A kind of malachite green hapten preparation method and its application
A malachite green and hapten technology, applied in the field of immunology, achieves the effects of high yield, large amount of processed samples, and simple and feasible preparation method
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Embodiment 1
[0035] Embodiment 1, preparation and identification of malachite green hapten
[0036] 1. Preparation of malachite green hapten
[0037] (1) Add 1g of p-hydroxybenzaldehyde and 2g of zinc chloride in a 25mL conical flask, dissolve in 20mL of absolute ethanol;
[0038] (2) Add 3 mL of N,N-dimethylaniline, reflux at 90°C for 24 hours, after the reaction is complete, remove the solvent under reduced pressure, and purify by column chromatography (ethyl acetate / petroleum ether, 1 / 2, v / v);
[0039] (3) It was dissolved in 20 mL of pyridine, and 0.74 g of succinic anhydride was added (the molar ratio to the substrate was 1:1.2). Reacted at 80°C for 20h, and the reaction was completed, and the solvent was removed under reduced pressure. Dilute hydrochloric acid was added to adjust the pH value to about 5, and ethyl acetate was added to extract 3 times. The organic phases were combined, dried over anhydrous magnesium sulfate, the solvent was removed under reduced pressure, and pur...
Embodiment 2
[0043] Embodiment 2, preparation and identification of malachite green artificial antigen
[0044] 1. Synthesis of malachite green antigen
[0045] (1) Take 10 mg of hapten and dissolve it in 1 mL of dimethylformamide (DMF);
[0046] (2) Take 15 mg of dichloroethane (EDC) and dissolve it fully with 0.2 mL of water, then add it to the hapten solution, stir at room temperature for 24 hours, and then obtain the reaction solution A;
[0047] (3) Weigh 40mg of carrier protein, fully dissolve it in 2.8mL PBS (pH 7.2), slowly add reaction solution A to the protein solution drop by drop, and stir at room temperature for 24h;
[0048] (4) Dialyze with 0.01mol / L PBS at 4°C for 3 days and change the dialysate 3 times a day to remove unreacted small molecular substances;
[0049] (5) Aliquot and store at -20°C for later use.
[0050] 2. Identification of malachite green artificial antigen
[0051]According to the ratio of hapten, carrier protein and coupling product used in syntheti...
Embodiment 3
[0052] Embodiment 3, monoclonal antibody preparation and sensitivity determination
[0053] 1. Preparation of malachite green monoclonal antibody
[0054] 1. Use 100 μg of the immunogen (MG-BSA) prepared above, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in equal volume, and inject subcutaneously on the back of the neck to immunize Balb / c females aged 6-8 weeks. On the 7th, 14th, and 28th day after the initial immunization, mix the immunogen and Freund's incomplete adjuvant in equal volumes, and each additionally immunize once, and 3 days before the fusion, 100 μg of the immune complex / rat, without Freund's adjuvant Add another immunization.
[0055] 2. Proceed according to the conventional method, take the splenocytes of the immunized mice and mix them with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (PEG4000) within 45 seconds for fusion , suspend evenly with HAT me...
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