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Human DNA
（cytosine-5）-methyltransferase 1
（DMNT1）ELISA Kit
（96T）
This immunoassay kit allows for the in vitro quantitative determination of human DMNT1
concentrations in serum, plasma.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to DMNT1. Standards or samples are then added to the
appropriate microtiter plate wells with a HRP-conjugated antibody
preparation specific for DMNT1 to each microplate well and incubated.
Then a TMB （3,3',5,5' tetramethyl-benzidine） substrate solution is added
to each well. Only those wells that contain DMNT1, HRP-conjugated
antibody 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 DMNT1 in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
156 ng/ml-10000 ng/ml. The standard curve concentrations used for the
ELISA’s were 10000 ng/ml, 5000 ng/ml, 2500 ng/ml, 1250 ng/ml, 625
ng/ml, 312 ng/ml, 156 ng/ml.
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SPECIFICITY
This assay recognizes recombinant and natural human DMNT1. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human DMNT1 is typically less than 39
ng/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.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 1
Sample Diluent 1 x 20 ml
HRP-antibody Diluent 1 x 10 ml
HRP-antibody 1 x 120μl
Wash Buffer
1 x 20 ml
（25×concentrate）
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
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STORAGE
1. Unopened test kits should be stored at 2-8°C upon receipt and the
microtiter plate should be kept in a sealed bag. The test kit may be
used throughout the expiration date of the kit. Refer to the package
label for the expiration date.
2. Opened test kits will remain stable until the expiring date shown,
provided it is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less and an
optical density range of 0-3 OD or greater at 450nm wavelength is
acceptable for use in absorbance measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm up to
room temperature and mix gently until the crystals have compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 500 ml of Wash Buffer.
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2. Standard Reconstitute the Standard with 1.0 ml of Sample
Diluent. This reconstitution produces a stock solution of 400 ng/ml.
Allow the standard to sit for a minimum of 15 minutes with gentle
agitation prior to making serial dilutions. The undiluted standard
serves as the high standard （10000 ng/ml）. The Sample Diluent
serves as the zero standard （0 ng/ml）.
3. HRP-antibody Dilute to the working concentration using
HRP-antibody Diluent（1:100）, respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye,
hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450 nm, with
the correction wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate washer.
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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 1000 g.
Remove serum and assay immediay or aliquot and store samples at
-20° C. Avoid repeated freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes
of collection. Assay immediay or aliquot and store samples at
-20°C. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended
that all samples, standards, and controls be assayed in duplicate.
1. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 30min at 37°C.
2. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash by filling each well with Wash Buffer
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（200μl） 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.
3. Add 100μl of HRP -antibody working solution to each well. Incubate
for 30min at 37°C. HRP-antibody working solution may appear
cloudy. Warm up to room temperature and mix gently until solution
appears uniform.
4. Repeat the aspiration and wash five times as before.
5. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes
at 37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
6. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
7. Determine the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm.
CALCULATION OF RESULTS
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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 y-axis
against the concentration on the x-axis and draw a best fit curve through
the points on the graph. The data may be linearized by plotting the log of
the DMNT1 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.
LIMITATIONS OF THE PROCEDURE
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.
It is important that the Calibrator Diluent selected for the standard
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curve be consistent with the samples being assayed.
If samples generate values higher than the highest standard, dilute the
samples with the appropriate Calibrator 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,
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.
TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoid
foaming.
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.
When using an automated plate washer, adding a 30 second soak
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period following the addition of wash buffer, and/or rotating the plate
180 degrees between wash steps may improve assay precision.
To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary.
Substrate Solution should remain colorless until added to the plate.
Keep Substrate Solution protected from light. Substrate Solution
should change from colorless to gradations of blue.
Stop Solution should be added to the plate in the same order as the
Substrate Solution. 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 Substrate Solution.
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