Rat ELISA kit test kit sample collection, processing and preservation method serum ---- avoid any cell stimulation during the operation. Use tubes containing pyrogen and endotoxin. After collecting the blood, the serum and red blood cells were quickly and carefully separated by centrifugation at 1000 x g for 10 minutes.

Plasma-----EDTA, citrate, heparin plasma can be used for detection. The pellet was removed by centrifugation at 1000 x g for 30 minutes.

The cell supernatant was centrifuged at 1000 x g for 10 minutes to remove particles and polymer.

Tissue homogenization ----- tissue is added to the appropriate amount of physiological saline. Centrifuge at 1000 × g for 10 minutes, and take the supernatant for storage. ------ If the sample is not used immediately, it should be divided into small portions - 70 ° C to avoid repeated freezing. Do not use hemolysis or hyperlipemia as much as possible. If there are large amounts of particles in the serum, centrifuge or filter before testing. Do not heat thaw at 37 ° C or higher. It should be thawed at room temperature and ensure that the sample is fully thawed evenly.

Kit contents and its preparation kit components (2-8 °C preservation) 96-well configuration 48-well configuration 96/48-person ELISA plate 1 plate (96T) half-plate (48T) ready-to-use plastic membrane cover One and a half block ready-to-use rat ELISA kit test kit standard: 80ng/ml 1 bottle (0.6ml) 1 bottle (0.3ml) according to the instructions for the diluted blank control 1 bottle (1.0ml) 1 bottle (0.5ml ) Ready-to-use standard dilution buffer 1 bottle (4.0ml) 1 bottle (2.0ml) ready-to-use biotinylated anti-CD3 antibody 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use affinity enzyme -HRP1 bottle (8.0ml) 1 bottle (4.0ml) ready-to-use washing buffer 1 bottle (20ml) 1 bottle (10ml) diluted according to the instructions Substrate A1 bottle (6.0ml) 1 bottle (3.0ml) ready to use Type substrate B1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use stop solution 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use sample dilution 1 bottle (12ml) 1 bottle (6.0ml) Distilled water with a self-contained material.

Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.

Oscillators and magnetic stirrers, etc.

Safety Avoid direct contact with the stop solution and substrates A, B. Once exposed to these liquids, rinse with water as soon as possible.

Do not eat, drink, smoke or use cosmetics during the experiment.

Do not use your mouth to take any ingredients from the kit.

Rat ELISA Kit Test Kit Operation Precautions Reagents should be stored according to the label instructions and returned to room temperature before use. The diluted standard should be discarded and cannot be stored.

The slats not used in the experiment should be immediately put back into the bag and sealed to avoid deterioration.

Other reagents that are not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.

Use a disposable tip to avoid cross-contamination. Avoid pipettes with metal parts when drawing stop solution and substrate A and B.

Use a clean plastic container to configure the wash solution. Mix all the ingredients and samples in the kit thoroughly before use.

Wash the enzyme plate should be fully patted dry, do not put the absorbent paper directly into the enzyme standard reaction well to absorb water.

Substrate A should be volatilized to avoid opening the lid for extended periods of time. Substrate B is sensitive to light and avoids prolonged exposure to light. Avoid contact with hands and be toxic. The OD value should be read immediately after the experiment is completed.

The order of addition of reagents should be consistent to ensure that all wells are incubated for the same time.

The incubation is carried out according to the time indicated in the instructions, the amount of the addition, and the order.

Preparation standards for reagents: Serial dilutions of standards should be prepared during the experiment and should not be stored. Mix the standard shakes before dilution. The dilution ratio is as follows:

The original concentration of 80 ng/ml (standard 6) was directly added to 50 ul without dilution.

40 ng/ml (No. 5 standard) 100 ul of the original standard. Add 100 ul of standard dilution 20 ng/ml (No. 4 standard) 100 ul of No. 5 standard. Add 100 ul of standard dilution 10 ng/ml (No. 3) Standard) 100 ul of Standard No. 4 Add 100 ul of standard dilution 5.0 ng/ml (No. 2 standard) 100 ul of Standard No. 3 Add 100 ul of standard dilution 2.5 ng/ml (No. 1 standard) 100 ul The No. 2 standard was added to 100 ul of the standard dilution 0 ng/ml (blank control). The original concentration was directly added to 50 ul without dilution.

Dilution of wash buffer (50 x): 50-fold dilution of distilled water.

Mix all reagents thoroughly before using. Do not allow the liquid to generate a large amount of foam, so as to avoid adding a large amount of air bubbles during the loading, resulting in errors in the loading.

The number of slats required is determined by the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and the double hole can be used as much as possible. The specimen was diluted 1:1 with the specimen dilution and 50 ul was added to the reaction well.

50 ul of the diluted standard was added to the reaction well, and 50 ul of the sample to be tested was added to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate for 1 hour at 37 °C.

Remove the liquid from the wells, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.

60 ul of affinity streptavidin-HRP was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 30 minutes.

Remove the liquid from the wells, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.

50 μl of each of the substrates A and B was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 10 minutes. Avoid lighting.

Remove the microplate and quickly add 50 ul of stop solution. Immediately after adding the stop solution, the results should be determined.

The OD value of each well was measured at a wavelength of 450 nm.

The results above the limit of standard 6 are non-linear, and accurate results cannot be obtained from this standard curve.

Kit Performance 1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. The linearity of the dilution. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value was 0.990.

2. Specificity: Does not react with other cytokines.

3. Repeatability: The coefficient of variation between the plates and the plates is less than 10%.

Result judgment and analysis 1. Instrument value: read the OD value of each hole on the microplate reader with wavelength of 450 nm, the OD value of the absorbance is the ordinate (Y), and the corresponding CD3 standard concentration is the abscissa (X). A corresponding curve is made, and the CD3 content of the sample can be converted into a corresponding concentration from the standard curve according to its OD value.

3. Detection range: 0-80ng/ml

4. Sensitivity: 0.1ng/ml


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