This kit is for research use only. Detection range: 96T0.4nmol / L-12nmol / L Purpose: This kit is used to determine the content of testosterone (T) in zebrafish serum, plasma and related liquid samples. Experimental principle The kit uses the double antibody sandwich method to determine the level of testosterone (T) in the specimen. Microporous plates were coated with purified zebrafish testosterone (T) antibodies to make solid phase antibodies. Testosterone (T) was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP labeled testosterone (T) antibodies to form The antibody-antigen-enzyme-labeled antibody complex is thoroughly washed and added with substrate TMB for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the testosterone (T) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of zebrafish testosterone (T) in the sample was calculated by a standard curve. Kit composition 1 30-fold concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle 2 enzyme label reagent 6ml × 1 bottle 8 standard product (24nmol / L) 0.5ml × 1 bottle 3 enzyme label coated plate 12 well × 8 strips 9 standard diluent 1.5ml × 1 bottle 4 sample diluent 6ml × 1 bottle 10 instruction manual 1 copy 5 developer A solution 6ml × 1 bottle 11 sealing film 2 sheets 6 developer B solution 6ml × 1 / Bottle 12 sealed bag 1 specimen requirement 1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. Procedure 1. Dilution of standard product: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart. 12nmol / L No. 5 standard 150μl original standard added 150μl standard dilution 6nmol / L No. 4 standard 150μl standard 5 added 150μl standard dilution 3nmol / L No. 3 standard 150μl No. 4 standard Add 150μl standard dilution 1.5nmol / L No. 2 standard 150μl No. 3 standard add 150μl standard dilution 0.75 nmol / L No. 1 standard 150μl No. 2 standard add 150μl standard dilution 2. Add Sample: Set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix. 3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes. 4. Mixing solution: Dilute the 30-fold concentrated washing solution with distilled water 30-fold and reserve for use. 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, and let it stand for 30 seconds before discarding , Repeat this 5 times, pat dry. 6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well. 7. Incubation: The operation is the same as 3. 8. Washing: The operation is the same as 5. 9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop color at 37 ° C in the dark 15 minutes. 10. Stop: Add 50μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time). 11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
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