use:
GA ELISA kit for plant extract GA. This kit can detect natural and recombinant GA.
This kit is designed for scientific research, not for clinical diagnosis.
Test principle:
The GA kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known GA concentrations and samples with unknown concentrations are added to microwell enzyme plates for detection. Incubate GA and biotin-labeled antibodies first. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of GA in the sample.
Kit contents and preparation kit components (stored at 2-8 ° C) 96-well configuration 48-well configuration Preparation
96/48 serving microplate 1 plate (96T) half plate (48T) ready-to-use plastic membrane plate cover 1 half-plate ready-to-use standard: 40nmol / L 1 bottle (0.6ml) 1 bottle (0.3 ml) Carry out dilute blank control according to the instructions 1 bottle (1.0 ml) 1 bottle (0.5 ml) ready-to-use standard dilution buffer 1 bottle (5 ml) 1 bottle (2.5 ml) ready-to-use biotin-labeled anti-GA antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use affinity streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use wash buffer 1 bottle (20ml) 1 bottle (10ml) according to the instructions Dilute Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use stop solution 1 bottle (6.0ml) 1 bottle (3.0 ml) ready to use
Bring your own material distilled water.
Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500u, 1000lul.
Oscillator and magnetic stirrer etc.
Safety The HbsAg and anti-HIV in the human blood products of this kit are negative, but there is no laboratory to fully guarantee that this blood product will not infect hepatitis, AIDS or other infectious diseases. Dispose of this kit according to local safety regulations Components and serum and plasma samples.
Avoid direct contact with the stop solution and substrates A and 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 absorb any ingredients in the kit.
Handling precautions Reagents should be stored according to label instructions, and returned to room temperature before use. The sparse standards should be discarded and cannot be stored.
The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.
Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and substrates A and B.
Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use.
When washing the enzyme-labeled plate, it should be fully patted dry. Do not put the absorbent paper directly into the enzyme-labeled reaction well to absorb water.
Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed.
The order of adding reagents should be the same to ensure that all wells are incubated for the same time.
Perform the incubation operation according to the time, the amount and order of the liquid indicated in the instructions.
Reagent preparation standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows:
40 nmol / L (Standard No. 6) The original concentration is directly added to 50ul without dilution.
20 nmol / L (No. 5 standard) 100ul of the original standard is added to 100ul of standard dilution
10 nmol / L (No. 4 standard) 100ul of No. 5 standard is added with 100ul of standard diluent
5.0 nmol / L (Standard No. 3) Add 100ul of Standard No. 4 to 100ul of Standard Diluent
2.5 nmol / L (Standard No. 2) Add 100ul of No. 3 standard to 100ul of standard dilution
1.25 nmol / L (No. 1 standard) 100ul of No. 2 standard is added with 100ul of standard diluent
0 nmol / L (blank control) The original concentration is directly added to 50ul without dilution.
Dilution of washing buffer (50 ×): 50-fold dilution with distilled water.
Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition.
The number of slats required is determined by the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible.
Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37 ° C for 1 hour.
Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation four times. If washing with a plate washer, the number of washes is increased once.
Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes.
Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation four times. If washing with a plate washer, the number of washes is increased once.
Add 50ul each of substrate A and B to each well, gently shake to mix, and incubate at 37 ° C for 15 minutes. Avoid light.
Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.
The OD value of each well was measured at a wavelength of 450 nm.
Recommended experimental protocol Standard concentration (nmol / L)
A 40 40 sample sample sample sample sample sample sample sample sample sample sample
B 20 20 sample sample sample sample sample sample sample sample sample sample sample
C 10 10 sample sample sample sample sample sample sample sample sample sample sample
D 5.0 ​​5.0 sample sample sample sample sample sample sample sample sample sample sample
E 2.5 2.5 sample sample sample sample sample sample sample sample sample sample sample
F 1.25 1.25 sample sample sample sample sample sample sample sample sample sample sample
G 0 0 sample sample sample sample sample sample sample sample sample sample sample
H sample sample sample sample sample sample sample sample sample sample sample sample sample
Result analysis: Absorbance OD value is the ordinate (Y), the corresponding GA standard concentration is the abscissa (X), the corresponding curve is made, and the GA content of the sample can be converted from the standard curve to the corresponding concentration according to its OD value.
Limitation
The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve.
Kit performance sensitivity: The minimum detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient R between the linear regression of the sample and the expected concentration is 0.990.
Specificity: Does not react with other human cytokines.
Repeatability: The coefficients of variation within and between plates are less than 10%.
placemat
Placemat,Place Mats,Table Mat,Ecofriendly Pvc Mat
Rongxin New Material (Jiangsu) Co., Ltd. , https://www.rongxinxcljs.com