A kind of tyramide hapten, antigen and antibody and its preparation method and application
A tyramine hapten, tyramine technology, applied in the preparation of carboxylic acid amide, chemical instruments and methods, cyanide reaction preparation and other directions, can solve the problems of poor repeatability and unreasonable antibody preparation scheme, and achieve high accuracy and linearity. Broad and specific effects
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Embodiment 1
[0063] The preparation method of embodiment 1 tyramide hapten
[0064] S11. Put 1g of tyramine and 1.197g of methyl 4-formylbenzoate into a 25mL round bottom flask, mix well, add 25mL of anhydrous methanol to fully dissolve, condense and reflux at 60°C for 20h
[0065] S12. Add 0.55g of sodium borohydride for reduction, and condense and reflux for 1 hour at 50°C;
[0066] S13. Methanol is spin-dried, dissolved in water, extracted 3 times with ethyl acetate, and then ethyl acetate is spin-dried;
[0067] S14. Redissolve the product with 20mL of methanol, and add 0.22g of potassium hydroxide, and reflux at 70°C for 3h;
[0068] S15. Spin dry and dissolve in saturated saline, adjust the pH to about 5-6 with hydrochloric acid, extract with n-butanol to obtain the target tyramine hapten, and its structure is shown in formula I.
[0069] Formula I.
[0070] The ESI-MS identification results of the above tyramide haptens are as follows: figure 1 As shown, the synthesized compou...
Embodiment 2
[0071] Embodiment 2 The preparation method of immunogen / coating original
[0072] In the preparation method of the immunogen and the coating source, the difference lies in the structural formula of the hapten, the use type of the carrier protein and the coupling method. The immunogen carrier protein mainly adopts bovine serum albumin (BSA), and the coating Chicken ovalbumin (OVA) was mainly used as the original carrier protein, and the coupling method used was carbodiimide method or glutaraldehyde two-step method.
[0073] A preparation method (immunogen (carbodiimide method)) of a tyramide antigen, which comprises the following steps:
[0074] S21. 9mg BSA is dissolved in the PBS buffer of 5mL PH7.4;
[0075] S22. Dissolve 11 mg of tyramide hapten (Tyr-271) in 400 μL of DMF and 500 μL of first-grade water, add it to the protein solution obtained in S21, and stir for 10 minutes;
[0076] S23. Dissolve 12 mg of EDC in 200 μL of water, then add dropwise to the protein-hapten m...
Embodiment 3
[0090] Example 3 Antibody preparation and identification
[0091] Emulsify the prepared immunogen with an equal amount of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) and immunize animals. BalB / C mice aged 6 to 7 weeks were immunized by subcutaneous injection in the back, subcutaneous injection in various parts, abdominal cavity and foot respectively. The second immunization was performed 2 weeks later, and the booster immunization was performed every 2 weeks thereafter. One week after the third booster immunization, blood was collected from the tail of the mice, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, intraperitoneal injection is used to boost the immunization. Three days later, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 4°C and 10,000 rpm for 15 minutes, and the supernatant was tak...
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