Rat keratinocyte growth factor (KGF) enzyme-linked immunosorbent assay kit instruction manual <br>This reagent is for research use only
Purpose: This kit is used to determine the content of keratinocyte growth factor (KGF) in rat serum, plasma, cell supernatant and related liquid samples.
Experimental principle:
The kit uses a double antibody sandwich assay to determine the level of rat keratinocyte growth factor (KGF) in the specimen. The microporous plate was coated with purified rat keratinocyte growth factor (KGF) antibody to prepare a solid phase antibody, and keratinocyte growth factor (KGF) was sequentially added to the microwell of the coated monoclonal antibody, and then labeled with HRP. KGF antibody binds to form an antibody-antigen-enzyme-labeled antibody complex,
After thorough washing, add substrate TMB to develop color. 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 keratinocyte growth factor (KGF) in the sample. Using a microplate reader
Absorbance (OD value) was measured at 450 nm, and rat keratinocyte growth factor (KGF) was calculated from the standard curve.
concentration.
Kit composition:
Kit Composition 48-well configuration 96-well configuration Storage instructions 1 copy 1 part sealing film 2 pieces (48) 2 pieces (96)
Sealed bag 1 piece of enzyme label package 1×48 1×96 2-8°C Preservation standard: 360ng/L 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 color Agent A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2 Store concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 8 ° C 2-8 ° C
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 anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge.
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 if a precipitate forms during storage, 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 in a sterile tube. Centrifuge 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).
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The concentration reaches 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). 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. 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:
1. Dilution and loading of standard products: 10 wells of standard wells on the enzyme label coating plate, 100 μl of standard in the first and second holes, and then add standard in the first and second holes. 50 μl of the dilution, mix; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells, respectively.
Mix well; then discard 50μl in each of the third and fourth wells, then add 50μl to each of the fifth and sixth wells, and then add 50ul of standard dilution in the fifth and sixth wells. Mix well; after mixing, take 50μl from each of the fifth and sixth holes and add them to the seventh and eighth holes respectively, then add 50μl of the standard dilution solution in the seventh and eighth holes respectively, and mix them from the first 7. Add 50 μl to the ninth and tenth holes in the eighth well, and then add 50 μl of the standard dilution to the ninth and tenth holes. After mixing, 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 240 ng/L, 160 ng/L, 80 ng/L, 40 ng/L, 20 ng/L, respectively.
2. Adding samples: Set blank holes separately (the blank control wells 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.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Dosing: Dilute 30 (20 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 50 μl 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: add 50 μl of color developer A50 to each well, then add 50 μl of color developer B, gently shake and mix, and avoid coloration at 37 °C.
15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned 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. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Precautions:
1. 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 unsealing, the strip 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. 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.
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 substance to be tested in the specimen is too high (sample OD value)
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If the OD value is larger than the first hole of the standard well, please dilute it with a certain dilution (n times) and then measure it. When calculating, multiply the total dilution factor (×n×5).
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. In the case of an English manual, the English manual shall prevail.
Calculation:
The concentration of the standard is the abscissa and the OD is the ordinate.
Draw a standard curve on the coordinate paper, depending on the OD of the sample
The value is determined from the standard curve by the corresponding concentration; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied by The dilution factor is the actual concentration of the sample.
(This picture is for reference only)
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:
5ng/L -300ng/L
Storage conditions and expiration date:
1. Kit storage: 2-8 ° C.
2. Validity: 6 months
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