Haptens and whole antigens of kinesin and their preparation methods and applications
The technology of hapten and glycosin, which is applied in the field of glycosin hapten and whole antigen and their preparation, can solve the problems of design and synthesis of unseen hapten, and achieves high sensitivity and broad application prospect. Effect
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Embodiment 1
[0036] Synthesis and identification of embodiment 1 kinesin hapten
[0037] 1. Synthesis of Kelkinin-Hhapten
[0038] Dissolve 50 mg of kelezin in 3 mL of anhydrous THF, add 3.0 eq of sodium borohydride, add 5 μL of anhydrous methanol every 3 hours, add a total of 15 μL, and stir at room temperature for 12 hours. Add 20 μL of glacial acetic acid to quench the reaction, pour the reaction system into a separatory funnel, add 10 mL of water and 60 mL of dichloromethane, and extract three times. The combined organic phases were concentrated and dried by column chromatography (ethyl acetate / petroleum ether system was used as a developing solvent, and the volume ratio was 1:1 to obtain about 20 mg of the target compound of formula (II). Subsequently, the reduced product of 20 mg of kelezin was dissolved in Add 2.0eq succinic anhydride to 0.4mL DMF, stir overnight at room temperature, quench the reaction system with water, extract with ethyl acetate, wash with a small amount of dilu...
Embodiment 2
[0041] The preparation of the whole antigen of embodiment 2 kiskinin
[0042] Conjugate I of hapten and BSA was used as immunogen. Conjugate II of hapten and OVA was used as coating agent.
[0043] 1. Preparation of kelecine immunogen
[0044] Weigh 20 mg of hapten and dissolve it in 1 mL of DMF to obtain a hapten solution; add 20 mg of EDC and 20 mg of NHS to the prepared hapten solution, place it on a magnetic stirrer, and react at room temperature at 400 rpm for 2 hours to obtain the product in the reaction; take 50 mg of BSA , dissolved in 10 mL of PBS buffer containing 10% (volume percentage) DMF to obtain a protein solution with a concentration of about 5 mg / mL.
[0045] The liquid phase of the product in the reaction was added dropwise to the prepared protein solution, stirred at 4°C, reacted overnight, and then transferred to a dialysis bag with a molecular weight cut-off of 7KDa, and then placed in PBS buffer for dialysis at 4°C for 3 days. After the dialysis is co...
Embodiment 3
[0052] The preparation of embodiment 3 kiskinin self-antiserum
[0053] The kinesin immunogen prepared in Example 2 was emulsified with an adjuvant, and five 6-8-week-old female Balb / c mice were immunized by subcutaneous multi-point injection on the back of the neck, and boosted once every 4 weeks.
[0054] The preparation method of the first immunization preparation is as follows: take the immunogen solution prepared in Example 2, dilute it to 1 mg / mL with PBS buffer, then mix and emulsify with an equal volume of Freund's complete adjuvant.
[0055] The preparation method of the booster preparation is as follows: take the immunogen solution prepared in Example 2, dilute it to 1 mg / mL with PBS buffer, then mix it with an equal volume of Freund's incomplete adjuvant and emulsify completely.
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