Human Matrix Metalloproteinase 26，MMP26 ELISA Kit;Human MMP26 elisakit 说明书
FOR IN VITRO USE AND RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
[ INTENDED USE ]
quantitative measurement of MMP26 in human serum,plasma.
[ TEST PRINCIPLE ]
The microtiter plate provided in this kit has been pre-coated with an antibody specific to MMP26. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for MMP26. Next, Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain MMP26, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of MMP26 in the samples is then determined by comparing
the O.D. of the samples to the standard curve.
[ ASSAY PROCEDURE ]
1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.Add 100μL each of dilutions of standard （read Reagent Preparation）, blank and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.
2. Remove the liquid of each well, don’t wash.
3. Add 100μL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer.
4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,
multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells compley by snapping the plate onto absorbent paper. Totally wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
5. Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after covering it with the Plate sealer.
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37oC （Don't exceed 30 minutes）. Protect from light. The liquid will turn blue by the addition of Substrate Solution.
8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediay.
[ TYPICAL DATA ]
In order to make the calculation easier, we plot the O.D. value of the standard （X-axis） against the known concentration of the standard （Y-axis）, although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance （e.g. operator, pipetting technique, washing technique or temperature effects）, plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
Typical Standard Curve for Human MMP26 ELISA.
[ DETECTION RANGE ]
0.156-10ng/mL. The standard curve concentrations used for the ELISA’s were 10ng/mL, 5ng/mL, 2.5ng/mL,1.25ng/mL, 0.625ng/mL, 0.312ng/mL, 0.156ng/mL.
[ SENSITIVITY ]
The minimum detectable dose of human MMP26 is typically less than 0.052ng/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. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
[ ASSAY PROCEDURE SUMMARY ]
1. Prepare all reagents, samples and standards;
2. Add 100μL standard or sample to each well. Incubate 2 hours at 37oC;
3. Add 100μL prepared Detection Reagent A. Incubate 1 hour at 37oC;
4. Aspirate and wash 3 times;
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
6. Aspirate and wash 5 times;
7. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37oC;
8. Add 50μL Stop Solution. Read at 450nm immediay.
[ REAGENTS AND MATERIALS PROVIDED ]
Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1
Plate sealer for 96 wells 4
Standard （lyophilized） 2
Standard Diluent 1×20mL
Detection Reagent A （green） 1×120μL
Assay Diluent A （2 × concentrate） 1×6mL
Detection Reagent B （red） 1×120μL
Assay Diluent B （2 × concentrate） 1×6mL
TMB Substrate 1×9mL
Stop Solution 1×6mL
Wash Buffer （30 × concentrate） 1×20mL
Instruction manual 1
[ STORAGE OF THE KITS ]
1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the others should be at 4 oC.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note:
It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
Note:
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC （≤1month） or -80oC （≤2 months） to avoid loss of bioactivity and contamination.
2. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
3. When performing the assay, bring samples to room temperature.
