Antigen, antibody and enzyme-linked immunoassay kit for amantadine
An enzyme-linked immunosorbent assay and amantadine technology, which is applied in measurement devices, color/spectral property measurement, instruments, etc., can solve the problem that the accuracy, sensitivity, and specificity cannot fully meet the detection requirements, the process is complex, and the diamond does not meet the requirements. Alkylamine testing requirements and other issues
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Embodiment 1
[0068] Preparation and identification of embodiment 1 amantadine hapten
[0069] 1. Preparation of amantadine hapten
[0070] (1) Accurately weigh 10 mg of N-(1-adamantane)hydrazine carboxamide and dissolve it in 125 μL of methanol, stir and dissolve to obtain liquid A;
[0071] (2) Dissolve 4.0 mg of glyoxylic acid in 125 μL of methanol to obtain liquid B;
[0072] (3) Mix A and B liquids and put them into a round-bottomed flask with a magnetic stirrer, stir and react at room temperature for 48 hours, centrifuge the reacted mixed solution at 4500r / min for 15min, then discard the supernatant solution, and The precipitate was vacuum-dried at 50°C, and finally a white solid powder was obtained, which was the amantadine hapten.
[0073] 2. Identification of amantadine hapten
[0074] The resulting hapten was identified by mass spectrometry, -MS: M-1 = 264.6, such as figure 2 shown. The results show that the chemical structural formula of the amantadine hapten is as shown in...
Embodiment 2
[0076] The preparation of embodiment 2 amantadine artificial antigen
[0077] 1. Synthesis of amantadine immune antigen
[0078] (1) Weigh 5.93 mg of hapten and dissolve it in 1 mL of DMF, add 4.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5.2 mg of N-hydroxysuccinate imide, stirred and reacted at room temperature for 2h to obtain solution C;
[0079] (2) 50 mg of BSA was dissolved in 5 mL of 0.1 mol / L sodium bicarbonate buffer to obtain a protein solution;
[0080] (3) Add the above solution C to the protein solution drop by drop, stir overnight at room temperature, dialyze with PBS solution at 4°C for 3 days, and change the dialysate 6 times during the period; Filter, aliquot into ampoules, and store at -20°C.
[0081] 2. Synthesis of Amantadine Coating Source
[0082] The amantadine coating agent was prepared in the same synthesis method as the above-mentioned amantadine immune antigen.
Embodiment 3
[0083] Example 3 Preparation and Specific Identification of Amantadine Monoclonal Antibody
[0084] 1. Preparation of amantadine monoclonal antibody
[0085] (1) Animal immunization: 150 μg / mouse of the immunogen prepared above was dissolved in normal saline, mixed with Freund's complete adjuvant in an equal volume, and subcutaneously injected into the back of the neck to immunize 6- and 8-week-old Balb / c female mice On the 7th, 14th, and 28th day after the initial immunization, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each additional immunization was given once. The immune complex was 150 μg per mouse 3 days before fusion, and no Freund's adjuvant was added. One booster immunization;
[0086] (2) Cell fusion: Take splenocytes from immunized mice and mix them with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add preheated fusion agent (PEG4000) for fusion within 45s. Suspend the HAT medium evenly, then ad...
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