Instruction Manual for Rabbit Matrix Metalloproteinase 2 (MMP-2) ELISA Kit

This kit is for in vitro research use only, not for clinical diagnosis!

Intended application

Quantitative determination of MMP-2 in rabbit serum, plasma or other related biological fluids by ELISA.

Experimental principle

Coat the microplate with purified MMP-2 antibody to make a solid-phase carrier, add specimens or standards, biotinylated MMP-2 antibody, and HRP-labeled avidin to the microwells in turn, after thorough washing Use substrate (TMB) for color development. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with MMP-2 in the sample. Measure the absorbance (value) at 450nm with a microplate reader to calculate the sample concentration.

Kit composition and reagent preparation

1. Enzyme microplate: one piece (96 wells)

2. Standard product (lyophilized product): 2 bottles, please prepare within 15 minutes before use. Each bottle is diluted to 1ml with the sample diluent. After capping, it is allowed to stand at room temperature for about 10 minutes. At the same time, it is inverted / rubbed repeatedly to help dissolve. Its concentration is 200 ng / ml. After dilution to 50 ng / ml, do it again. Serial multiple dilutions (Note: Do not directly perform multiple dilutions in the plate), respectively prepared into 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml , 0.78 ng / ml, the sample dilution is directly used as a blank well 0 ng / ml. For example, to prepare a 25 ng / ml standard: take 0.5ml (not less than 0.5ml) and add 50 ng / ml of the above standard to an Eppendorf tube containing 0.5ml of sample diluent, mix well, and the rest of the concentration can be deduced by analogy.

3. Sample diluent: 1 × 20ml.

4. Detection of diluent A: 1 × 10ml.

5. Detection of diluent B: 1 × 10ml.

6. Detection solution A: 1 × 120 / bottle (1: 100). Before use, dilute with Test Diluent A 1: 100 (eg: 10 Test Solution A / 990 Test Diluent A), mix thoroughly, and prepare according to the pre-calculated total amount required for each experiment before dilution (100 / Hole), more 0.1-0.2ml should be prepared during actual preparation.

7. Detection solution B: 1 × 120 / bottle (1: 100). Before use, dilute with Test Diluent B 1: 100. The method of dilution is the same as that of Test Solution A.

8. Substrate solution: 1 × 10ml / bottle.

9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.

10. Stop solution: 1 × 10ml / bottle (2 mol / L H2SO4).

11. Lamination: 5 sheets

12. Instruction manual: 1 copy

Bring your own items

1. Microplate reader (It is recommended to preheat the instrument before use)

2. Micro-filler and pipette tip, EP tube

3. Distilled or deionized water, filter paper

Collection and preservation of specimens

1. Serum: Whole blood samples should be left at room temperature for 2 hours or 4 overnight. After centrifugation at 1000 g for 20 minutes, take the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated freezing and thawing. .

2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 1000 g for 15 minutes within 30 minutes after collection. Take the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated Freeze and thaw.

3. Other biological specimens: centrifuge at 1000 g for 20 minutes, take the supernatant for testing, or store the supernatant at -20 or -80, but avoid repeated freezing and thawing.

4. Sample processing: serum or plasma specimens are recommended to be diluted 20 times. The specimen was diluted with 0.1 M PBS (PH = 7.0-7.2).

Note: The above specimens should be sealed and stored, 4 should be stored for less than 1 week, -20 should not exceed 1 month, -80 should not exceed 2 months; specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested .

Steps

Before starting the experiment, all reagents should be equilibrated to room temperature, and the reagents should not be dissolved directly at 37; when preparing reagents or samples, they must be thoroughly mixed, and try to avoid foaming when mixing. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiplied by the corresponding dilution factor when calculating.

1. Sample adding: set blank hole, standard hole and sample hole to be tested respectively. Add 100% of the sample diluent to the blank well, and 100% of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the enzyme plate when adding the sample. Try not to touch the wall of the well. Cover or cover the target plate and incubate at 37 for 2 hours. To ensure that the experimental results are valid

For each experiment, please use a new standard solution.

2. Discard the liquid and dry it without washing. Add 100 working solutions of detection solution A (prepared before use) to each well, add the microplate to the membrane, and incubate at 37 for 1 hour.

3. Discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, about 400 per hole, spin dry (you can also pat the liquid in the hole dry).

4. Add testing solution B working solution (prepared before use) 100 to each well, add the coating, and incubate at 37 for 1 hour.

5. Discard the liquid in the hole, spin dry, wash the plate 5 times, the method is the same as step 3.

6. Add substrate solution 90 per well, enzyme label plate and film 37 to avoid color development (the reaction time is controlled at 15-30 minutes, when the first 3-4 wells of the standard wells have obvious blue gradient, the last 3 -4 When the pore gradient is not obvious, it can be terminated).

7. Add 50% stop solution to each well to stop the reaction. At this time, the blue color turns to yellow. The order of adding the stop solution should be the same as that of the substrate solution.

8. Immediately measure the optical density (value) of each well with a microplate reader at 450nm wavelength.

Note:

1. Reagent preparation: All reagents should be equilibrated to room temperature before use. Please save the reagents according to the instructions immediately after use. Please use disposable tips during the experiment to avoid cross contamination.

2. Sample addition: when adding samples or adding reagents, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which will obviously affect the measured value. Accuracy and repeatability. The time of one sample injection (including standard and all samples) is best controlled within 10 minutes. It is recommended to set up multiple holes for experiments.

3. Incubation: In order to prevent the sample from evaporating, the enzyme-labeled plate with a cover or film is placed in the wet box during the experiment to avoid liquid evaporation; the next step should be carried out as soon as possible after washing the plate, and should be avoided at any time The microplate is in a dry state; at the same time, the given incubation time and temperature should be strictly observed.

4. Washing: The washing liquid remaining in the reaction well during the washing process should be patted dry on the filter paper. Do not put the filter paper directly into the reaction well to absorb water. At the same time, the remaining liquid and fingerprints on the bottom of the plate should be eliminated to avoid affecting the final enzyme label. Instrument reading.

5. Reagent preparation: Detection A and Detection B should be shaken a few times or centrifuged for a short time before use, so that the liquid on the tube wall or bottle cap is deposited on the bottom of the tube. The standard product, the test solution A working solution, and the test solution B working solution should be prepared and used according to the required amount, and should be prepared using the corresponding diluent, which should not be confused. Please accurately prepare the standard products and working solutions, and try not to prepare them in a small amount (for example, when drawing the test solution A, it should not be less than 10 at a time) to avoid concentration errors due to inaccurate dilution; do not reuse the diluted standard products and test Solution A working fluid, detection solution B working fluid.

6. Control of reaction time: Please observe the color change of the reaction well regularly after adding the substrate (for example, every 10 minutes). If the color is darker, please add the stop solution in advance to stop the reaction to avoid the reaction being too strong and affecting the microplate reader. Optical density reading.

7. Substrate: Please keep the substrate away from light, and avoid direct exposure to strong light during storage and incubation.

Washing method

1. Manual plate washing method: Inject at least 0.4ml of the recommended washing buffer into the well, soak for 1-2 minutes, aspirate (do not touch the plate wall) or shake off the liquid in the enzyme plate, and lay a few layers on the experimental table Absorbent paper, with the microtiter plate down and vigorously pat several times; repeat this process several times as needed.

2. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used proficiently.

Specificity

This kit can detect recombinant or natural rabbit MMP-2 at the same time, and does not cross-react with other related proteins.

Calculation

Each standard and sample value is deducted from the blank hole value (7-point diagram). If multiple holes are set, the average value should be used for calculation. Taking the concentration of the standard product as the ordinate (logarithmic coordinate) and the value as the abscissa (logarithmic coordinate), draw a standard curve on the logarithmic coordinate paper. It is recommended to use professional curve making software for analysis, such as curve expert 1.3, according to the sample value, find the corresponding concentration from the standard curve and multiply it by the dilution factor; or use the concentration and value of the standard to calculate the regression equation of the standard curve The value of is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample.

Detection range: 0.78 ng / ml-50 ng / ml, please use the following concentration values ​​for drawing standard curve: 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml, 0.78 ng / ml.

Minimum detection limit: 0.195 ng / ml

Explanation

1. Due to the existing conditions and the level of science and technology, it is not possible to conduct a comprehensive identification and analysis of all raw materials provided by all suppliers,

This product may have certain quality and technical risks.

2. The final experimental results are closely related to the effectiveness of the reagents, the relevant operations of the experimenter and the experimental environment at that time, please be sure

Prepare sufficient specimens for backup.

3. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.

4. Storage of the kit: Please store the standard, detection solution A and detection solution B at -20 as soon as possible after receiving the kit, and store the remaining reagents at 4 for short-term storage and -20 for long-term storage. After opening, the microplate should be sealed with desiccant and stored at -20 to avoid moisture.

5. Salt will be precipitated from the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted.

6. There may be a little water-like substance in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not have any impact on the experimental results.

7. Validity: 6 months.

8. These operating instructions apply to the 48T kit, but all reagents in the 48T kit are halved.

Detailed Description:
Product  Name :Disposable Raincoat
Disposable raincoat is made of PE material.
Usage: It is very suitable for outdoor work. It`s very useful in the rain weather/  emergency raincoat
Service: ODM, OEM and buyer label service available.


Size

Each size is available  standard size is 78x120cm

Material

PE/PEVA material ,0.015~0.04mm . LDPE /HDPE 

Design

Designs can be choosen, customized works also are welcomed./ With or  

without  sleeve bottons

Feature

high quality with lower price.

Color

Blue,yellow, black, red etc. customized

Logo

Offset printing, screen printing

Package

Plastic bag

Sample Cost

According to item`s work and value

Sample Time

In 7days.

leading time

30 days


Process of the disposable raincoat:


Disposable Raincoat

Disposable Raincoat, Disposable Plastic Raincoats, PE Raincoat

Zhejiang CC Industrial And Trading Co., Ltd , http://www.ccraincoats.com