Cetilistat hapten, artificial antigen, antibody and preparation method and application thereof
A technology of new listatin and artificial antigen, which is applied in the field of new listatin hapten, antibody and its preparation, and artificial antigen, to achieve the effects of high titer, high degree of skeleton structure overlap and high affinity
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Embodiment 1
[0112] Example 1 Synthesis and Identification of New Lisstat Hapten
[0113] 1. Synthesis and identification of new listatin hapten CET1
[0114] 1.1 Synthesis of the new lisstat hapten CET1
[0115] Take 2-amino-5-methylbenzoic acid (1mmoL) and pyridine (3mmoL), dissolve in dichloromethane, stir until clear, cool down to 15°C, add dropwise 3-chloropropyl chloroformate (1.2mmoL), Control the temperature below 20°C, and react at room temperature for 2 hours after the dropwise addition is complete. After the reaction, dichloromethane and unreacted pyridine were removed by rotary evaporation, 750g of dichloromethane and 250g of water were added, cooled with ice water, the temperature was controlled at 5°C, the volume fraction was 10% hydrochloric acid, and the pH was adjusted to 1-2 , a large amount of solid was precipitated, filtered, the solid was washed with water, and dried to intermediate product 1.
[0116] Add intermediate product 1 (1mmoL) to the reaction flask, dissol...
Embodiment 2
[0133] Synthesis and identification of embodiment 2 new lixistat artificial antigen
[0134] 1. Synthesis of new likistat artificial antigen
[0135] The hapten CET1 or hapten CET2 prepared in Example 1 was coupled to lactoferrin (LF) or chicken ovalbumin (OVA) by the active ester method.
[0136] Weigh 3.3 mg (0.008 mmol) of the hapten CET1 prepared in Example 1, dissolve it in 50-200 μL DMF with 0.014 mmol NHS and 0.018 mmol EDC, and stir at room temperature for 2-4 hours in the dark to obtain the hapten activation solution; LF was added to 1 mL of PBS buffer (0.01mol / L, pH=7.4); the hapten activation solution was slowly added dropwise to the PBS buffer solution of LF, and reacted at 4°C for 12 hours; then dialyzed with PBS buffer for 3 days, Three times a day, the artificial antigen CET1 was obtained after the dialysis, which was divided into centrifuge tubes and stored at -20°C for use.
[0137] Wherein, the formula of PBS buffer solution: Na 2 HPO 4 12H 2 O 2.90g, Na...
Embodiment 3
[0144] The preparation of embodiment 3 antibody
[0145] 1. Preparation of Polyclonal Antibody
[0146]The CET1-LF prepared in Example 2 was used as the immunogen and immune adjuvant (incomplete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations) in a volume ratio of 1:1 to emulsify evenly, The body weight of immunization is 2.5~3kg New Zealand white rabbits. The neck and back were injected subcutaneously at multiple points, and the second immunization was performed 4 weeks later, and the immunization was boosted every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the heart, and the method of obtaining serum from the collected blood was as follows: i...
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