seal plate and incubate for 3 hours at 37 °C. Linearity may be accomplished with more plasma and a shorter incubation time.
    Notes: The microplate incubator must be able to rapidly raise the assay temperature to 37 °C. Large, humidified air incubators may cause problems by slowly increasing the temperature from 25 °C to only 34 °C after three hours. Floating the plate in a water bath is recommended, rather than using an air incubator.

    Never incubate the plate in the microplate reader.

    Fluorescent assays are highly sensitive and will respond to slight changes in assay volume. Microplate incubations must be placed in a sealed container with standing water to prevent evaporation. Microplates should be sealed as tightly as possible with plate sealers.

  2. Measure the increase in fluorescence of samples using a fluorimeter (λex = 465/λem = 535 nm). Determine the fluorescence intensity in the plasma or serum samples by subtracting the fluorescence intensity of the sample blank from each sample.
    Note: The fluorescence of the sample blank should NOT increase over time. It is normal for the sample blank to become slightly lower in intensity in the first 15 minutes, but never higher.

Validation Assay Reaction
Torcetrapib is a cholesteryl ester transfer protein (CETP) inhibitor.

  1. Dissolve torcetrapib, 6.00 mg, in 1 mL of freshly opened DMSO to generate a 10.00 mM Stock Solution.
    Note: Give extra consideration to the compound’s hydrophobic properties and the tendency of DMSO to absorb water from the atmosphere.

  2. Dilute the 10.00 mM Stock Solution 100-fold by adding 5 µL of the Stock Solution to 495 µL of DMSO generating a 100.0 µM solution.

  3. Add 50 µl of the 100.0 µM solution to 750 µl of DMSO to prepare a Torcetrapib Working Solution of 6.25 µM. Serial dilutions are made to give the following concentrations: 6.25, 3.12, 1.56, 0.78, and 0.078 µM. Use DMSO as 0 µM (inhibitor blank).

  4. Dilute plasma sample 10-fold with buffer and store on ice.

  5. Prepare Master Reaction Mix according to Table 1 (188 µL of Master Reaction Mix is required for each reaction well). Pipette 188 µL of the Master Reaction Mix into each reaction well of a black plate.

  6. Table 1. Master Reaction Mix

    Reagent Volume
    CETP Assay Buffer 180 mL
    Donor Particle 4 mL
    Acceptor Particle 4 mL


  7. Add 2 µL of each serial dilution of the Torcetrapib Working Solution and the inhibitor blank to separate wells, mixing well by pipetting. Add 10 µL of the diluted plasma, again mixing well by pipetting. This will give final torcetrapib concentrations of 62.5, 31.2, 15.6, 7.8, 0.78, and 0 nM per well.

  8. For a sample blank, add 10 µL of buffer and 2 µL of DMSO, in place of torcetrapib and sample solutions.
    Note: It is suggested to run negative control with torcetrapib solutions and buffer (in place of plasma sample) to make certain results are not an artifact from destruction of the donor/acceptor particles or a DMSO effect. The assay tolerates up to 10% DMSO.

  9. Seal plate and incubate for 3 hours at 37 °C.

  10. Measure the increase in fluorescence of samples using a fluorimeter (λex = 465/λem = 535 nm). Determine the fluorescence intensity in the plasma or serum samples by subtracting the sample blank fluorescence intensity from each sample.

Results

Temperature is a possible cause of difficulty in obtaining reproducible results. CETP activity will be reduced at temperatures below 37 °C. IC50 results will be impacted by temperature.

Multiple assay results from the same sample should be tight. Variability indicates evaporation, inaccurate pipetting, or incomplete mixing of assay components.

Assay results may be expressed in terms of pmoles of fluorescent substrate transferred. The substrate concentration of the Donor Particle solution (nmoles/mL) is printed on the label of the donor particle vial.

Calculate the pmoles of fluorescent substrate transferred from the standard curve using the fluorescence intensity values in the assay. Be sure to subtract the sample blank fluorescence intensity from the sample fluorescence intensity before attempting to enter the values into the regression or the values from the assay will be higher than the standard.

Human Plasma Assay

Table 2.
Typical Standard Curve

pmoles Fluorescence Intensity
duplicates
Average of duplicates
minus 0 (Blank)
Regression
Statistics
 
130 1873 1987 1619 Multiple R 0.999677
65 1145 1162 842 R2 0.999355
32.5 730 758 433 Adjusted R2 0.999193
16.25 489 557 212 Intercept 20
8.1 467 423 134 X Variable 1 12.4
0 (Blank) 313 310 0    

5 µL of Donor Particle solution dispersed in 2 mL of 2-propanol. Mixture is serially diluted 2-fold and 200 µL of each dilution is read. Concentration of Donor Particle solution = 260 nmoles/mL

Table 3.
Typical Sample Values

Plasma
Samples
Fluorescence Intensity, triplicates Average Average of
Fluorescence Intensity
minus Sample Blank
pmole
transferred
Sample Blank 1005 1035 1026 1022    
Sample 1 2094 2113 2065 2091 1069 84.6
Sample 2 1849 1730 1738 1772 750 58.9
Sample 3 1611 1652 1652 1638 616 48

Human plasma samples assayed with CETP activity kit. The plasma samples were incubated for 3 hours at 37 °C in a total assay volume of 200 µL (mixture of 4 µL each of Donor and Acceptor Particle solutions and 187 µL of buffer with 5 µL of 10-fold diluted plasma).

Figure 1.
CETP Activity in human plasma

CETP Activity in human plasma

Figure 2.
Torcetrapib Inhibition of CETP Activity

Torcetrapib Inhibition of CETP Activity

This product is supplied by Roar Biomedical, Inc. and covered by several patents including U.S. Pat. Nos. 5,585,235; 5,618,683; 5,770,355, and related US and foreign patents.

Materials

     
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