Acid orange i hapten, artificial antigen and its synthesis and application
An artificial antigen, acid orange technology, applied in the field of artificial antigen and its synthesis and application, acid orange I hapten, can solve the health hazards of consumers and other problems, and achieve the effect of simple synthesis steps
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Embodiment 1
[0046] The synthesis of embodiment 1 hapten AOI
[0047] The synthetic route is as follows:
[0048]
[0049] 1.1. After dissolving p-aminobenzenesulfonic acid (1.73g, 10.0mmol) in 2mL of DMF, add 3mL of HCl (1mol / L) solution, stir at room temperature for 15min, and add 10% NaNO dropwise 2 , until the potassium iodide test paper turns blue, and then continue to stir at 4°C for 30 min to obtain liquid A.
[0050] 1.2. Weigh 1-hydroxy-2-naphthoic acid (1.88g, 10.0mmol) into saturated 3mL Na 2 CO 3 solution, cooled to 4°C to obtain liquid B.
[0051] 1.3. Slowly add solution A to solution B, adjust the pH to 8-9, stir at 4°C for 2 hours to obtain a red precipitate; finally, adjust the pH of the solution to 3.0 with hydrochloric acid, continue to stir at 4°C for 30 minutes, and filter under reduced pressure , to obtain a red residue, which was dried at 37°C to obtain a crude sample.
[0052] 1.4. The crude sample was purified by recrystallization, and 3.2 g of the crude sa...
Embodiment 2
[0054] The preparation of embodiment 2 complete antigen
[0055] 2.1. Weigh 1.6 mg of the hapten AOI obtained in Example 1, 2.5 mg of EDC, and 1.5 mg of NHS, dissolve in 300 μL of DMF, stir at room temperature for 4-6 hours, and obtain solution I. Weigh 10.0 mg of BSA (the molar ratio of BSA to AOI is 1:30), and add 3 mL of carbonate buffer solution to obtain solution II. At room temperature, add solution I dropwise to solution II, adjust the pH of the mixture to 8-9 with 1mol / L NaOH solution, and react overnight at room temperature to obtain the conjugate AOI-EDC-BSA; replace BSA into OVA to obtain the conjugate AOI-EDC-OVA.
[0056] Adjust the dosage of hapten AOI to 3.3 mg, the dosage of EDC to 5.1 mg, and the dosage of NHS to 3.1 mg, so that the molar ratio of BSA to AOI is 1:60; or
[0057] Adjust the amount of hapten AOI to 5.0 mg, EDC to 7.7 mg, and NHS to 4.6 mg, so that the molar ratio of BSA to AOI is 1:90, and the conjugate AOI-EDC-BSA can also be prepared.
[00...
Embodiment 3
[0060] The immunization of embodiment 3 mice
[0061] For the first immunization, mix and emulsify the complete antigen of Acid Orange I with the same amount of complete Freund's adjuvant in the amount of 100 μg / mouse, and immunize BALB / c mice by multi-point subcutaneous injection on the back of the neck; after 4 weeks For booster immunization, the dose of the complete antigen was halved (50 μg / monkey), mixed and emulsified with incomplete Freund’s adjuvant, and the time interval between multiple booster immunizations was 3 weeks; the dose was halved again (25 μg / bird) for sprint immunization, The complete antigen was diluted with saline and injected intraperitoneally into mice. After the third immunization of the mice, blood can be collected by docking the tail, and the titer and IC of the mouse serum can be detected by indirect competitive enzyme-linked immunoassay (ic-ELISA) 50 , choose high potency, IC 50 Low mice undergo fusion.
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