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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 DMNT1concentrations in serum, plasma.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.2PRINCIPLE OF THE ASSAYThe microtiter plate provided in this kit has been pre-coated with anantibody specific to DMNT1. Standards or samples are then added to theappropriate microtiter plate wells with a HRP-conjugated antibodypreparation specific for DMNT1 to each microplate well and incubated.Then a TMB (3,3,5,5 tetramethyl-benzidine) substrate solution is addedto each well. Only those wells that contain DMNT1, HRP-conjugatedantibody will exhibit a change in color. The enzyme-substrate reaction isterminated by the addition of a sulphuric acid solution and the colorchange is measured spectrophotometrically at a wavelength of 450 nm 2nm. The concentration of DMNT1 in the samples is then determined bycomparing the O.D. of the samples to the standard curve.DETECTION RANGE156 ng/ml-10000 ng/ml. The standard curve concentrations used for theELISA s were 10000 ng/ml, 5000 ng/ml, 2500 ng/ml, 1250 ng/ml, 625ng/ml, 312 ng/ml, 156 ng/ml.3SPECIFICITYThis assay recognizes recombinant and natural human DMNT1. Nosignificant cross-reactivity or interference was observed.SENSITIVITYThe minimum detectable dose of human DMNT1 is typically less than 39ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD)was defined as the lowest protein concentration that could bedifferentiated from zero.MATERIALS PROVIDEDReagent QuantityAssay plate 1Standard 1Sample Diluent 1 x 20 mlHRP-antibody Diluent 1 x 10 mlHRP-antibody 1 x 120 lWash Buffer1 x 20 ml(25 concentrate)TMB Substrate 1 x 10 mlStop Solution 1 x 10 ml4STORAGE1. Unopened test kits should be stored at 2-8 C upon receipt and themicrotiter plate should be kept in a sealed bag. The test kit may beused throughout the expiration date of the kit. Refer to the packagelabel 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 anoptical density range of 0-3 OD or greater at 450nm wavelength isacceptable for use in absorbance measurement.REAGENT PREPARATIONBring all reagents to room temperature before use.1. Wash Buffer If crystals have formed in the concentrate, warm up toroom temperature and mix gently until the crystals have completelydissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized ordistilled water to prepare 500 ml of Wash Buffer.52. Standard Reconstitute the Standard with 1.0 ml of SampleDiluent. This reconstitution produces a stock solution of 400 ng/ml.Allow the standard to sit for a minimum of 15 minutes with gentleagitation prior to making serial dilutions. The undiluted standardserves as the high standard (10000 ng/ml). The Sample Diluentserves as the zero standard (0 ng/ml).3. HRP-antibody Dilute to the working concentration usingHRP-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, withthe 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.6SAMPLE COLLECTION AND STORAGE
Serum Use a serum separator tube (SST) and allow samples to clotfor 30 minutes before centrifugation for 15 minutes at 1000 g.Remove serum and assay immediately or aliquot and store samples at-20 C. Avoid repeated freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as ananticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutesof collection. Assay immediately 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 PROCEDUREBring all reagents and samples to room temperature before use. It is recommendedthat all samples, standards, and controls be assayed in duplicate.1. Add 100 l of Standard, Blank, or Sample per well. Cover with theadhesive strip. Incubate for 30min at 37 C.2. Aspirate each well and wash, repeating the process three times for atotal of three washes. Wash by filling each well with Wash Buffer7(200 l) using a squirt bottle, multi-channel pipette, manifolddispenser or autowasher. Complete removal of liquid at each step isessential to good performance. After the last wash, remove anyremaining Wash Buffer by aspirating or decanting. Invert the plateand blot it against clean paper towels.3. Add 100 l of HRP -antibody working solution to each well. Incubatefor 30min at 37 C. HRP-antibody working solution may appearcloudy. Warm up to room temperature and mix gently until solutionappears uniform.4. Repeat the aspiration and wash five times as before.5. Add 90 l of TMB Substrate to each well. Incubate for 10-30 minutesat 37 C. Keeping the plate away from drafts and other temperaturefluctuations in the dark.6. Add 50 l of Stop Solution to each well. If color change does notappear uniform, gently tap the plate to ensure thorough mixing.7. Determine the optical density of each well within 30 minutes, using amicroplate reader set to 450 nm.CALCULATION OF RESULTS8Average the duplicate readings for each standard, control, and sample andsubtract the average zero standard optical density. Create a standard curveby reducing the data using computer software capable of generating a fourparameter logistic (4-PL) curve-fit. As an alternative, construct a standardcurve by plotting the mean absorbance for each standard on the y-axisagainst the concentration on the x-axis and draw a best fit curve throughthe points on the graph. The data may be linearized by plotting the log ofthe DMNT1 concentrations versus the log of the O.D. and the best fit linecan be determined by regression analysis. This procedure will produce anadequate but less precise fit of the data. If samples have been diluted, theconcentration read from the standard curve must be multiplied by thedilution 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 orsources.
It is important that the Calibrator Diluent selected for the standard9curve be consistent with the samples being assayed.
If samples generate values higher than the highest standard, dilute thesamples 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 cancause variation in binding.
This assay is designed to eliminate interference by soluble receptors,binding proteins, and other factors present in biological samples. Untilall factors have been tested in the Quantikine Immunoassay, thepossibility of interference cannot be excluded.TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoidfoaming.
To avoid cross-contamination, change pipette tips between additionsof each standard level, between sample additions, and betweenreagent additions. Also, use separate reservoirs for each reagent.
When using an automated plate washer, adding a 30 second soak10period following the addition of wash buffer, and/or rotating the plate180 degrees between wash steps may improve assay precision.
To ensure accurate results, proper adhesion of plate sealers duringincubation steps is necessary.
Substrate Solution should remain colorless until added to the plate.Keep Substrate Solution protected from light. Substrate Solutionshould change from colorless to gradations of blue.
Stop Solution should be added to the plate in the same order as theSubstrate Solution. The color developed in the wells will turn fromblue to yellow upon addition of the Stop Solution. Wells that aregreen in color indicate that the Stop Solution has not mixedthoroughly with the Substrate Solution.

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