Preparation method of albuterol hapten and antigen and its application in quantum dot immunofluorescence kit
An albuterol and immunofluorescence technology, which is applied in the field of immunological detection, can solve the problems of unsuitable production, basic unit and on-site detection, high requirements for the operation process of instruments and equipment, complicated processing, etc., and achieves a simple and feasible preparation method, low cost, and processing. The effect of large sample volumes
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Embodiment 1
[0035] Embodiment 1, the preparation of immunogen and coating former
[0036] 1. Preparation of hapten
[0037] 1. Synthesis of intermediate sal-1: Add 2-bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethan-1-one 1000mg, dichloromethane 200ml, p-toluenesulfonate into a 250ml three-necked flask Acid 3.8mg, cooled to 0-5°C in an ice bath, added dropwise 2,2 methoxypropane 2208ul, reacted overnight at room temperature, TLC detected that the reaction was complete, post-treatment, washed the organic phase with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, It was filtered and concentrated under reduced pressure to obtain 1211 mg of off-white solid (sal-1).
[0038] 2. Synthesis of intermediate sal-2: Add intermediate sal-1, 1140mg, 40ml of anhydrous methanol to a 100ml three-necked flask, cool down to 0°C in an ice bath, add 227mg of sodium borohydride, react at 0-5°C for 2 hours, and detect by TLC After the reaction was complete, the reaction solution was...
Embodiment 2
[0052] Embodiment 2, the preparation of polyclonal antibody
[0053] The immunogen solution prepared in Example 1 was diluted with PBS buffer solution with pH 7.4 and 0.01 M to obtain the immunogen dilution, which was used for the preparation of polyclonal antibodies. New Zealand white rabbits were used as immunized animals.
[0054] The immunization process is as follows (the immunization dose is based on the amount of protein):
[0055] First immunization: Mix the immunogen dilution with an equal volume of Freund's complete adjuvant to make an emulsifier, inject it subcutaneously at multiple points on the back of the neck, and the immune dose is 1 mg / kg b.w.;
[0056] Booster immunization: 4 weeks after the first immunization, 8 weeks later and 12 weeks later, a booster immunization was carried out each time. The immunogen dilution was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple points on the back of the...
Embodiment 3
[0059] Embodiment 3, the assembly of quantum dot immunofluorescence detection kit
[0060] Quantum dots were purchased from Shenzhen Taylor, Si Technology Co., Ltd., the product catalog number is TLS ® LumiQD TM 20. The quantum dots are characterized as follows: the particle size is 20nm, the CV of the particle size is 15%, the quantum yield is 60%, and the surface carboxyl content is 5×10 -3 mmol / mg, water-soluble, CdSe / ZnS core-shell structure, excitation wavelength is 345nm, emission wavelength is 620nm; red fluorescent quantum dots. A scan of a quantum dot is shown in figure 1 shown. The carboxyl groups on the quantum dots form peptide bonds with the amino groups on the protein and connect them.
[0061] Quantum dot immunofluorescence detection kit includes the following components:
[0062] 1. Microwell plate coated with the original coating
[0063] Take the original coating solution prepared in Example 1 and dilute it with the coating buffer (the coating buffer i...
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