Name: Human Adipsin ELISA Kit
Brand: Biological test kits, ELISA (enzyme-linked immunosorbent assay) kits, chemical reagents-Shanghai IDEAL MEDICAL TECH Co. Ltd.
SKU: ADL-EL-HM01869
Availability: InStock
Rating: 5 (1 reviews)

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Researchuse ELISA kit

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Home > ELISA Kit > Researchuse ELISA kit > Human Elisa Kit

Description

Human Adipsin ELISA Kit

This kit is intended for in vitro studies only and is not intended for clinical diagnosis.

 This kit is for the in vitro quantitative analysis of Human Adipsin in serum, plasma, tissue homogenates and related fluid samples.

 Validity: 6 months

 Storage condition: 2-8°C

Kit components

Component	48-well configuration	96-well configuration	Remarks
Pre-coated enzyme labeling plate	48T	96T	/
Standard	0.3mL*6 tubes	0.3mL*6 tubes	/
Diluent	3 ml	6ml	/
HRP Labeled Antibody	5 ml	10 ml	/
Colorimetric Substrate A	3 ml	6ml	/
Colorimetric Substrate B	3 ml	6ml	/
Termination solution	3 ml	6ml	/
20× Wash Solution	25 ml	25 ml	Operate according to instructions
Plate Sealing Membrane	2 sheets	2 sheets	/
Instruction manual	1 copy	1 copy	/
Self-sealing bag	1 piece	1 piece	/

Remarks

1. (96T/48T) After opening the package, please check whether all items are complete in time.

2. The concentration of standard is 80, 40, 20, 10, 5, 2.5 ng/mL.

3. After testing a large number of normal specimens, the normal concentration of the specimen values are within the detection range provided by the kit, and 50 μL of the sample can be sampled directly during the experiment. When the value of some samples exceeds the maximum concentration of the standard, the sample diluent can be used to dilute the specimen appropriately and then carry out the experiment.

Preparation before the test:

1. Remove the kit from the refrigerator 20 minutes in advance and equilibrate to room temperature. 2.

2. It is normal that crystals may form in the concentrated wash solution removed from the refrigerator; leave it at room temperature, shake gently and evenly, and allow the crystals to dissolve completely before preparing the wash solution. 20 ml of concentrated wash solution can be diluted with distilled or deionized water to 400 ml of working concentration of wash solution, and the unused amount can be put back to 4°C. 3. Dilute 20 x wash solution with distilled or deionized water.

3. Dilution of 20 × washing solution

Principle of the experiment:

The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the pre-coated microtiter wells pre-coated with human adipsin capture antibody, the specimen, standard, and HRP-labeled detection antibody are added sequentially, after warming and thorough washing. The color is developed with the substrate TMB, which is converted to blue by catalysis of peroxidase and to final yellow by acid. There is a positive correlation between the shade of color and the amount of human adipsin in the sample. The absorbance (OD) was measured at 450 nm using an enzyme meter and the concentration of the sample was calculated.

The equipment for the experiment was prepared by the laboratory:

1. Enzyme labeling instrument (450nm)

2. High-precision sampler and gun: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL.

3.37℃ constant temperature box

4. Distilled water or deionized water

Sample processing and requirements:

1. serum: whole blood specimen collected in serum separator tube should be placed at room temperature for 2 hours or overnight at 4℃, then centrifuged at 1000×g for 20 minutes, and the supernatant should be taken, or the supernatant should be stored at -20℃ or -80℃, but repeated freezing and thawing should be avoided.

2. Plasma: use EDTA or heparin as anticoagulant to collect the specimen, and centrifuge the specimen at 1000×g for 15 minutes at 2-8℃ within 30 minutes after collection, and then take the supernatant to be tested, or put the supernatant at -20℃ or -80℃ for storage, but should avoid repeated freezing and thawing.

3. Tissue homogenization: Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (lysed erythrocytes in the homogenate will affect the measurement results), weigh the tissue and then cut it into pieces. Mix the minced tissue with the corresponding volume of PBS (generally 1:9 weight-to-volume ratio, e.g., 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted according to the experimental needs, and make a record. It is recommended to add protease inhibitors to PBS) into a glass homogenizer and grind it thoroughly on ice. In order to further lyse the tissue cells, the homogenate can be ultrasonically broken, or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5~10 minutes and take the supernatant for testing.

4. Cell culture supernatant: Please centrifuge the supernatant at 1000×g for 20 minutes, and take the supernatant for detection, or place the supernatant at -20℃ or -80℃ for storage, but should avoid repeated freezing and thawing.

5. Other biological specimens: centrifuge at 1000×g for 20 minutes and take the supernatant for testing.

6. Sample appearance: the sample should be clear and transparent, and the suspension should be removed by centrifugation.

7. Sample preservation: If the sample is collected and tested within 1 week, it can be stored at 4℃; if it can not be tested in time, please divide the sample into portions according to the amount to be used at one time, and freeze and store at -20℃ (tested within 1 month) or -80℃ (tested within 6 months), avoiding repeated freezing and thawing, as hemolysis of the specimen will affect the final test results, so hemolysed specimens are not suitable for this test.

Precautions:

1. Follow strictly the specified time and temperature for warming to ensure accurate results. All reagents must reach room temperature 20-25°C before use. Keep reagents refrigerated immediately after use.

2. Incorrect plate washing can lead to inaccurate results. Ensure that the wells are drained of as much liquid as possible before adding substrate. Do not let the microtiter wells dry out during warming.

3. Eliminate residual liquid and fingerprints on the bottom of the plate, otherwise the OD value will be affected.

4. The substrate chromogenic solution should be colorless or very light in color, the substrate solution that has turned blue cannot be used.

5. Avoid cross contamination of reagents and specimens to avoid false results.

6. Avoid direct exposure to strong light during storage and incubation.

7. Equilibrate to room temperature before opening the sealed bag to prevent water droplets from condensing on the cold slats.

8. Do not expose any of the reaction reagents to bleaching solvents or strong gases emitted by bleaching solvents. Any bleaching components will destroy the biological activity of the reaction reagents in the kit.

9. Do not use expired products.

10. If disease transmission is possible, all samples should be managed and samples and detection devices should be handled in accordance with prescribed procedures.

Operating Procedure:

1. Remove the desired plate from the aluminum foil pouch after equilibrating for 60min at room temperature, and return the remaining plate to 4℃ by sealing it with a self-sealing bag.

2. Set up the standard wells and sample wells, and add 50 μL of different concentrations of standards to each standard well.

3. Add 50 μL of the sample to be tested into the sample wells; the blank wells are not added.

4. In addition to the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard and sample wells, seal the reaction wells with a sealing film, and incubate for 60min at 37℃ in a water bath or thermostat.

5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and so repeat the plate washing for 5 times (also use plate washer to wash the plate).

6. Add 50μL of substrate A and B to each well and incubate at 37℃ for 15min.

7. Add 50μL of termination solution to each well, and measure the OD value of each well at 450nm within 15min.

Experimental results were calculated:

Plotting the standard curve: in the Excel worksheet, with the concentration of the standard as the horizontal coordinate and the corresponding OD value as the vertical coordinate, the linear regression curve of the standard was plotted, and the concentration value of each sample was calculated according to the curve equation.

ELISA kit performance:

1.Detection range: 2.5 ng/mL - 80 ng/mL.

2. Sensitivity: the lowest detectable concentration is less than 0.1 ng/mL.

3. Specificity: no cross-reactivity with other soluble structural analogs.

4. Repeatability: intra-plate coefficient of variation less than 10%, inter-plate coefficient of variation less than 15%.

Technical tips:

1. When mixing or re-solubilizing protein solutions, try to avoid foaming.

2. In order to avoid cross-infection, it is necessary to change the tip of the gun when configuring standards of different concentrations, loading samples and adding different reagents. In addition, please use different pipetting tanks for different reagents.

3. Whenever incubating, please use the sealing gel correctly to ensure the accuracy of the results.

4. The mixed substrate should be colorless before loading onto the plate, please keep it away from light; if the plate is microtiter plate, it will change from color to different depths of blue.

5. The order of the termination solution on the plate should be the same as the order of the chromogenic substrate on the plate; after adding the termination solution, the color of the wells will change from blue to yellow; if there is green color in the wells, it indicates that the liquid in the wells has not been mixed well; please mix it well.

Instructions:

1. Due to the existing conditions and the level of science and technology is not yet able to all suppliers of all raw materials to carry out a comprehensive identification and analysis, this product may have certain quality and technical risks.

2. The final results of the experiment are closely related to the effectiveness of the reagents, the relevant operation of the experimenter and the experimental environment at the time, please be sure to prepare sufficient specimen backup.

3. There may be slight differences between different batches of the same product, such as: detection limit, sensitivity and color development time, etc., please follow the instructions in the kit to perform the experiments, the electronic version of the manual on the website is only for reference.

4. Only using all the reagents in this kit can ensure the detection effect, and cannot mix the products of other manufacturers. The best results can only be obtained by strictly following the instructions in the kit.

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Human Adipsin ELISA Kit

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