Descripción del producto
Intended use This immunoassay kit allows for the in vitro
quantitative determination of Human Intestinal trefoil factor, ITF
concentrations in cell culture supernates, serum, plasma, tissue
homogenates and other biological fluids. Test principle The
microtiter plate provided in this kit has been pre-coated with an
antibody specific to ITF. Standards or samples are then added to
the appropriate microtiter plate wells with a biotin-conjugated
polyclonal antibody preparation specific for ITF. Next, Avidin
conjugated to Horseradish Peroxidase (HRP) is added to each
microplate well and incubated. Then a TMB substrate solution is
added to each well. Only those wells that contain ITF,
biotin-conjugated antibody and enzyme-conjugated Avidin will
exhibit a change in color. The enzyme-substrate reaction is
terminated by the addition of a sulphuric acid solution and the
color change is measured spectrophotometrically at a wavelength of
**0 nm ± 2 nm. The concentration of ITF in the samples is then
determined by comparing the O.D. of the samples to the standard
curve. Materials and components Reagent Quantity Assay plate 1
Standard 2 Sample Diluent 1 × *0ml Assay Diluent A 1 × *0ml Assay
Diluent B 1 × *0ml Detection Reagent A 1 × **0μl Detection Reagent
B 1 × **0μl Wash Buffer(*5 x concentrate) 1 × *0ml Substrate 1 ×
*0ml Stop Solution 1 × *0ml Plate sealer for *6 wells 5 Instruction
1 Other supplies required Luminometer. Pipettes and pipette tips.
EP tube Deionized or distilled water. Sample collection and storage
Tissue homogenates - The preparation of tissue homogenates will
vary depending upon tissue type. For this assay, tissue was rinsed
with 1X PBS to remove excess blood, homogenized in 5~*0 mL of 1X
PBS and stored overnight at ≤ **0° C. After two freeze-thaw cycles
were performed to break the cell membranes, the homogenates were
centrifuged for 5 minutes at ***0 x g. Remove the supernate and
assay immediately or aliquot and store at ≤ **0° C. Other
biological fluids - Remove particulates by centrifugation and assay
immediately or aliquot and store samples at **0 or **0 . Avoid
repeated freeze-thaw cycles. Note: Tissue homogenates and other
biological fluids to be used within 7 days may be stored at **8 ,
otherwise samples must stored at **0 ( 1 month) or **0 ( 2 months)
to avoid loss of bioactivity and contamination. Avoid freeze-thaw
cycles. When performing the assay slowly bring samples to room
temperature. DO NOT USE HEAT-TREATED SPECIMENS. Limitations of the
procedure 1. The kit should not be used beyond the expiration date
on the kit label. 2. Do not mix or substitute reagents with those
from other lots or sources. 3. If samples generate values higher
than the highest standard, further dilute the samples and repeat
the assay. Any variation in standard diluent, operator, pipetting
technique, washing technique, incubation time or temperature, and
kit age can cause variation in binding. 4. This assay is designed
to eliminate interference by soluble receptors, ligands, binding
proteins, and other factors present in biological samples. Until
all factors have been tested in the Quantikine Immunoassay, the
possibility of interference cannot be excluded. 5. Limited by the
current condition and scientific technology, we can't completely
conduct the comprehensive identification and analysis on the raw
material provided by suppliers. So there might be some qualitative
and technical risks to use the kit. Reagent preparation Bring all
reagents to room temperature before use. Wash Buffer - If crystals
have formed in the concentrate, warm to room temperature and mix
gently until the crystals have completely dissolved. Dilute *0 mL
of Wash Buffer Concentrate into deionized or distilled water to
prepare **0 mL of Wash Buffer. Standard - Reconstitute the Standard
with 1.0 ml of Sample Diluent. This reconstitution produces a stock
solution of **0 pg/mL. Allow the standard to sit for about *0
minutes with gentle agitation prior to making serial dilutions
(Making serial dilution in the wells directly is not permitted).
The undiluted standard serves as the highest standard (**0 pg/mL).
The Sample Diluent serves as the zero standard (0 pg/mL). pg/mL **0
**0 **0 *0 *5 *2.5 6.*5 0 Detection Reagent A and B - Dilute to the
working concentration using Assay Diluent A or B (1:**0),
respectively. Assay procedure Allow all reagents to reach room
temperature (Please do not dissolve the reagents at *7 directly.).
All the reagents should be mixed thoroughly by gently swirling
before pipetting. Avoid foaming. Keep appropriate numbers of strips
for 1 experiment and remove extra strips from microtiter plate.
Removed strips should be resealed and stored at 4 until the kits
expiry date. Prepare all reagents, working standards and samples as
directed in the previous sections. 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. 1. Add **0 of Standard,
Blank, or Sample per well. Cover with the Plate sealer. Incubate
for two hours at *7 . 2. Remove the liquid of each well, don’t
wash. 3. Add **0 μl of Detection Reagent A working solution to each
well. Cover with the Plate sealer. Incubate for 1 hour at *7 .
Detection Reagent A working solution may appear cloudy. Warm to
room temperature and mix gently until solution appears uniform. 4.
Aspirate each well and wash, repeating the process three times for
a total of three washes. Wash by filling each well with Wash Buffer
(approximately **0 μl) using a squirt bottle, multi-channel
pipette, manifold dispenser or autowasher. Complete removal of
liquid at each step is essential to good performance. After the
last wash, remove any remaining Wash Buffer by aspirating or
decanting. Invert the plate and blot it against clean paper towels.
5. Add **0 μl of Detection Reagent B working solution to each well.
Cover with a new Plate sealer. Incubate for one hour at *7 . 6.
Repeat the aspiration/wash process for five times as conducted in
step 4. 7. Add *0 μl of Substrate Solution to each well. Cover with
a new Plate sealer. Incubate for *5 - *0 minutes at *7 . Protect
from light. 8. Add *0 μl of Stop Solution to each well. If color
change does not appear uniform, gently tap the plate to ensure
thorough mixing. 9. Determine the optical density of each well at
once, using a microplate reader set to **0 nm. Important Note: 1.
Absorbance is a function of the incubation time. Therefore, prior
to starting the assay it is recommended that all reagents should be
freshly prepared prior to use and all required strip-wells are
secured in the microtiter frame. This will ensure equal elapsed
time for each pipetting step, without interruption. 2. Please
carefully reconstitute Standards or working Detection Reagent A and
B according to the instruction, and avoid foaming and mix gently
until the crystals have completely dissolved. The reconstituted
Standards can be used only once. This assay requires pipetting of
small volumes. To minimize imprecision caused by pipetting, ensure
that pipettors are calibrated. It is recommended to suck more than
*0μl for once pipetting. 3. 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. 4. For each
step in the procedure, total dispensing time for addition of
reagents to the assay plate should not exceed *0 minutes. 5. 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. 6. The
wash procedure is critical. Insufficient washing will result in
poor precision and falsely elevated absorbance readings. 7.
Duplication of all standards and specimens, although not required,
is recommended. 8. Substrate Solution is easily contaminated.
Please protect it from light. Specificity This assay recognizes
recombinant and natural human ITF. No significant cross-reactivity
or interference was observed. Sensitivity The minimum detectable
dose of human ITF is typically less than 1.0 pg/mL. The sensitivity
of this assay, or Lower Limit of Detection (LLD) was defined as the
lowest protein concentration that could be differentiated from
zero. Detection Range 6.*****0 pg/mL. The standard curve
concentrations used for the ELISA’s were **0 pg/ml, **0 pg/ml, **0
pg/ml, *0 pg/ml, *5 pg/ml, *2.5 pg/ml, 6.*5 pg/ml. Calculation of
results Average the duplicate readings for each standard, control,
and samples and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer
software capable of generating a four parameter logistic (*-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 ITF concentrations versus the log of the
O.D. and the best fit line can be determined by regression
analysis. It is recommended to use some related software to do this
calculation, such as curve expert *3.0. 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. Storage of test kits and
instrumentation 1. The Standard, Detection Reagent A and Detection
Reagent B should be stored at **0 upon being received. Other
reagents are kept according to the labels on vials. But for long
term storage, please keep the whole kit at **0 . The unused strips
should be kept in a sealed bag with the desiccant provided to
minimize exposure to damp air. The test kit may be used throughout
the expiration date of the kit (six months from the date of
manufacture). Opened test kits will remain stable until the
expiring date shown, provided it is stored as prescribed above. 2.
There may be some foggy substance in the wells when the plate is
opened at the first time. It will not have any effect on the final
assay results. 3. Do not remove microtiter plate from the storage
bag until needed. 4. A microtiter plate reader with a bandwidth of
*0nm or less and an optical density range of **3 OD or greater at
**0nm wavelength is acceptable for use in absorbance measurement.
5. Use fresh disposable pipette tips for each transfer to avoid
contamination. 6. Do not substitute reagents from one kit lot to
another. Use only the reagents supplied by manufacturer. 7. The
final experimental results will be closely related to validity of
the products, operation skills of the end users and the
experimental environments. Please make sure that sufficient samples
are available. 8. Valid period: six months. Precaution The Stop
Solution suggested for use with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
País: |
China |
N º de Modelo: |
E0748h
|
Precio FOB: |
(Negociable)
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Lugar de origen: |
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Precio de pedido mínimo: |
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Cantidad de pedido mínimo: |
1 |
Detalle de embalaje: |
- |
El tiempo de entrega: |
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Capacidad de suministro: |
50 |
Tipo de pago: |
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Grupo de productos : |
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