Preparation method of ractopamine hapten and antigen and its application in quantum dot immunofluorescence kit
A technology of ractopamine and immunofluorescence, which is applied in the field of immunological detection, can solve the problems of inapplicability to large-scale sample screening and detection, cumbersome operation steps, and high cost, and achieve the effect of simple and feasible preparation method, large amount of processed samples, and low cost
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Embodiment 1
[0038] Embodiment 1, the preparation of immunogen and coating former
[0039] 1. Preparation of hapten
[0040] 1. Take 500mg of ractopamine, dissolve it in 45ml of methanol, add 348.8mg of 6-bromohexanoic acid and 233mg of potassium hydroxide, heat to 80°C and reflux for 40~72 hours. Spot plate to confirm most conversion of starting material.
[0041] 2. Concentrate under reduced pressure. Reconstitute with 15ml, separate and purify using preparative thin-layer chromatography plate, developing agent DCM:ME=4:1, locate with ractopamine standard, take the point that is more polar than it, extract with methanol, and concentrate to obtain RAC-BBAR. The resulting dry matter is the hapten.
[0042] The structural formula of the hapten is shown in formula (I).
[0043] Formula (Ⅰ).
[0044] 2. Preparation of immunogen
[0045] 1. Take 160 mg of RAC-BHAR hapten and dissolve it in 10.7 ml of DMF, stir to fully dissolve it. Add 21.75mg of EDC2 and 191.4mg of NHS, and react at...
Embodiment 2
[0053] Embodiment 2, the preparation of polyclonal antibody
[0054] 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.
[0055] The immunization process is as follows (the immunization dose is based on the amount of protein):
[0056] 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.;
[0057] 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
[0060] Embodiment 3, the assembly of quantum dot immunofluorescence detection kit
[0061] 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.
[0062] Quantum dot immunofluorescence detection kit includes the following components:
[0063] 1. Microwell plate coated with the original coating
[0064] Take the original coating solution prepared in Example 1 and dilute it with the coating buffer (the coating buffer i...
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