2. Microplate: Select the number of plates needed for the experiment. The remaining unused parts are put back in the original bag together with the desiccant seal. 1. Anti-inhibin B-coated microplate coated with anti-inhibin bB subunit antibody: 96 wells, 2-8 & ordm; C sealed and preserved.
2. Standard A/ sample diluent: 2ml× 1 bottle, fetal bovine serum containing 0pg/mL dimer inhibin B. 2-8&; Ordm is saved under C. 2-8&; Ordmc can be kept for 2 weeks. on-20 & ordm； C can be kept for a long time.
3. Standard B-G (inhibinbstandsb-g):1ml× 6 bottles, fetal bovine serum, containing dimers with concentrations of 10, 30, 100, 250, 500, 1000 picograms/ml. 2-8 before use&; Ordm is saved under C. 2-8&; Ordmc can be kept for 2 weeks. on-20 & ordm； C can be kept for a long time.
4. Inhibin control: 1 ml× 2 bottles, concentrations I and II, fetal bovine serum, containing low and high concentrations of Inhibin A dimer. 2-8 before use&; Ordm is saved under C. 2-8&; Ordmc can be kept for 2 weeks. on-20 & ordm； C can be kept for a long time.
5. Sample diluent A: 10 ml× 1 bottle, 2-8 & ordm; Save it under C.
6. Sample diluent B: 10 ml× 1 bottle, 2-8 & ordm; Save it under C.
7. Antibody-biotin conjugate (ready-to-use): 10ml× 1 bottle, containing biotin-labeled antibody against inhibin A subunit. 2-8 & amp； Ordm is saved under C.
8. Streptoavidin-enzyme conjugate (ready-to-use): 10ml× 1 bottle, containing the combination of Streptomyces and horseradish peroxidase. 2-8 & amp； Ordm is saved under C.
9.TMB substrate solution: 15ml× 1 bottle, 2-8&; Ordm is saved under C.
10. Termination solution: 15ml× 1 bottle, containing 0.2M sulfuric acid. 2-8 & amp； Ordm is saved under C.
1 1. washing concentrate: 100 ml× 1 bottle, 2-8&; Ordm is saved under C. All reagents should be balanced to room temperature (~ 25&; Ordmc) is thoroughly mixed. Standard, quality control and sample to be tested shall be made in duplicate at the same time.
1. Take out the slats needed for the experiment and record the position of each hole.
2. Add 50 microliters of standard, quality control and sample to be tested into the corresponding holes.
3. Add 25ul of sample diluent a to each well.
4. Add 25ul of sample diluent b to each well.
5. Seal the board, shake it at the speed of 300-400rpm, and stay overnight at room temperature (14- 18 hours).
6. Wash the board three times and pat it dry with absorbent paper.
7. Add 50 microliters of antibody-biotin conjugate to each well.
8. Seal the plate, shake at the speed of 500-700rpm, and incubate at room temperature for 65438 0.5 hours.
9. Wash the board 6 times, and then pat it dry on absorbent paper.
10. Add 50 microliters of a mixture of Streptomyces and horseradish peroxidase to each well.
1 1. Seal the plate, shake at 500-700rpm, and incubate at room temperature for 20 minutes.
12. Wash the board 6 times. After the last rinse, leave the lotion in the hole 15 minutes before taking it out. Pat dry with absorbent paper.
13. Add 100ulTMB substrate solution to each well.
14. Shake at a speed of 500-700 rpm and incubate at dark room temperature 15-30 minutes.
15. Add 100ul termination solution to each well.
Read 450nm in 16.30 minutes.
Note: Zero standard must be blank. If dual-wavelength measurement can be realized, it can be read at 600 or 620nm, and the optical error can be reduced by subtracting the absorption value of 600(620)nm from 450nm. Blood was collected by venipuncture using serum samples. Samples were taken from 2-8&; Ordmc can be stored for 24 hours,-20&; Ordmc or below can be kept for 30 days. Avoid repeated freezing and thawing of samples. Hemolytic or lipid samples should not be used. Before the experiment, frozen samples should be thawed and thoroughly mixed.