Mouse type I collagen α2 enzyme-linked immunosorbent assay kit instruction manual-华强电子网

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Mouse type I collagen

α

2 (COL1a2)

ELISA kit

user's Guide

Product ID:

E-EL-M0318

(This kit is for in vitro studies only, not for clinical diagnosis.

!

)

statement:

Dear customers, thank you for choosing our products. This product uses the raw materials of world famous manufacturers, adopting

profession

ELISAkit

Production technology manufacturing. Suitable for in vitro quantitative testing

Mouse

Serum, plasma, tissue homogenate or cell culture

Natural and reconstituted in clear liquid

COL1a2

concentration.

Please read the instructions carefully and check the reagent components before use.

! If in doubt, please

Contact Elariot Biotech Co., Ltd.

Kit composition:

name

-

Chinese

name

-

English

specification

Storage Conditions

ELISA

Enzyme plate

MicroELISAPlate

8×

12/8×

6*

4

°C

Freeze-dried standard

ReferenceStandard

2/1

support

*

4

°C

Standard

&

Sample diluent

ReferenceStandard&SampleDiluent

1

bottle

20mL/12mL*

4

°C

Concentrated biotinylated antibody

ConcentratedBiotinylatedDetectionAb

1

support

120μ

L

/70μ

L*

4

°C

Biotinylated antibody dilution

DiluentforBiotinylatedDetectionAb

1

bottle

10mL/6mL*

4

°C

concentrate

HRP

Enzyme conjugate

ConcentratedHRPConjugate

1

support

120μ

L

/70μ

L*

4

°C

(

Protected from light

)

Enzyme conjugate dilution

DiluentforHRPConjugate

1

bottle

10mL/6mL*

4

°C

Concentrated washing solution

25×

)

ConcentratedWashBuffer

(

25×

)

1

bottle

30mL/16mL*

4

°C

Substrate solution

TMB

)

SubstrateReagent

1

bottle

10mL/6mL*

4

°C

(

Protected from light

)

Reaction stop solution

StopSolution

1

bottle

10mL/6mL*

4

°C

Sealing film

PlateSealer

5/3

Zhang

*

Product Manual

ProductDescription

1

Share

*:[96T/48T]

(Please check all items in time after opening the package)

Mouse type I collagen

α

2 (COL1a2)

ELISA kit

user's Guide

Product ID:

E-EL-M0318

(This kit is for in vitro studies only, not for clinical diagnosis.

!

)

statement:

Dear customers, thank you for choosing our products. This product uses the raw materials of world famous manufacturers, adopting

profession

ELISAkit

Production technology manufacturing. Suitable for in vitro quantitative testing

Mouse

Serum, plasma, tissue homogenate or cell culture

Natural and reconstituted in clear liquid

COL1a2

concentration.

Please read the instructions carefully and check the reagent components before use.

! If in doubt, please

Contact Elariot Biotech Co., Ltd.

Kit composition:

name

-

Chinese

name

-

English

specification

Storage Conditions

ELISA

Enzyme plate

MicroELISAPlate

8×

12/8×

6*

4

°C

Freeze-dried standard

ReferenceStandard

2/1

support

*

4

°C

Standard

&

Sample diluent

ReferenceStandard&SampleDiluent

1

bottle

20mL/12mL*

4

°C

Concentrated biotinylated antibody

ConcentratedBiotinylatedDetectionAb

1

support

120μ

L

/70μ

L*

4

°C

Biotinylated antibody dilution

DiluentforBiotinylatedDetectionAb

1

bottle

10mL/6mL*

4

°C

concentrate

HRP

Enzyme conjugate

ConcentratedHRPConjugate

1

support

120μ

L

/70μ

L*

4

°C

(

Protected from light

)

Enzyme conjugate dilution

DiluentforHRPConjugate

1

bottle

10mL/6mL*

4

°C

Concentrated washing solution

25×

)

ConcentratedWashBuffer

(

25×

)

1

bottle

30mL/16mL*

4

°C

Substrate solution

TMB

)

SubstrateReagent

1

bottle

10mL/6mL*

4

°C

(

Protected from light

)

Reaction stop solution

StopSolution

1

bottle

10mL/6mL*

4

°C

Sealing film

PlateSealer

5/3

Zhang

*

Product Manual

ProductDescription

1

Share

*:[96T/48T]

(Please check all items in time after opening the package)

Mouse Type I Collagen α2 (COL1a2) ELISA Kit Instructions (This kit is for in vitro studies only, not for clinical diagnosis!)
Disclaimer: Dear customers, thank you for choosing our products. This product is made from the raw materials of world famous manufacturers and is manufactured by professional ELISA kit production technology. It is suitable for quantitative detection of natural and recombinant COL1a2 concentrations in mouse serum, plasma, tissue homogenate or cell culture supernatant in vitro. Please read the instructions carefully and check the reagent components before use! If you have any questions, please contact Shanghai Jinma Biotechnology Co., Ltd. in time.
Kit composition:
1. Assay plate: one piece (96 holes).
2. Standard: 2 bottles (lyophilized product).
3. Sample Diluent: 1 x 20 ml / bottle.
4. Biotin-antibody Diluent: 1 x 10 ml/vial.
5. Horseradish peroxidase-labeled avidin dilution (HRP-avidin Diluent): 1 x 10 ml / bottle.
6. Biotin-antibody: 1 × 120 μl / bottle (1:100)
7. Horseradish peroxidase-labeled avidin (HRP-avidin): 1 × 120 μl / bottle (1: 100)
8. Substrate solution (TMB Substrate): 1 x 10 ml / bottle.
9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution: 1 x 10 ml / bottle (2N H2SO4).
*: [96T/48T] (Please check all items in time after opening the package)
Detection principle:
This kit uses a double antibody sandwich ELISA method. The anti-mouse COL1a2 antibody was coated on the plate, and COL1a2 in the specimen or standard was bound to the coated antibody during the experiment, and the free component was washed away. Biotinylated anti-mouse COL1a2 antibody and horseradish peroxidase-labeled avidin were sequentially added. The anti-mouse COL1a2 antibody binds to mouse COL1a2 bound to the coated antibody, and biotin binds to avidin to form an immune complex, and the free component is washed away. Adding chromogenic substrate (TMB), TMB is blue under the catalysis of horseradish peroxidase, and turns yellow after adding stop solution. The OD value was measured by a microplate reader at a wavelength of 450 nm, and the concentration of COL1a2 was proportional to the OD450 value. The concentration of COL1a2 in the specimen was determined by drawing a standard curve.
Specimen collection:
1. Serum: Whole blood samples are allowed to stand at room temperature for 2 hours or at 4 ° C overnight and then centrifuged at 1000 × g for 20 minutes. The supernatant can be detected. The tube for collecting blood should be a disposable pyrogen-free, endotoxin-free tube.
2. Plasma: EDTA.Na2 is recommended for anticoagulant. The specimen is centrifuged at 1000×g for 15 minutes within 30 minutes after collection. The supernatant can be detected. Avoid using hemolysis, hyperlipidemia specimens.
3. Tissue homogenate: Wash the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (cleaved red blood cells in the homogenate will affect the measurement), and weigh the tissue after weighing. The shredded tissue and the corresponding volume of PBS (generally corresponding to a weight ratio of 1:9, such as 1g of tissue sample corresponding to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and record. Recommended to add in PBS The protease inhibitor) was added to a glass homogenizer and thoroughly ground on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly thawed. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was taken for detection.
4. Cell culture supernatant: The cell culture supernatant was centrifuged at 1000 x g for 20 minutes to remove impurities and cell debris. Take the supernatant test.
5. Other biological specimens: centrifuge at 1000 × g for 20 minutes, take the supernatant to detect (the specific treatment method can call Shanghai Jinma Bio)
6. Specimens should be clear and transparent, and the suspension should be removed by centrifugation.
7. If the specimen is not detected in time after collection, please pack it once and store it in the refrigerator at -20°C/-80°C to avoid repeated freezing and thawing. It should be tested within 1-6 months and stored at 4°C. Testing is performed during the week.
8. If the concentration of the test substance in your sample is higher than the highest value of the standard, please make a suitable multiple dilution according to the actual situation (it is recommended to do a preliminary experiment to determine the dilution factor).
Self-prepared items required for the test:
1. Microplate reader (450nm wavelength filter)
2. High precision pipette, EP tube and disposable tip: 0.5-10μL, 2-20μL, 20-200μL, 200-1000μL
3. 37 ° C incubator, double distilled water or deionized water
4. Prepare the absorbent paper before testing:
1. Remove the kit from the refrigerator 20 minutes in advance and equilibrate to room temperature.
2. Dilute the concentrated wash with double distilled water (1:25). Return unused 4 °C. The concentrated washing liquid taken out from the refrigerator may be crystallized, which is a normal phenomenon. It can be completely dissolved in a 40 ° C water bath to completely dissolve the crystals, and then the washing liquid is prepared (the heating temperature should not exceed 50 ° C, and the washing liquid should be room temperature when used).
3. Standards: Add 1.0 mL of Standard & Sample Diluent to the lyophilized standard and let stand for 10 minutes. After it is fully dissolved, mix gently (concentration 10 ng/mL). Then dilute the dilution as needed (Note: Do not directly dilute in the well). It is recommended to prepare the following concentrations: 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0 ng/mL, and the sample diluent is directly used as a blank well of 0 ng/mL. For example, prepare a 5 ng/mL standard product: 0.5 mL 10 ng/mL of the above standard is added to an EP tube containing 0.5 mL of the sample diluent, and the mixture can be mixed, and the other concentrations are similar.
4. Biotinylated antibody working solution: Calculate the amount required for the next experiment (in 100 μL/well) before the experiment, and prepare 100-200 μL for the actual preparation. Concentrated biotinylated antibody (1:100) was diluted to a working concentration with biotinylated antibody dilution 15 minutes prior to use. Used on the day.
5. Enzyme conjugate working solution: Calculate the amount required for the next experiment (in 100μL/well) before the experiment, and prepare 100-200μL for the actual preparation. The concentrated HRP enzyme conjugate (1:100) was diluted to the working concentration with the enzyme conjugate dilution 15 minutes before use. Used on the day.
cleaning method:
1. Automatic plate washer: 350μL of washing solution is added to each well, and the injection and aspiration intervals are 60 seconds.
2. Manually wash the plate: Drain the liquid in the hole, pat dry on the clean absorbent paper, add 350 μL of the washing solution to each well, soak for 1-2 minutes, aspirate (not touch the wall) or pry off the liquid in the plate. , pat dry on thick absorbent paper.
Steps:
Before the start of the experiment, each reagent should be equilibrated to room temperature; when preparing the reagent or sample, mix thoroughly and avoid foaming as much as possible.
1. Adding samples: blank holes, standard holes, and sample holes to be tested. Add 100 μL of the sample dilution to the blank well, and add 100 μL of the standard or the sample to be tested. Be careful not to have air bubbles. Add the sample to the bottom of the plate when adding the sample. Try not to touch the wall of the well and gently shake it to mix. The plate was plated and incubated at 37 ° C for 90 minutes. To ensure the validity of the results, use a new standard solution for each experiment.
2. Discard the liquid, dry it, and do not wash it. 100 μL of biotinylated antibody working solution (prepared within 15 minutes before use) was added to each well, and the plate was coated with a membrane and incubated at 37 ° C for 1 hour.
3. Discard the liquid in the well, dry it, wash the plate 3 times, soak for 1-2 minutes each time, about 350 μL/well, dry and pat dry on the absorbent paper to pat dry the liquid in the well.
4. Add 100 μL of each well to the enzyme conjugate working solution (prepared within 15 minutes before use), add the membrane, and incubate at 37 ° C for 30 minutes.
5. Discard the liquid in the well, dry it, and wash the plate 5 times. The method is the same as step 3.
6. Add 100 μL of substrate solution (TMB) per well, incubate the plate with a plate at 37 ° C for 15 minutes in the dark (depending on the actual coloration, shorten or extend as appropriate, but not more than 30 minutes. When the standard hole appears obvious When the gradient is over, it can be terminated).
7. Add 50 μL of stop solution to each well to stop the reaction, and the blue color turns yellow. The order of addition of the stop solution should be as close as possible to the order in which the substrate solution is added.
8. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader. The microplate reader should be turned on in advance, preheat the instrument, and set up the test procedure.
9. After the experiment is completed, return the unused reagents to the refrigerator at the specified storage temperature.
Precautions:
1. Storage: Please store the reagents in the kit according to the instructions. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightened to prevent evaporation and microbial contamination, otherwise erroneous results may occur.
2. Enzyme plate: There may be a little water-like substance in the well of the newly opened ELISA plate. This is normal and will not affect the experimental results.
3. Loading: When adding or adding reagents, if the loading interval between the first hole and the last hole is too large, it will lead to different “pre-incubation” time, which will obviously affect the accuracy of the measured value. Sex and repetitive. The loading time is preferably controlled within 10 minutes. It is recommended to set the double hole.
4. Incubation: In order to prevent the sample from evaporating, the enzyme labeling plate must be coated during the experiment; the next step should be carried out as soon as possible after washing, to avoid the enzyme plate being in a dry state; strictly comply with the given incubation time and temperature.
5. Washing: The washing liquid remaining in the reaction well during the washing process should be patted on the absorbent paper. Do not put the filter paper directly into the reaction well to absorb water. Pay attention to the residual liquid and fingerprints at the bottom before reading to avoid affecting the microplate reader reading.
6. Reagent preparation: Concentrated Biotinylated Detection Ab and Concentrated HRP Conjugate are small in size. The transportation process will cause the liquid to adhere to the tube wall or the cap. Therefore, 1000 rpm/separation of the heart for 1 min before use, so as to adhere the tube wall or the cap liquid. Deposited to the bottom of the tube. Before using, please use a pipette to carefully blow the solution 4-5 times to mix the solution. Standard, biotinylated antibody working solution, and enzyme conjugate working solution should be prepared according to the required dosage and prepared with the corresponding dilution solution, which should not be confused. Please accurately prepare the standard and working fluid, try not to prepare in a small amount (such as not to be less than 10μL when Concentrated Biotinylated Detection Ab is taken) to avoid concentration error caused by inaccurate dilution; do not reuse diluted standards, Biotinylated antibody working solution, enzyme conjugate working solution. If it is necessary to use the standard products in batches, they should be packed according to each dosage and stored at -20~-80 °C. Avoid repeated freezing and thawing.
7. Control of color development time: Please observe the color change of the reaction well after adding the substrate (for example, every 5 minutes). If the gradient is obvious, please stop the reaction by adding the stop solution in advance to avoid the color too deep to affect the microplate reader. reading.
8. Substrate: Keep the substrate away from light and avoid direct exposure to strong light during storage and incubation.
9. Mixing: Fully light mixing is especially important for the reaction results. It is best to use a micro-oscillator (using the lowest frequency). If there is no trace oscillator, you can manually tap the microplate frame before mixing.
10. Safety: Wear a lab coat and latex gloves for protection during the test. In particular, when testing blood or other body fluid samples, please follow the National Biological Laboratory Safety Protection Regulations.
11. Kit components of different batches cannot be mixed (except washing solution and reaction stop solution)
12. The EP tube and the tip used in the test are all used once, and it is forbidden to mix it, otherwise it will affect the test result!
The result is judged:
1. The OD value of each standard is subtracted from the OD value of the blank hole. If the double hole is set, the average value should be taken. The standard curve is drawn by plotting the concentration of the standard as the abscissa and the OD as the ordinate. Alternatively, the OD value may be plotted on the abscissa and the concentration of the standard product on the ordinate as a standard curve.
2. It is recommended to use professional curve making software, such as curve expert 1.3. In the software interface, the corresponding concentration can be found from the standard curve according to the sample OD value, multiplied by the dilution factor; the OD value of the sample can also be substituted into the standard curve. Fit the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample.
3. If the OD value of the specimen is higher than the upper limit of the standard curve, it should be diluted and retested. The concentration should be multiplied by the dilution factor.
Sensitivity, detection range, specificity and repeatability:
● Sensitivity: The minimum measurement is 0.09ng/mL.
● Detection range: 0.16 – 10ng/mL.
● Specificity: Recombinant or natural mouse COL1a2 can be detected and has no cross-reactivity with other related proteins.
● Repeatability: within the board, the coefficient of variation between the boards is <10%.
Description
1. Limited to existing conditions and scientific and technological level, it is not possible to conduct comprehensive identification and analysis of all raw materials. 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 the time. Please be sure to prepare sufficient samples to be tested.
3. Only use ElabTM reagents to ensure the test results. Do not mix products from other manufacturers. The best test results will only be obtained if the experimental instructions for ElabTM reagents are strictly followed.
4. Validity period: 6 months.
5. These instructions also apply to the 48T kit.