Melamine antigen, related antibody and preparation method thereof
A technology of melamine and antigen, applied in related antibody and its preparation, melamine antigen containing polyaldehyde dextran-linked carrier protein, melamine antigen, antigen field, can solve the problem of not really replacing protein, increasing protein content, and melamine has no nutrition Questions such as value
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[0159] The preparation method of the melamine antigen monoclonal antibody of the present invention specifically comprises:
[0160] (1) Immunization of mice
[0161] Mice were immunized by conventional methods, specifically:
[0162] Use the melamine-PAD-carrier protein conjugate product as an immunogen to immunize male BALB / C mice for 6-8 weeks. 2 to 3 mice. The melamine-PAD-carrier protein was mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and intraperitoneally injected into each mouse. Booster immunizations were carried out every two weeks, and a total of 5 booster immunizations were carried out. During the first 4 booster immunizations, melamine-PAD-carrier protein was fully emulsified with an equal volume of Freund's incomplete adjuvant, and each mouse was intraperitoneally injected with 0.1 mg of melamine-PAD-carrier protein antigen. The last immunization was done by tail vein injection without adding adjuvant. After 3 days, the splenocyt...
Embodiment 1
[0239] Embodiment 1, the preparation of melamine antigen
[0240] (1) Prepare the following solutions:
[0241] a. 0.25g PAD (made from 2W molecular weight dextran) was dissolved in 5ml 0.1M PB (pH=7.2) buffer
[0242]b. Dissolve 0.25g OVA in 2.55ml 0.1M PB (pH=7.2) buffer
[0243] c. 50mg melamine dissolved in 1ml 20% ethanol solution
[0244] d. Dissolve 50mg of sodium trihydroborocyanide in 0.5ml of 0.1M PB (pH=7.2) buffer solution, the solution is ready for immediate use
[0245] (2) Mix solution a with solution b, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer, test the dialysate with 2,4-dinitrophenylhydrazine color test, and dialyze When complete, set aside.
[0246] (3) Mix solution c with the solution obtained after complete dialysis, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer solution, and test the dialysate without free melamine small molecules to obtain melamine - PAD-OVA antigen.
[0247] ...
Embodiment 2
[0248] Embodiment 2, the preparation of melamine antigen
[0249] (1) Prepare the following solutions:
[0250] a. 0.25g PAD (made from 4W molecular weight dextran) was dissolved in 5ml 0.1M PB (pH=7.2) buffer
[0251] b. Dissolve 0.25g OVA in 2.55ml 0.1M PB (pH=7.2) buffer
[0252] c. 50mg melamine dissolved in 1ml 20% ethanol solution
[0253] d. Dissolve 50mg of sodium trihydroborocyanide in 0.5ml of 0.1M PB (pH=7.2) buffer solution, the solution is ready for immediate use
[0254] (2) Mix solution a with solution b, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer, test the dialysate with 2,4-dinitrophenylhydrazine color test, and dialyze When complete, set aside.
[0255] (3) Mix solution c with the solution obtained after complete dialysis, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer solution, and test the dialysate without free melamine small molecules to obtain melamine - PAD-OVA antigen.
[0256]...
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