Instruction Manual of Mouse Estradiol (E2) ELISA Kit

Instruction Manual of Mouse Estradiol (E2) ELISA Kit
This kit is for research use only
Detection range: 20 pg / ml -1000 pg / ml
Specificity: This kit can detect natural or recombinant estradiol at the same time, and there is basically no cross reaction with other related substances.
Validity: 6 months (store at 2-8 ° C in the dark)
Intended application: ELISA method for quantitative determination of estradiol in mouse 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.
Experimental principle
The content of estradiol was detected by competitive enzyme-linked immunoassay. First coat the microplate with sheep anti-rabbit to prepare a solid-phase secondary antibody, then add the test specimen, horseradish peroxidase-labeled estradiol and anti-estradiol antibody to form a coated secondary antibody Anti-estradiol antibody-estradiol (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 content of estradiol in the sample to be tested is calculated in reverse.
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
S1
S2
S3
S4
S5
concentration
(pg / ml)
20pg / ml
80pg / ml
200pg / ml
550pg / ml
1000pg / ml
3. Enzyme conjugate (HRP-conjugate): 1 × 6ml / bottle.
4. Antibody: 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
Reagents and equipment needed but 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. Distilled water, volumetric flask, etc.
Collection and preservation of specimens
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.
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 50ul of enzyme conjugate to each well (except blank control wells), then add 50ul of antibody to each well in the same order, mix well, attach a self-adhesive seal, and incubate at 37 ° C for 2 hours.
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, put back at 2-8 ℃ and protected 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.

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