Human High Velocity Protein 17 (HMG-17) ELISA Kit Instructions

Human High Velocity Protein 17 (HMG-17) ELISA Kit Instructions

Elisa kit specifications: 48-well configuration / 96-well configuration

Standard dilution: 1.5ml × 1 bottle

Enzyme standard reagent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)

[ Human Hyperkinetic Protein 17 (HMG-17) ELISA Kit ] This reagent is for research use only.

Calculation:

Taking the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the concentration of the standard Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample.

Kit composition:

Sealing film: 2 pieces (48) / 2 pieces (96)

Instructions: 1 copy

Sealed bag: 1

Standard: 2700ng/L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C preservation

Enzyme label coated plate: 1×48 1×96 2-8°C preservation

Sample dilution: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation

Reagent A: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation

Developer B solution: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation

Stop solution: 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C preservation

Concentrated washing solution: (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ° C preservation

[Human high-speed kinesin 17 (HMG-17) ELISA kit] experimental principle:

This kit uses the double antibody sandwich method to determine the level of human ferritin (FE) in the specimen. The microplate was coated with purified human ferritin (FE) antibody to prepare a solid phase antibody, and ferritin (FE) was sequentially added to the microcapsules of the coated monoclonal antibody, and then combined with HRP-labeled ferritin (FE) antibody. The antibody-antigen-enzyme-labeled antibody complex is formed, and after thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with ferritin (FE) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the concentration of human ferritin (FE) in the sample was calculated from a standard curve.

Purpose: This kit is used to determine the content of ferritin (FE) in human serum, plasma and related fluid samples.

Pledge:

Delivery period: payment to delivery.

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Provide customers with sample testing services to maximize the effectiveness of experimental results (free proxy).

Storage conditions and expiration date:

Kit storage: 2-8 ° C | Validity: 6 months

[Human High Velocity Protein 17 (HMG-17) ELISA Kit] Sample Processing and Requirements:

1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected and precipitated if it is formed during the preservation process. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If a precipitate forms during storage, it should be centrifuged again.

5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.

7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps

Dilution and loading of standard products: 10 wells of standard wells were placed on the enzyme-labeled plate, 100 μl of standard was added to the first and second wells, and then diluted with standard in the first and second wells. was 50 μl, mix; 100 μl are applied from each of the third and fourth apertures from the first hole, the second hole, and then the third and fourth holes each standard dilution 50 μl, mixed Then, 50 μl of each of the third and fourth wells is discarded, and 50 μl of each is added to the fifth and sixth wells, respectively, and a standard dilution of 50 ul is added to the fifth and sixth wells. Mix well; add 50 μl from each of the fifth and sixth wells to the seventh and eighth wells, and add 50 μl of the standard dilution in the seventh and eighth wells . Mix well. Then, 50 μl from the seventh and eighth holes were added to the ninth and tenth holes, and then 50 μl of the standard dilution was added to the ninth and tenth holes, respectively , and then mixed from the ninth and tenth holes. Take 50 μl and discard it. (The amount of each well was 50 μl after dilution , and the concentrations were 1800 ng/L, 1200 ng/L, 600 ng/L, 300 ng/L, 150 ng/L, respectively).

Adding samples: Set blank holes separately (the blank control holes are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate , and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.

Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.

Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used.

Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.

Add enzyme: 50 μl of enzyme labeling reagent was added to each well , except for blank wells.

Incubation: The operation is the same as 3.

Washing: The operation is the same as 5.

Color development: Add A50 μl of color developer to each well , then add B50 μl of color developer , mix gently by shaking, and color for 15 minutes at 37 °C.

Termination: 50 μl of stop solution was added to each well to terminate the reaction (the blue color turned yellow).

Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.

[Human High Velocity Protein 17 (HMG-17) ELISA Kit] Note:

The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after opening, the strip should be stored in a sealed bag.

Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.

The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.

Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).

The sealing film is intended for single use only to avoid cross-contamination.

Please keep the substrate away from light.

Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.

All samples, washings and various wastes should be treated as infectious materials.

The different batch components of this reagent must not be mixed.

10. In the case of an English manual, the English manual shall prevail.

Kit performance:

1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.95 or more.

2. Within and within the batch should be less than 9% and 11% respectively

examination range:

0.2IU/L - 6IU/L