Human mevalonate kinase (MVK) enzyme-linked immunosorbent assay (ELISA) kit instruction manual

Human mevalonate kinase (MVK) enzyme-linked immunosorbent assay (ELISA) kit instruction manual

This reagent is for research use only

Purpose: This kit is used to determine human mevalonate kinase in human serum, tissue and related fluid samples.

(MVK) content.

Experimental principle:

This kit uses a double antibody sandwich assay to determine human mevalonate kinase (MVK) levels in specimens. Purified human hydroxyl

The valerate kinase (MVK) antibody is coated with a microplate to prepare a solid phase antibody, and the mevalonate is sequentially added to the microwell of the coated monoclonal antibody.

Kinase (MVK), which binds to HRP-labeled mevalonate kinase (MVK) antibody to form antibody-antigen-enzyme-labeled antibodies

The body complex was thoroughly washed and then added with the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and

It is converted to the final yellow color by the action of an acid. The color depth is positively correlated with mevalonate kinase (MVK) in the sample. use

The microplate reader measures the absorbance (OD value) at a wavelength of 450 nm, and calculates the human mevalonate kinase in the sample by a standard curve.

(MVK) Concentration.

Kit composition:

Kit Composition 48-well configuration 96-well configuration Save

Instructions 1 copy 1 copy

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

Sealed bag 1 1

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

Standard: 450pg/mL 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C preservation

Standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle 2-8 ° C preservation

Enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation

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

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

Developer B Liquid 3 ml × 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

Sample processing and requirements:

1. Serum: The blood is naturally coagulated at room temperature for 10-20 minutes and centrifuged for about 20 minutes (2000-3000 rpm). Carefully collect

Clear, if precipitation occurs during storage, centrifuge again.

2. Plasma: EDTA, sodium citrate or heparin should be selected as anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes,

Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant, if there is a precipitate formed during the preservation process, it should be

Centrifugation.

3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant and save it

If a precipitate forms, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them with a sterile tube. Centrifugation for about 20 minutes (2000-3000 rpm /

Minute). Collect the supernatant carefully. When measuring the components in the cells, dilute the cell suspension with PBS (pH 7.2-7.4).

The degree reaches about 1 million / ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for 20 minutes

Left and right (2000-3000 rpm). Collect the supernatant carefully. 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. Rapid freezing and storage with liquid nitrogen

use. The specimen still maintains a temperature of 2-8 ° C after melting. Add a certain amount of PBS (pH 7.4), using a manual or homogenizer

Specimen homogenate is sufficient. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After the split, one is to be tested.

The rest is frozen for later use.

Steps

1. Dilution and loading of standard products: Set 10 holes of standard holes on the enzyme labeling board, and add the labels in the first and second holes respectively.

100 μl of the standard product, then add 50 μl of the standard dilution in the first and second wells, mix; then from the first hole, the second

100 μl of each well was added to the third and fourth wells, and 50 μl of the standard dilution was added to the third and fourth wells, respectively.

Mix well; then take 50 μl each in the third and fourth wells, and then take 50 μl each to the fifth and sixth holes.

In the fifth and sixth holes, add 50 ul of the standard dilution, and mix; after mixing, from the fifth and sixth holes.

Add 50μl to the seventh and eighth wells, and add 50μl of the standard dilution in the seventh and eighth wells.

After mixing, take 50μl from the seventh and eighth holes, add them to the ninth and tenth holes, and add the standard in the ninth and tenth holes respectively.

50 μl of the product dilution, mix and take 50 μl from each of the ninth and tenth holes and discard. (The amount of each well after dilution is 50μl,

The concentrations were 300 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL).

2. Adding samples: set blank holes separately (the blank control hole is not added with sample and enzyme standard reagent, the other steps are the same), sample to be tested

Pinhole. 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 product 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 gently shake the mixture.

uniform.

3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes.

4. Dosing: Dilute 30 (48 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and set aside.

5. 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.

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

7. Incubation: The operation is the same as 3.

8. Wash: Operate the same as 5.

9. Color development: first add 50μl of color developer to each well, then add 50μl of color developer B, gently shake and mix, avoid light and develop color at 37 °C

15 minutes.

10. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color turns yellow).

11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. Determination should be terminated

It can be done within 15 minutes after the liquid.

Precautions:

1. The kit should be taken out of the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed,

When used up, the slats should be stored in a sealed bag.

2. 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.

3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. The best time for one sample