Kit as well as preparation method and application thereof
A kit and inhibitor technology, applied in the field of medical diagnosis, can solve problems such as difficulty in obtaining, increasing market demand, and high price
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[0052] The present invention further proposes a preparation method of the kit as described above, comprising the following steps:
[0053] S10. Coupling the biotin to the coating antibody, removing excess biotin, and then diluting with the first buffer solution to obtain the first working solution.
[0054] For specific operations, the following methods can be used:
[0055] Take 60 μg of coated antibody, and dilute the antibody to a final concentration of 2 mg / mL with biotinylated activation buffer (the biotinylated activation buffer is 50 mM phosphate buffer, containing 50 mM disodium hydrogen phosphate and a mass fraction of 0.9 %NaCl, pH value is 8.0), weighed 0.3mg of biotin, and added 300μL of DMSO (dimethyl sulfoxide) to prepare a 10mg / mL biotin solution, and added 4-10μL of biotin solution to the coating antibody solution Incubate at 22°C in the dark for 0.5-3 hours, purify with a desalting column to remove excess unreacted biotin, and dilute to 50-500 mL with biotiny...
Embodiment 1
[0084] Example 1 Kit
[0085] The first working solution: biotinylated coating antibody (coating antibody: the monoclonal antibody whose article number is YM0213, Shenzhen Yawei Century Technology Co., Ltd.), the first buffer solution [50mM disodium hydrogen phosphate, 0.9% NaCl (w / w), 1% BSA (w / w), 0.5mM CaCl 2 , 0.05% Triton X-100 (w / w), 0.05% Tween-20 (w / w), 1% glycine (w / w)], wherein the pH value of the first buffer is 7.2;
[0086] The second working solution: superparamagnetic particles coated with streptavidin, the second buffer [50mM Tris, 0.9% NaCl (w / w), 0.05% Tween-20 (w / w), 0.05% Triton X-100 (w / w), 0.05% Proclin300 (w / w)], wherein the pH value of the second buffer is 7.2;
[0087] The third working solution: labeled antibody labeled with enzyme (labeled antibody: monoclonal antibody with product number YM0212, Shenzhen Yawei Century Technology Co., Ltd.), the third buffer solution [0.05M MES, 0.15M NaCl, 0.1% Tween- 20(w / w), 0.05% Proclin300(w / w), 1% BSA(w / w),...
Embodiment 2
[0090] The preparation method of embodiment 2 kit
[0091] The material preparation kit according to embodiment 1 may further comprise the steps:
[0092] 1. Preparation of various buffer solutions
[0093] Second buffer: 50mM Tris, 0.9% NaCl (w / w), 0.05% Tween-20 (w / w), 0.05% TritonX-100 (w / w), 0.05% Proclin300 (w / w), pH is 7.2;
[0094] The third buffer: 0.05M MES, 0.15M NaCl, 0.1% Tween-20 (w / w), 0.05% Proclin300 (w / w), 1% BSA (w / w), 1% glycine (w / w) , 10% Glycerol (w / w), 5mM MgCl 2 , 0.1 mM ZnCl 2 , the pH value is 6.0;
[0095] First buffer: 50 mM disodium phosphate, 0.9% NaCl (w / w), 1% BSA (w / w), 0.5 mM CaCl 2 , 0.05% Triton X-100 (w / w), 0.05% Tween-20 (w / w), 1% glycine (w / w), pH value is 7.2;
[0096] Biotinylation activation buffer: 50mM phosphate buffer, containing 50mM disodium hydrogen phosphate, and 0.9% NaCl (w / w), pH 8.0;
[0097] Enzyme blocking solution: 0.1M disodium hydrogen phosphate, 0.15mM NaCl, 50mM hydroxylamine hydrochloride, pH value is 7.2.
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