Manual of Human Matrix Metalloproteinase 2 (MMP-2) ELISA Test Kit

Human matrix metalloproteinase 2 (MMP-2) ELISA detection kit This reagent is for research use only Specimens: serum or plasma

Test principle:
The MMP-2 kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known MMP-2 concentrations and samples with unknown concentrations are added to microwell enzyme plates for detection. Incubate MMP-2 and biotin-labeled antibody first. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of MMP-2 in the sample.

Kit contents and preparation kit components (stored at 2-8 ° C) 96-well configuration 48-well configuration Preparation
96/48 serving microplate 1 plate (96T) half plate (48T) ready-to-use plastic membrane plate cover 1 half-plate ready-to-use standard: 800ng / ml 1 bottle (0.6ml) 1 bottle (0.3 ml) Carry out dilute blank control according to the instructions 1 bottle (1.0 ml) 1 bottle (0.5 ml) ready-to-use standard dilution buffer 1 bottle (5 ml) 1 bottle (2.5 ml) ready-to-use biotin-labeled anti-MMP- 2 Antibodies 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use affinity streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use wash buffer 1 bottle (20ml) 1 bottle (10ml) Dilute according to the instructions Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) Ready-to-use substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) Ready-to-use stop solution 1 bottle (6.0ml) 1 bottle (3.0ml) Ready-to-use

Bring your own materials
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500ul, 1000ul.
3. Oscillator and magnetic stirrer etc.

safety
1. Avoid direct contact with the stop solution and substrates A and B. Once exposed to these liquids, rinse with water as soon as possible.
2. Do not eat, drink, smoke or use cosmetics during the experiment.
3. Do not use your mouth to absorb any ingredients in the kit.

Operation notes
1. Reagents should be stored according to the label instructions and returned to room temperature before use. The sparse standards should be discarded and cannot be stored.
2. The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.
3. Unused Other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
4. Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and substrates A and B.
5. Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use.
6. When washing the enzyme-labeled plate, it should be fully patted dry. Do not put the absorbent paper directly into the enzyme-labeled reaction well to absorb water.
7. Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed.
8. The order of adding reagents should be the same to ensure that all wells are incubated for the same time.
9. Perform the incubation operation according to the time, the amount and order of the liquid indicated in the instructions.

Sample collection, processing and storage methods
1. Serum-Avoid any cell stimulation during the operation. Use test tubes free of pyrogens and endotoxins. After collecting blood, centrifuge at 1000 × g for 10 minutes to separate the serum and red blood cells quickly and carefully.
2. Plasma ----- EDTA, citrate and heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles.
3. The cell supernatant --- 1000 × g centrifuged for 10 minutes to remove particles and polymers.
4. Storage ------ If the sample is not used immediately, it should be divided into small parts and stored at -70 ℃ to avoid repeated freezing. If possible, do not use hemolysis or hyperlipidemia. If there are a large number of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 ° C or higher. Thaw at room temperature and ensure that the sample is thawed evenly and adequately.

Reagent preparation
1. Standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows:
800 ng / ml (standard No. 6) The original concentration is added directly to 50ul without dilution.
400 ng / ml (Standard 5) 100ul of the original standard plus 100ul of standard diluent
200 ng / ml (No. 4 standard) 100ul of No. 5 standard is added with 100ul of standard diluent
100 ng / ml (Standard No. 3) 100ul No. 4 Standard plus 100ul Standard Diluent
50 ng / ml (Standard No. 2) 100ul No. 3 Standard plus 100ul Standard Diluent
25 ng / ml (standard 1) 100ul of standard 2 is added with 100ul of standard diluent
0 ng / ml (blank control) The original concentration is directly added to 50ul without dilution.

2. Dilution of washing buffer (50 ×): 50-fold dilution with distilled water.

Steps
1. Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition.
2. The number of slats required is determined by the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible.
3. Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37 ° C for 1 hour.
4. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
5. Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes.
6. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
7. Add 50ul of substrate A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid light.
8. Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.
9. The OD value of each well was measured at a wavelength of 450 nm.

Recommended experimental protocol Standard concentration (ng / ml)
A 800 800 sample sample sample sample sample sample sample sample sample sample sample
B 400 400 sample sample sample sample sample sample sample sample sample sample sample
C 200 200 sample sample sample sample sample sample sample sample sample sample sample
D 100 100 sample sample sample sample sample sample sample sample sample sample sample
E 50 50 sample sample sample sample sample sample sample sample sample sample sample
F 25 25 sample sample sample sample sample sample sample sample sample sample sample
G 0 0 sample sample sample sample sample sample sample sample sample sample sample
H sample sample sample sample sample sample sample sample sample sample sample sample sample


Limitation
The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve.

Kit performance
1. Sensitivity: The smallest detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient R between the linear regression of the sample and the expected concentration is 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficients of variation within and between plates are less than 10%.

Judgment and analysis of results
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450nm

2. Take the absorbance OD value as the ordinate (Y) and the corresponding MMP-2 standard concentration as the abscissa (X), and make the corresponding curve. The MMP-2 content of the sample can be converted from the standard curve according to its OD value The corresponding concentration.

3. Detection value range: 0-800ng / ml

4. Sensitivity: 1.0 ng / ml

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