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Human 1,25-dihydroxyvitamin D3 (DVD/DHVD 3)
ELISA Kit
Catalog Number. CSB-E05120h
For the quantitative determination of human 1,25-dihydroxyvitamin D3
(DVD/DHVD 3) concentrations in serum.
This package insert must be read in its entirety before using this product.
In order to obtain higher efficiency service, please ready to supply the lot number
of the kit to us (found on the outside of the box).2
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme immunoassay technique.
The microtiter plate provided in this kit has been pre-coated with an antibody.
Standards or samples are added to the appropriate microtiter plate wells with
Horseradish Peroxidase (HRP) conjugated DHVD 3 Hapten. The competitive
inhibition reaction is launched between with HRP-conjugated DHVD 3 Hapten
and DHVD 3 in samples. A substrate solution is added to the wells and the color
develops in opposite to the amount of DHVD 3 in the sample. The color
development is stopped and the intensity of the color is measured.
DETECTION RANGE
1000 fmol/L-5000 fmol/L.
SENSITIVITY
The minimum detectable dose of human DHVD 3 is typically less than 250
fmol/L.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as
the lowest human DHVD 3 concentration that could be differentiated from zero. It
was determined the mean O.D value of 20 replicates of the zero standard added
by their three standard deviations.
SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of human
DHVD 3. No significant cross-reactivity or interference between human DHVD 3
and analogues was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete
the cross-reactivity detection between human DHVD 3 and all the analogues,
therefore, cross reaction may still exist.3
PRECISION
Intra-assay Precision (Precision within an assay): CV%<10%
Three samples of known concentration were tested twenty times on one plate to
assess.
Inter-assay Precision (Precision between assays): CV%<15%
Three samples of known concentration were tested in twenty assays to assess.
LIMITATIONS OF THE PROCEDURE
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES.
? The kit should not be used beyond the expiration date on the kit label.
? Do not mix or substitute reagents with those from other lots or sources.
? If samples generate values higher than the highest standard, dilute the
samples with Sample Diluent and repeat the assay.
? Any variation in Sample Diluent, operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can cause variation
in binding.
? This assay is designed to eliminate interference by soluble receptors,
binding proteins, and other factors present in biological samples. Until all
factors have been tested in the Immunoassay, the possibility of
interference cannot be excluded.4
MATERIALS PROVIDED
Reagents Quantity
Assay plate 1(96 wells)
Standard (Freeze dried) 1
High Value Control (Freeze dried) 1
Middle Value Control (Freeze dried) 1
Sample Diluent 1 x 30 ml
HRP-conjugate 1 x 7 ml
Wash Buffer (20 x concentrate) 1 x 30 ml
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Adhesive Strip (For 96 wells) 4
Instruction manual 1
STORAGE
Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date.
Opened kit
Coated assay
plate
May be stored for up to 1 month at 2 - 8°C.
Try to keep it in a sealed aluminum foil bag,
and avoid the damp.
Standard
May be stored for up to one week at 2 - 8°C.
High Value
Control
Middle Value
Control
HRP-conjugate
May be stored for up to 1 month at 2 - 8°C.
Sample Diluent
Wash Buffer
Substrate A
Substrate B
Stop Solution
*Provided this is within the expiration date of the kit.5
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with the
correction wavelength set at 630 nm.
? An incubator which can provide stable incubation conditions up to
37°C±0.5°C.
? Squirt bottle, manifold dispenser, or automated microplate washer.
? Absorbent paper for blotting the microtiter plate.
? 100ml and 500ml graduated cylinders.
? Deionized or distilled water.
? Pipettes and pipette tips.
? Test tubes for dilution.
PRECAUTIONS
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face,
and clothing protection when using this material.
SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube (SST) and allow samples to clot for
two hours at room temperature or overnight at 4°C before centrifugation
for 15 minutes at 1000 ×g. Remove serum and assay immediately or
aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw
cycles.
SAMPLE PREPARATION
? Serum samples require a 10-fold dilution into Sample Diluent. The
suggested 10-fold dilution can be achieved by adding 25μl sample to
225μl of Sample Diluent. The recommended dilution factor is for reference
only. The optimal dilution factor should be determined by users according
to their particular experiments.6
Note:
1. CUSABIO is only responsible for the kit itself, but not for the samples
consumed during the assay. The user should calculate the possible
amount of the samples used in the whole test. Please reserve sufficient
samples in advance.
2. Samples to be used within 5 days may be stored at 2-8°C, otherwise
samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid
loss of bioactivity and contamination.
3. Grossly hemolyzed samples are not suitable for use in this assay.
4. If the samples are not indicated in the manual, a preliminary experiment to
determine the validity of the kit is necessary.
5. Please predict the concentration before assaying. If values for these are
not within the range of the standard curve, users must determine the
optimal sample dilutions for their particular experiments.
6. Tissue or cell extraction samples prepared by chemical lysis buffer may
cause unexpected ELISA results due to the impacts of certain chemicals.
7. Influenced by the factors including cell viability, cell number and also
sampling time, samples from cell culture supernatant may not be detected
by the kit.
8. Fresh samples without long time storage are recommended for the test.
Otherwise, protein degradation and denaturalization may occur in those
samples and finally lead to wrong results.
REAGENT PREPARATION
Note:
? Kindly use graduated containers to prepare the reagent. Please don't
prepare the reagent directly in the Diluent vials provided in the kit.
? Bring all reagents to room temperature (18-25°C) before use for 30min.
? Prepare fresh standard for each assay. Use within 4 hours and discard
after use.
? Making serial dilution in the wells directly is not permitted.
? Distilled water is recommended to be used to make the preparation for
reagents or samples. Contaminated water or container for reagent
preparation will influence the detection result.7
1. Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to
room temperature and mix gently until the crystals have completely
dissolved. Dilute 30 ml of Wash Buffer Concentrate (20 x) into deionized
or distilled water to prepare 600 ml of Wash Buffer (1 x).
2. Standard
Centrifuge the standard vial at 6000-10000rpm for 30s before opening.
Reconstitute the Standard with 0.5 ml of ddH2O. Do not substitute other
diluents. This reconstitution produces a stock solution of 200000 fmol/L.
Mix the standard to ensure complete reconstitution and allow the standard
to sit for a minimum of 15 minutes with gentle agitation prior to making
dilutions.
Dilute the 200000 fmol/L stock solution with Sample Diluent(1:40) to 5000
fmol/L(S4). The suggested 40-fold dilution can be achieved by adding 25μl
stock solution to 975μl of Sample Diluent.
Use S4 to dilute to follow concentrations (S1-S3). Mix each tube
thoroughly before the next transfer. S0 contain only Sample Diluent.
Tube S4 S3 S2 S1 S0
Concentration
(fmol/L)
5000 4000 2000 1000 0
S4 (μl) - 160 80 40 0
Sample Diluent (μl) - 40 120 160 2008
3. High Value Control - Centrifuge the High Value Control vial at
6000-10000rpm for 30s before opening. Reconstitute the High Value
Control with 0.5 ml of ddH2O. Mix the High Value Control to ensure
complete reconstitution and allow the High Value Control to sit for a
minimum of 15 minutes with gentle agitation prior to making dilution.
Dilute the High Value Control with Sample Diluent(1:40) before test. The
suggested 40-fold dilution can be achieved by adding 5μl High Value
Control to 195μl of Sample Diluent.
4. Middle Value Control - Centrifuge the Middle Value Control vial at
6000-10000rpm for 30s before opening. Reconstitute the Middle Value
Control with 0.5 ml of ddH2O. Mix the Middle Value Control to ensure
complete reconstitution and allow the Middle Value Control to sit for a
minimum of 15 minutes with gentle agitation prior to making dilution.
Dilute the Middle Value Control with Sample Diluent(1:40) before test.
The suggested 40-fold dilution can be achieved by adding 5μl Middle
Value Control to 195μl of Sample Diluent.9
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. Centrifuge
the sample again after thawing before the assay. It is recommended that all
samples, standards and controls be assayed in duplicate.
1. Prepare all reagents, working standards, controls and samples as directed
in the previous sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be
used and put any remaining wells and the desiccant back into the pouch
and seal the ziploc, store unused wells at 4°C.
3. Set a Blank well without any solution.
4. Add 50μl of standard, diluted control and diluted sample per well. Add
50μl HRP-conjugate to each well immediately (not to Blank well). Mix well
with the pipette or shake the plate gently for 60 seconds. A plate layout is
provided to record standards and samples assayed.
5. Cover with the adhesive strip provided. Incubate for 60 minutes at 37°C.
6. Aspirate each well and wash, repeating the process four times for a total of
five washes. Wash by filling each well with Wash Buffer (300μl) using a
squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,
and let it stand for 10 seconds, complete removal of liquid at each step is
essential to good performance. After the last wash, remove any remaining
Wash Buffer by aspirating ordecanting. Invert the plate and blot it against
clean paper towels.
7. Add 50μl of Substrate A and 50μl of Substrate B to each well. Incubate
for 15 minutes at 37°C. Protect from light.
8. Add 50μl of Stop Solution to each well, gently tap the plate to ensure
thorough mixing.
9. Determine the optical density of each well within 5 minutes, using a
microplate reader set to 450 nm. If wavelength correction is available, set
to 630 nm. Subtract readings at 630 nm from the readings at 450 nm. This
subtraction will correct for optical imperfections in the plate. Readings
made directly at 450 nm without correction may be higher and less
accurate.
*Samples may require dilution. Please refer to Sample Preparation section.10
Note:
1. The final experimental results will be closely related to validity of the
products, operation skills of the end users and the experimental
environments.
2. Samples or reagents addition: Please use the freshly prepared Standard.
Please carefully add samples to wells and mix gently to avoid foaming. Do
not touch the well wall as possible. For each step in the procedure, total
dispensing time for addition of reagents or samples to the assay plate
should not exceed 10 minutes. This will ensure equal elapsed time for each
pipetting step, without interruption. Duplication of all standards and
specimens, although not required, is recommended. To avoid
cross-contamination, change pipette tips between additions of each
standard level, between sample additions, and between reagent additions.
Also, use separate reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary. Do not allow wells to sit uncovered for
extended periods between incubation steps. Once reagents have been
added to the well strips, DO NOT let the strips DRY at any time during the
assay. Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at each
step is essential to good performance. After the last wash, remove any
remaining Wash Solution by aspirating or decanting and remove any drop of
water and fingerprint on the bottom of the plate. Insufficient washing will
result in poor precision and falsely elevated absorbance reading. When
using an automated plate washer, adding a 30 second soak period following
the addition of wash buffer, and/or rotating the plate 180 degrees between
wash steps may improve assay precision.
5. Controlling of reaction time: Observe the change of color after adding TMB
Substrate (e.g. observation once every 10 minutes), TMB Substrate should
change from colorless or light blue to gradations of blue. If the color is too
deep, add Stop Solution in advance to avoid excessively strong reaction
which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. TMB Substrate should remain
colorless or light blue until added to the plate. Please protect it from light.
7. Stop Solution should be added to the plate in the same order as the TMB
Substrate. The color developed in the wells will turn from blue to yellow
upon addition of the Stop Solution. Wells that are green in color indicate that
the Stop Solution has not mixed thoroughly with the TMB Substrate.11
CALCULATION OF RESULTS
Using the professional soft "Curve Expert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard and sample and subtract the
average optical density of Blank.
Create a standard curve by reducing the data using computer software capable
of generating a four parameter logistic (4-PL) curve-fit. As an alternative,
construct a standard curve by plotting the mean absorbance for each standard
on the x-axis against the concentration on the y-axis and draw a best fit curve
through the points on the graph. The data may be linearized by plotting the log of
the DHVD 3 concentrations versus the log of the O.D. and the best fit line can be
determined by regression analysis. This procedure will produce an adequate but
less precise fit of the data.
If samples have been diluted, the concentration read from the standard curve
must be multiplied by the dilution factor.
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