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Human medium factor (MK) enzyme-linked immunoassay (ELISA)
Kit instruction manual
This reagent is for research use only
Purpose: This kit is used to determine the content of midkine (MK) in human serum, plasma and related liquid samples.
Kit composition:
Kit composition
48 hole configuration
96-well configuration
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Instructions
1 serving
1 serving
Sealing film
2 pieces (48)
2 pieces (96)
sealed bag
1
1
Enzyme coated plate
1 × 48
1 × 96
Store at 2-8 ℃
Standard product: 2700ng / L
0.5ml × 1 bottle
0.5ml × 1 bottle
Store at 2-8 ℃
Standard dilution
1.5ml × 1 bottle
1.5ml × 1 bottle
Store at 2-8 ℃
Enzyme reagent
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Sample diluent
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Developer A liquid
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Developer B liquid
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Stop solution
3ml × 1 bottle
6ml × 1 bottle
Store at 2-8 ℃
Concentrated washing liquid
(20ml × 20 times) × 1 bottle
(20ml × 30 times) × 1 bottle
Store at 2-8 ℃
Experimental principle:
This kit uses the double antibody sandwich method to determine the level of human midkine (MK) in the specimen. Microporous plates were coated with purified human midkine (MK) antibody to make solid-phase antibodies. Midkine (MK) was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP labeled midkine (MK) antibody To form an antibody-antigen-enzyme-labeled antibody complex, and after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the medium-term factor (MK) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human midkine (MK) in the sample was calculated by a standard curve.
Sample processing and requirements:
1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.
2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
7. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Steps
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of the standard products in the first and second wells, and then add the standard products in the first and second wells. 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 1800ng / L, 1200ng / L, 600ng / L, 300ng / L, 150ng / L)
2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the test sample (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing liquid with distilled water 30 times (20 times of 48T) and then use.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Precautions:
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. 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 test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is above 0.95.
2. The batch and approval shall be less than 9% and 11% respectively
examination range:
100ng / L -2000ng / L
Calculation:
Taking the concentration of the standard as the abscissa and the OD value as the ordinate,
Draw a standard curve on coordinate paper, according to the OD of the sample
The value is determined by the standard curve; then multiplied by the dilution
Multiple; or calculate the standard using the concentration and OD value of the standard
The linear regression equation of the quasi-curve, the OD value of the sample
Substitute into the equation, calculate the sample concentration, and multiply by the dilution
The multiple is the actual concentration of the sample.
Storage conditions and validity period:
1. Store the kit: 2-8 ℃.
2. Validity: 6 months
Essential cosmetics:
First, eye cream.
This is what we use every day. It is a care and protection for the delicate skin on the eyes. It can also be used to make a pre-makeup care on the skin of the eyes before we make up.
Concealer.
Covers pigmentation and uneven skin tone around the eyes. So that the eyeshadow can show its color, it can show the layering and gradation of the eyeshadow cleanly when drawing.
Third, sponge puff.
For the makeup around the eyes, you must use a sponge puff. You can evenly press the loose powder around the eyes. Because the makeup requirements of the eyes are longer, the makeup is the key.
Fourth, thin eyeliner.
Find a thin eyeliner to complete the outline of the eyeliner. The texture of the eyeliner used for different makeup is different, but whether it is powder, cream or liquid, do not use thick lines. Will affect the delicate sense of makeup, and feel that Eye Makeup cannot be integrated.
Fifth, a color Eye Shadow tri-color powder.
The three colors are chosen to create a sense of layering and gradation. The lightest one is used as the base, and the closer to the eyelashes, the deeper the color will be.
6. Eye shadow stick or eye shadow brush.
In general, the eye shadow can be used with an eye shadow stick, depending on personal habits, some people prefer to use a brush, or they prefer to use a finger.
Seven, eyelash curler.
It is best to prepare two types of eyelash curlers, one is our ordinary eyelash curler, and the other is a local clip, which can be clipped to the corner or the place where the ordinary clip does not tilt.
Eight or two mascaras.
Two types are also used. One is slightly sticky, which is used to brush the roots and waist of the eyelashes, which is where the clips are clamped. The other is to brush the eyelashes slightly. Just brush evenly.
Essential tools:
First, Makeup Tools
1, sponge: used to apply foundation. There are generally round, triangular and cylindrical (conical) shapes. The round sponge is characterized by a slightly hard texture and large area, which is suitable for basking on a large area on the forehead and cheeks. The other two textures should be more detailed, suitable for partial priming at the corners of the eyes, nose and mouth. Using different shaped sponges to make the base makeup more detailed.
2. Puff: Round puff and honey powder brush are commonly used. Round puffs also come in many sizes. Large powder puff is suitable for large area use, small powder puff is suitable for partial makeup. The powder brush is a large cylindrical brush, which is the largest of Makeup Brushes. Smearing powder on the face and sweeping it on the face can evenly sweep the powder onto the face, the effect is more natural.
3, blush brush: slightly smaller than the honey powder brush, there are bevel and flat two, the top of the bristles are semi-circular. The bevel is suitable for the modification of T-shaped and cheekbones, also called facial contour brush. Large brushes can be used for large areas to paint and brush away excess powder.
Eye makeup tools
1. Eyebrow plucking: Trim the shape of the eyebrows by removing the excess hair on the eyebrows. By pulling the eyebrows, the hairs grow out more slowly and the eyebrows remain longer.
2, eyebrow trimmer: used to trim the shape of the eyebrows and remove large areas of hair, fast and painless.
3, eyebrow trimmer: trim eyebrows that are too short. Eyelash curler: used to make the eyelashes curl. If the eyelashes are not easy to shape, use an electric eyelash curler.
4, eyebrow brush: most of the hard brush made of nylon or artificial fiber with oblique brush head. You can use the eyebrow brush to trim the eyebrows before trimming and thrushing. After the eyebrows, swipe the eyebrows in the direction of the eyebrows to make the eyebrows look natural and the eyebrows shape.
5, Eyebrow Pencil: makeup tools that everyone is familiar with, used to improve the shape of the eyebrows. Skilled sisters have also started using eyebrow powder.
6, eyebrow powder brush: The brush head is inclined and flat, in addition to taking the eyebrow powder to draw a suitable and clear eyebrow shape, used to draw eyeliner is also easy to grasp.
7, false eyelashes: a great weapon to enhance the charm of the eyes! There are more and more shapes.
8. Eyeshadow Brush: a flat-bodied round head brush, which is divided into large, medium and small. The large one is generally used to apply the background color. It can evenly apply the color at a time to cover the entire eye socket position. Can be drawn carefully, used to draw eyeliner more accurately. More suitable for powdery eyeshadow.
9, eye shadow stick: a bit similar to a cotton swab, used for partial color and multi-color eye shadow smudge, can make eye makeup natural. It can also be used for germanium gel and creamy eyeshadows. The elliptical head is suitable for coloring and spreading over a large area, and the pointed eye shadow stick is suitable for small area drawing.
10, eyeliner: used to draw eyeliner, can make the outline of the eye more clear. Some sisters who are more proficient in makeup techniques use eyeliner to draw eyeliner.
11, eyeliner brush: the shape is a bit similar to the lip brush, but the brush head is more delicate and the hair is softer, suitable for eyeliner.
12, eyelash brush: Some looks like a small comb, while others are similar to the mascara brush head, spiral. After applying the mascara, use it to brush away the mascara that sticks together to make the lashes look more neat.
Third, lip makeup tools
Lip Brush: The hair is stiffer, so you can easily control the brush point. Whether using lipstick or Lip Gloss , using a lip brush can help you draw detailed lines and modify the shape of your lips.
Facial Cosmetics,Cosmetic Set,Face Cream,Face Make Up Set
HENAN BON INDUSTRIAL(COSMETIC) CO.,LTD , https://www.boncosmetic.com