This kit is for research use only Detection range: 0.2 ng / ml-100 ng / ml Nature Material Nature Materials,Synthetic Leather,Polyurethane Leather,Pu Faux Leather DONGGUAN SHENGYUAN INDUSTRIAL CO,.LTD. , https://www.elementpuleather.com
Minimum detection limit: 0.1 ng / ml
Specificity: This kit can detect natural or recombinant PROG at the same time, and there is basically no cross reaction with other related proteins.
Validity: 6 months (stored at 2-8 ° C, protected from light)
Intended application: ELISA method for quantitative determination of PROG content in chicken serum, plasma and other related biological fluids.
Explanation
1. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.
2. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
The experimental principle uses competitive enzyme-linked immunoassay to detect the content of PROG. First, coat the microplate with a single antibody to prepare a solid-phase antibody, then add the test specimen or standard, horseradish peroxidase-labeled PROG and anti-PROG antibody to form a coating antibody-anti-PROG Antibody-PROG (HRP) complex. After color development, the absorbance value (OD value) is measured in a microplate reader, the concentration-absorbance curve is fitted by computer or drawing, and the PROG content in the sample to be tested is calculated inversely.
Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Standard product (Standard): 5 bottles (lyophilized product).
The standard product is a lyophilized product, which is dissolved in 0.5ml of distilled water before use, and the concentration after dissolution is:
Standard 1 Standard 2 Standard 3 Standard 4 Standard 4
0.2 ng / ml 1ng / ml 5 ng / ml 20 ng / ml 100 ng / ml
3. Antibody: 1 × 6ml / bottle.
4. Enzyme conjugate (HRP-conjugate): 1 × 6ml / bottle.
5. Developer A (Substrate A): 1 × 7ml / bottle.
6. Developer B (Substrate B): 1 × 7ml / bottle.
7. Wash Buffer: 1 × 15ml / bottle, each bottle is diluted 20 times with distilled water.
8. Stop Solution (Stop Solution): 1 × 7ml / bottle needed reagents and equipment not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Collection and preservation of specimens such as distilled water and volumetric flasks
1. Serum: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes. Supernatant can be taken for detection, or the specimens should be stored at -20 ° C or -80 ° C, but avoid repeated Freeze and thaw.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ° C or -80 ° C, but avoid repeated freezing melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
Note: The above specimens should be stored at 4 ℃ for less than 1 week, -20 ℃ or -80 ℃ should be sealed and stored, -20 ℃ should not exceed 1 month, -80 ℃ should not exceed 2 months; specimen hemolysis will affect the final The test results, so hemolytic specimens should not be tested.
Steps
1. Equilibrate various reagents to room temperature [18-25 ° C] for half an hour, take concentrated washing solution, dilute 1:20 with distilled water according to the number of batches tested, mix well and set aside.
2. Standard 1- Standard 5, dissolve in 0.5ml of distilled water before using for the first time.
3. Take out the enzyme labeling plate, set a blank control well without adding any liquid; set two wells in each standard point in turn, add 50ul of the corresponding standard to each well; add 50ul of the specimen to be tested directly to each remaining well.
4. Add 50 ul of enzyme conjugate to each well (except blank control wells), then add 50 ul of each antibody, mix well, attach a self-adhesive seal, and incubate at 37 ℃ for 1 hour.
5. Manually wash the plate and discard the liquid in the hole. Fill the holes with the washing solution, let it dry for 10 seconds, and then pat dry after repeating three times; wash the plate with a plate washer, choose three washing procedures, and pat dry after washing.
6. Add 50μl of developer A solution and 50μl of developer B solution to each well. After shaking and mixing, develop color at 37 ° C in the dark for 15 minutes. Add 50μl of stopper solution to each well.
7. Read with a microplate reader, take a wavelength of 450nm, first adjust the zero point with a blank hole, and then measure the OD value of each hole.
data processing
1. Manual work diagram: using double logarithmic graph paper, with the standard concentration as the horizontal axis, and the corresponding 0D value as the vertical axis, draw a smooth curve or straight line, and find the corresponding concentration on the curve according to the serum OD value value.
2. Computer: Using the linear fitting function, the logarithm (Log (concentration)) of the concentration of the standard products S1-S5 should be taken as X, and the logarithm (Log ( OD value-NSB)) As Y, linear fitting is performed. Then calculate the serum concentration to be measured from the fitted line.
Precautions
1. All the bottled reagents and the required pre-coated strips in the kit taken from the refrigerated environment should be kept at room temperature
(18-25 ℃) It can be used after being balanced for 30 minutes. The rest should be sealed in time and put back at 2-8 ℃ to protect from light for future use.
2. Reagents should be shaken well before use.
3. The result judgment must be completed within 10 minutes after the termination of the reaction.
4. Reagents of different batches cannot be mixed.
5. When adding samples, care should be taken to avoid contamination and contamination between the reagents and samples used.
6. During operation, use one tip for each reagent in the kit, one tip for each standard, and one tip for each sample. It is best to use the tip once.