Human Atrial Natriuretic Peptide, ANP ELISA Kit

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Human Atrial Natriuretic Peptide, ANP ELISA Kit
Catalog No: E0225h
96 Tests
Operating instruction
www.eiaab.com
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
Intended use
This immunoassay kit allows for the in vitro quantitative determination of human Atrial
Natriuretic Peptide, ANP concentrations in cell culture supernates, serum, plasma and
other biological fluids.
Introduction
Atrial natriuretic peptide (ANP), atrial natriuretic factor (ANF), or atriopeptin, is a
polypeptide hormone involved in the homeostatic control of body water, sodium, and
adiposity. It is released by atrial myocytes, muscle cells in the atria of the heart, in
response to high blood pressure. ANP acts to reduce the water, sodium and adipose
loads on the circulatory system, thereby reducing blood pressure.
ANP is a 28 amino acid peptide with a 17 amino acid ring in the middle of the molecule.
The ring is formed by a disulfide bond between two cysteine residues at positions 7 and
23. ANP is closely related to BNP (brain natriuretic peptide) and CNP (C-type natriuretic
peptide) which all share the same amino acid ring. ANP was discovered in 1981 by a team
in Ottawa led by Adolfo J. de Bold after they made the seminal observation that injection
of atrial (but not ventricular) tissue extracts into rats caused copious natriuresis.
ANP is produced, stored and released by atrial myocytes, muscle cells in the atria of the
heart. It is released in response to a variety of signals induced by hypervolaemia, exercise
or caloric restriction. The hormone is constitutively expressed in the ventricle in response
to stress induced by increased afterload (eg. increased ventricular pressure from aortic
stenosis) or injury (eg. myocardial infarction).
Neutral endopeptidase (NEP)is the enzyme that metabolizes natriuretic peptides. Several
inhibitors of NEP are currently being developed to treat disorders ranging from
hypertension to heart failure. Most of them are dual inhibitors. Omapatrilat (dual inhibitor
of NEP and Angiotensin Converting Enzyme) developed by BMS did not receive FDA
approval due to angioedema safety concerns. Other dual inhibitors of NEP with ACE /
angiotensin receptor are currently being developed by pharmaceutical companies.
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to
ANP. Standards or samples are then added to the appropriate microtiter plate wells with a
biotin-conjugated polyclonal antibody preparation specific for ANP and Avidin conjugated
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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 ANP,
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 450 nm ± 2
nm. The concentration of ANP in the samples is then determined by comparing the O.D.
of the samples to the standard curve.
Materials and components
Reagent
Assay plate
Standard
Sample Diluent
Assay Diluent A
Assay Diluent B
Detection Reagent A
Detection Reagent B
Wash Buffer(25 x concentrate)
Substrate
Stop Solution
Plate sealer for 96 wells
Instruction
Quantity
1 × 20ml
2
1 × 20ml
1 × 10ml
1 × 10ml
1 × 120μl
1 × 120μl
1 × 30ml
1 × 10ml
1 × 10ml
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1
Other supplies required
Luminometer.
Pipettes and pipette tips.
EP tube
Deionized or distilled water.
Sample collection and storage
Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes
before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay
immediately or aliquot and store samples at -20℃ or -80℃.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples
for 15 minutes at 1000 × g at 2 - 8℃ within 30 minutes of collection. Store samples at
-20℃ or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates by
centrifugation and assay immediately or aliquot and store samples at -20℃ or -80℃.
Avoid repeated freeze-thaw cycles.
Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may
be stored at 2-8 ℃, otherwise samples must stored at -20℃ (≤ 1 months) or -80℃ (≤ 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.
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Limitations of the procedure
1.
2.
3.
4.
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, further dilute the
samples with the Assay Diluent 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.
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
exANPded.
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 30 mL of Wash Buffer
Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution
produces a stock solution of 20,000 pg/mL. Allow the standard to sit for a minimum of 15
minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the
wells directly is not permitted). Please firstly dilute the stock solution to 2,000 pg/mL and
the diluted standard serves as the high standard (2,000 pg/mL). The Sample Diluent
serves as the zero standard (0 pg/mL).
pg/mL 20,000 2,000
1,000
500
250
125
62.5
31.2
0
Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A
and B (1:100), respectively.
Assay procedure
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37℃
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
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optimal sample dilutions for their particular experiments.
1. Add 100 μl of Standard, Blank, or Sample per well. Cover with the Plate sealer.
Incubate for 2 hours at 37℃.
2. Remove the liquid of each well, don’t wash.
3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the
4.
5.
6.
7.
8.
9.
Plate sealer. Incubate for 1 hour at 37℃. Detection Reagent A working solution may
appear cloudy. Warm to room temperature and mix gently until solution appears
uniform.
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 400 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.
Add 100 μl of Detection Reagent B working solution to each well. Cover with a new
Plate sealer. Incubate for 1 hours at 37℃.
Repeat the aspiration/wash as in step 4.
Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer.
Incubate within 30 minutes at 37℃. Protect from light.
Add 50 μl of Stop Solution to each well. If color change does not appear uniform,
gently tap the plate to ensure thorough mixing.
Determine the optical density of each well at once, using a microplate reader set to
450 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
10μl 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 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each
standard level, between sample additions, and between reagent additions. Also, use
4
6.
7.
8.
separate reservoirs for each reagent.
The wash procedure is critical. Insufficient washing will result in poor precision and
falsely elevated absorbance readings.
Duplication of all standards and specimens, although not required, is recommended.
Substrate Solution is easily contaminated. Please protect it from light.
Specificity
This assay recognizes recombinant and natural human ANP. No significant
cross-reactivity or interference was observed.
Sensitivity
The minimum detectable dose of human ANP is typically less than 15.6 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
31.2-2,000 pg/mL. The standard curve concentrations used for the ELISA’s were 2,000
pg/mL, 1,000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL.
Calculation of results
Average the duplicate readings for each standard, control, and sample 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 (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 ANP
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 13.0. 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. Unopened test kits should be stored referring to the package label for frequent use,
and stored at -20℃ for long time storage. The unused strips should be kept in a
sealed bag and stored at 2-8℃ in their pouch 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.
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4. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range
of 0-3 OD or greater at 450nm 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. 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.
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