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当前位置:首页 > 产品列表 > 【ELISA专栏】 > 人1,25二羟基维生素D3(1,25 DHVD3)ELISA试剂盒
人1,25二羟基维生素D3(1,25 DHVD3)ELISA试剂盒
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Human 1,25-dihydroxyvitamin D3 (DVD/DHVD 3) 
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
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.
1000 fmol/L-5000 fmol/L.
The minimum detectable dose of human DHVD 3 is typically less than  250
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.
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
Intra-assay Precision (Precision within an assay): CV%<10%
Three samples of known concentration were tested twenty times on one plate to 
Inter-assay Precision (Precision between assays): CV%<15%
Three samples of known concentration were tested in twenty assays to assess.
? 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
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
Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date.
Opened kit
Coated assay 
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.
May be stored for up to one week at 2 - 8°C. 
High Value 
Middle Value 
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
? 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 
? 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.
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, 
and clothing protection when using this material.
? 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 
? 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
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.
? 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 
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
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
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 
*Samples may require dilution. Please refer to Sample Preparation section.10
1. The final experimental results will be closely related to validity of the 
products, operation skills of the end users and the experimental 
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
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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