Biomarker for detecting non-obstructive azoospermia and application thereof
A biomarker, azoospermia technology, applied in the field of biomarkers for the detection of non-obstructive azoospermia, can solve problems such as difficulty in quantitative detection
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Embodiment 1
[0043] This embodiment provides a diagnostic kit for non-obstructive azoospermia, which contains an antibody to TNF-α protein (NOVUS, NB600-587), a TNF-α protein standard product (NOVUS, NBC1-18460), 96-well plate, diluent, chromogenic reagent and stop solution, wherein the diluent is PBS, chromogenic reagent (substrate chromogenic solution A: take 13.6 g of sodium acetate, 1.6 g of citric acid and 0.3 mL of 30% hydrogen peroxide, add distilled water to 500mL; Substrate Chromogenic Solution B: Take 0.2g disodium edetate, 0.95g citric acid, 50mL glycerin and 0.15g TMB (dissolved in 3mL DMSO), add distilled water to 500mL, ready to use) , the stop solution is sulfuric acid.
[0044] In addition, the 96-well plate needs to be coated. The coating method is: use phosphate buffer (pH 7.2) to dilute the TNF-α protein antibody, dilute the antibody to a concentration of 500ng / mL, and add the diluted antibody to the 96-well plate , placed overnight in a refrigerator at 4°C, sealed with...
Embodiment 2
[0046] In this example, the kit in Example 1 is used to detect the semen sample.
[0047] The semen of 10 NOA patients and the semen of 10 normal men were respectively taken, and the concentration of TNF-α protein in each semen sample was detected using the kit in Example 1, including the following steps:
[0048] (1) The 80pg / mL TNF-α protein standard was diluted by comparison to obtain 80pg / mL, 40pg / mL, 20pg / mL, 10pg / mL, 5pg / mL and 0pg / mL standard solution for making a standard curve ;
[0049] (2) The new type of semen sample obtained by masturbation of the patient was placed in a 37°C water bath for 30 minutes for liquefaction;
[0050] (3) After the semen is completely liquefied, centrifuge at 3000g / 5min, and take the supernatant seminal plasma;
[0051] (4) Take 100mL of seminal plasma and standard products of various concentrations, place them in a 96-well plate coated with antibodies, and incubate at 37°C for 30min;
[0052] (5) After incubation, wash 3 times with was...
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