Data Sheet
APOLIPOPROTEIN D (GCDFP-24)
ELISA KIT
Assay Protocol
Specimen collection and storage:
CAUTION: Handle the serum samples as if capable of transmitting infectious disease. Masks, gloves and eye
protection should always be worn when handling potentially infectious human samples.
REAGENTS:
Reagents are standardized by lot number and must not be interchanged with reagents from another kit with a different lot number. Do not use components after the expiration date shown on the label.
Kit Components
12 Assay Stripwells (1 pkg.); 8-well polystyrene strips coated with monoclonal antibody (96 wells).
1 Plate frame for strip wells.
1 Bottle (250mL) PAT buffer.
2 O-Phenylenediamine (OPD) tablets (10mg each).
1 Vial Conjugate Concentrate; 50µl monoclonal antibody conjugated to horseradish peroxidase with preservative.
7 Vials of GCDFP-24 Standards (0, 15, 30, 60, 90, 120 and 150µg/mL) at 50µl per vial.
1 Vial High Level Control (65 ± 5 µg/mL) at 50µl per vial.
1 Vial Low Level Control (16 ± 2 µg/mL) at 50µl per vial.
1 Vial (250µl) 3.0% Hydrogen Peroxide.
1 PBS packet.
1 Bottle 200X Tween 20 (6ml).
1 Insert Booklet.
FOR RESEARCH USE ONLY. NOT FOR IN VITRO DIAGNOSTIC USE.
Equipment and materials required but not provided:
1.
5ml and 10ml graduated serological pipettes.
2.
20µl, 200µl, and 1000µl, adjustable single-channel pipettes (Glison Pipetman or equivalent).
3.
300µl adjustable multichannel pipette (Flow Laboratories or equivalent).
4.
200µl and 1000µl disposable pipette tips.
6.
Conical polystyrene tubes, 15ml with caps (Corning or equivalent).
9.
Dual wavelength microtiter plate reader capable of reading wavelengths 492nm and 620 (Titertek Multiscan Plus or equivalent).
10.
Glass-distilled or deionized water.
11.
1 L graduated cylinder
12.
13.
14.
15.
16.
5.0 molar sulfuric acid.
Reagent reservoirs for multichannel pipetters (Costar or equivalent).
Absorbent paper.
Latex or polyethylene gloves (Lab Safety Supply or equivalent).
Particle face mask (Lab Safety Supply or equivalent.).
Precautions (please read thoroughly):
1.
Gloves and a face mask should be worn at all times while performing the assay to prevent assay contamination from human saliva and protection from potentially infectious human material.
2.
O-phenylenediamine (OPD) is a potential carcinogen. Handle with care. Wear gloves when handling the
OPD tablets. Avoid contact with eyes, skin or clothing as OPD may cause irritation or an allergic skin
reaction. Avoid contact of the OPD tablets with non-metallic surfaces.
3.
All pipettes must be calibrated regularly.
4.
Use glass-distilled or deionized water for reagent preparation. Store the water in nonmetallic containers.
5.
Disposable plastic should be used for preparation of the conjugate and substrate solutions.
6.
Cross-contamination between reagents may invalidate the test results. Do not interchange the vial caps.
7.
Use a new pipette tip for each specimen to be assayed. When using the multichannel pipette use new tips
for each reagent to be added.
8.
All reagents and serum samples must be equilibrated at room temperature (20-25°C) prior to use.
9.
When preparing 5.0M sulfuric acid from concentrated (18M) sulfuric acid, always add the acid to the water, never the reverse. Sulfuric acid is extremely corrosive; wear safety glasses and handle with care.
10.
Wash assay wells thoroughly during the washing steps. Improper or insufficient washing at any stage of
the procedure may result in either false positive or false negative results.
11.
During all washing procedures wear disposable gloves and discard all reagents and washing solutions into
an appropriate container. It is recommended that household bleach be added to the container after the
waste material has been disposed in it. Do not add bleach to the container before discarding washing solutions because of possible splash back.
12.
Do not allow the wells to dry during the test. Drying of the wells may result in falsely high absorbance
values.
13.
Do not touch the bottom surface of the wells. Fingerprints or scratches may interfere with the reading of
the wells.
14.
Adherence to the humidity and temperature conditions and time periods for incubation is essential for accurate results.
Preparation of Reagents:
Note: Gloves and a face mask should be worn at all times while performing this assay to prevent assay contamination from human saliva and for the protection against potentially infectious samples.
1. Preparation of Standards and Controls: All standards and controls should be diluted 1/60 in PAT
buffer prior to use. Standards and controls are prediluted 1/50 prior to packaging. As such, the final
working dilution for controls and standards is 1/3000.
2. Preparation of Working Conjugate: Add 40µL of Conjugate Concentrate to 12ml of PAT buffer for a
1/300 dilution. This volume is sufficient for approximately 96 wells.
3. Preparation of Samples: Dilute samples 1:3000 (Dilution 2) in PAT buffer.
Dilution 1 (1:50): 10µL patient sample + 490 µL of PAT buffer.
Dilution 2 (1:60): 10µL from dilution1 + 590 µL of PAT buffer.
4. Note: The average level of GCDFP-24 in serum has been reported to be approximately 55µg/ml in serum
(1) and 15mg/ml in cyst fluid derived from needle aspirates(2).
5. Preparation of PBS: Rehydrate one package of PBS provided in the kit with 1 liter of distilled or deionized water.
6. Preparation of PT: To the liter of prepared PBS, add 5.0ml of 200X Tween 20 and equilibrate.
7. Preparation of Substrate (10 minutes before use): Add 1 OPD tablet to 10ml of deionized water and
mix to solubilize. Immediately before addition to the assay wells add 50µl of 3% hydrogen peroxide to
the substrate solution (10.0ml), mix by inverting the tube several times and pour the solution into a reagent reservoir.
Test Procedure
Figure 1. A representative microtitration plate or stripwell pattern.
1
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A
0
90
L .C .
U
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B
0
90
L .C .
U
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U
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C
15
120
H .C .
U
U
U
U
U
U
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D
15
120
H .C .
U
U
U
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U
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E
30
150
U
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F
30
150
U
U
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G
60
B la n k
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H
60
B la n k
U
U
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U
U
U
U
U
1.
After equilibrating to room temperature, remove the pre-coated microtiter strip wells to be used from their
protective foil wrapper immediately prior to use. Unused strips may be resealed into the foil pouch along
with the desiccant packets. Prepare standards, controls, and samples in accordance with the Preparation
of Reagents section.
2. Beginning at the upper left hand corner of the pre-coated wells and working down each eight well column, add 100µl per well in duplicate of each of the following: seven calibration standards, two controls,
followed by samples (see Figure 1 for example). Cover the assay wells with a plate cover or Parafilm and
incubate the wells overnight at 4oC. The plate should be incubated under conditions of no light.
3. Empty the contents of the wells into an appropriate waste container according to your biohazard regulations (see ‘Precautions’). Wash the wells three times with PT at a volume of 250µl per well. Prepare the
conjugate in accordance with the Preparation of Reagents section.
4. Dispense the diluted conjugate into a disposable reagent reservoir. Using a multichannel pipette, add
100µl of the diluted conjugate to all assay wells. Incubate for 2 hours at room temperature (20-25o C).
This should also be performed under no light conditions.
5. Prepare OPD substrate solution 10 minutes before the end of the conjugate incubation period as described
in the Preparation of Reagents section.
6. Wash each well three times with PT as described in step 3.
7. Dispense the OPD substrate solution into a clean disposable reagent reservoir. Using a multichannel pipette, add 150µl of substrate solution to all assay wells. The addition should be completed within two
minutes.
8. Cover the assay wells with a plate cover or Parafilm and incubate at room temperature (20-25o C) for
20minutes.
9. At the end of the incubation period, add 50µl of 5.0 molar sulfuric acid to each well using a multichannel
pipette. Wipe off any water remaining on the underside of the microtitration wells. Air bubbles can cause
misleading results.
10. Set the microtiter plate reader at 492nm for the test wavelength (T) and 620nm for the reference wavelength (R). Read the wells using the dual wavelength setting (T- R). Blank the reader on well A1 or B1
containing the 0 ng/ml standard. Refer to the manufacturer's instructions for blanking the reader. Measure the color intensity in all wells. In order to check for reagent control, blank the reader again without
the wells in the light path, and directly measure wells A1 and B1.
11. Any serum sample having an O.D. reading greater than the 120µg/ml standard needs to be diluted and
retested in another assay. Note: A 150µg/ml standard is included as an assay reference point.
12. After the wells have been run, dispose of them in accordance with your hazardous waste regulations. Sulfuric acid is extremely corrosive. Do not pour the liquid contents down a drain or into a container to
which bleach will be added.
Calculation of Results:
Preparation of the Standard Curve and Determination of the GCDFP-24 Concentration for each
Sample.
Manual Graphic Method:
1. First average the O.D. values for each standard, control and unknown. On linear graph paper label the xaxis "GCDFP-24 concentration (µg/ml)" and the y-axis "O.D. (T- R)". Now plot each standard using the
O.D. values from your assay.
2. Starting at the origin draw a point to point curve through each of the standards.
3. Locate on the y-axis the point corresponding to the O.D. value of each control and unknown. For the controls and undiluted unknowns read the corresponding concentration (µg/ml) off the x-axis. For diluted
unknowns multiply the concentration on the x-axis by the dilution factor.
4. The resulting control values must be checked against their specific range values included with each kit. If
these values are not within their respective ranges, the test is invalid and must be repeated.
Automated Plate Reader Method:
Several microtitration plate readers with built-in statistical packages are available. The curve fit of choice
is the four parameter logistic model. If an option for multiplying patient results is not available, this must
be done manually. Please call SIGNET Technical Services for alternative data reduction recom mendations.
Illustration of Standard Curves using linear graph paper and the four parameter model.
These curves are for illustration purposes only and cannot be used to derive test results. Each
laboratory must prepare a new Standard Curve for each assay.
Test Controls.
Reagent Control:
When the plate reader is blanked against air and the wells (A1 & B1) containing the 0µg/mL standard are
measured, the resulting absorbance values provide the reagent control. The mean absorbance must be
less than 0.1 O.D. If the O.D. values are equal to or greater than 0.1, the test should be repeated using
freshly prepared reagents. In particular, check the color of the OPD solution before use. It should be
clear or only slightly yellow.
Low and High Controls:
The calculated values of the supplied controls must be checked against the ranges supplied with each kit.
If the calculated values are not within their respective ranges, the test is invalid and should be repeated.
Appendix
1.
Albers, J.J., et al., Characterization and immunoassay of apolipoprotein D.
Athero sclerosis 39: 395 - 409, 1981.
2.
Sanchez, Luis,M., Diez-Itza, Irene, Francisco, Vizoso, and Lopez-Otin, Carlos.
Cholestrol and Apolipoprotein D in Gross Cystic Disease of the Breast. Clin.
Chem. 38(5):695-698, 1992