| Product Description |
This reagent kit is used to determine the content of phenylalanine (PHE) in samples.
Experimental principle:
This kit uses enzyme-linked immunosorbent assay to determine the level of phenylalanine (PHE) in the sample. Purified phenylalanine (PHE) antibody was coated on a microplate to prepare a solid-phase antibody. Phenylalanine (PHE) was added to the microplate of the coated monoclonal antibody, along with HRP labeled Phenylalanine (PHE) antigen, to compete for binding. After thorough washing, TMB substrate was added for color development. The depth of sample color is negatively correlated with the content of phenylalanine (PHE) in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the content of phenylalanine (PHE) in the sample through a standard curve.
Specimen requirements:
1. Sample processing: (1) After water sample collection, it is repeatedly freeze-thawed three times at -20 ℃, and then filtered with glass fiber for future reference
(2) The tissue samples should be extracted using butanol: methanol: water (5:25:70 V: V: V), or extracted according to relevant literature. The experiment should be conducted as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃ for future reference
2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).
Operation steps:
1. Sample addition: Set up standard wells, blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the other steps are the same), and sample wells to be tested. Add 50 microliters to the standard well on the enzyme-linked immunosorbent assay (ELISA) plate, and first add 40 diluents to the sample well to be tested μ l. Then add 10 more samples to be tested μ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.
2. Enzyme addition: Add 50 enzyme labeled reagents to each well μ l. Excluding blank holes.
3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 60 minutes.
4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use
5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.
6. Color development: Add color development agent A50 to each well first μ l. Add color developer B50 again μ l. Gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes
7. Termination: Add 50% termination fluid to each hole μ l. Terminate the reaction (at this point, the blue color immediately turns yellow).
8. Measurement: Zero the blank hole and sequentially measure the absorbance (OD value) of each hole at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.
Calculation:
Draw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.
Notes:
1. The kit shall be used after being taken out of the cold storage environment and balanced at room temperature for 1 hour. If the enzyme coated plate is not used up after opening, the Flat noodles shall be stored in a sealed bag.
2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.
3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.
4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple (x n x 5).
5. The sealing film is only for one-time use to avoid cross contamination.
6. Please store the substrate in dark.
7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader 8. All samples, washing liquids, and various waste should be treated as infectious substances.
9. The components of this reagent with different batch numbers shall not be mixed.
Detection range:
three μ Mol/L-150 μ Mol/L
Specifications:
96T
Storage conditions and validity period:
1. Kit storage: 2-8 ℃.
2. Validity period: 6 months
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E2430405
