Melamine hapten and its preparation method and application
A technology of melamine and hapten, which is applied to the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of high detection cost by instrumental methods, cumbersome pretreatment methods, expensive instruments, etc., and achieve a simple method. Feasible, high hapten yield and purity, high affinity effect
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Embodiment 1
[0021] Example 1: Synthesis and identification of melamine hapten
[0022] 1. Synthesis of melamine hapten
[0023] Take 0.64g of ammeline and 1.0g of succinic anhydride, add 10ml of pyridine, stir at room temperature for 12-24h, remove the solvent and recrystallize in ethanol-water to obtain white powdery crystals, which are semi-succinic amber of ammeline Ester, that is, melamine hapten, molecular structure is as follows, synthetic route is as follows: figure 1 Shown:
[0024]
[0025] 2. Identification of melamine hapten
[0026] Get above-mentioned formula I compound and carry out structural analysis through proton nuclear magnetic resonance spectrum, as figure 2 As shown, it is found that the peak near 12ppm (mg / L) in the spectrum is the H on the carboxylic acid, and the peak around 2.7ppm (mg / L) is the two methylene groups on the hemisuccinic anhydride, indicating that the melamine hapten is successfully synthesized .
Embodiment 2
[0027] Embodiment two, the preparation of melamine antigen
[0028] 1. Synthesis of immunogen
[0029] Take 30mg of melamine hapten, dissolve it in 1.5ml of DMF to obtain solution I, then take 20mg of EDC and fully dissolve it with 0.2m water, add it to solution I, stir at room temperature for 24 hours, and then obtain solution II; weigh 50mg of BSA, make it fully Dissolve in 8.3ml of PBS with a pH of 7.2, slowly add solution II to the above BSA solution drop by drop, stir at room temperature for 24h to obtain solution III; dialyze with 0.02mol / LPBS at 4°C for 3 days to remove unreacted of small molecules. The obtained immunogen was aliquoted and stored at -20°C for future use.
[0030] 2. Synthesis of Coatogen
[0031] Take 30mg of melamine hapten, dissolve it in 1.5ml of DMF to obtain solution I, then take 20mg of EDC and dissolve it fully with 0.2ml of water, add it to solution I, stir at room temperature for 24h, and obtain solution II; weigh 40mg of OVA to fully dissol...
Embodiment 3
[0035] Embodiment three, melamine monoclonal antibody
[0036] 1. Preparation of melamine monoclonal antibody
[0037] Animal immunization: 8-10 week old Balb / c mice were immunized with the conjugate of melamine hapten and carrier protein.
[0038] Cell fusion and cloning: take immunized mouse splenocytes, fuse with SP2 / 0 myeloma cells under the action of fusion agent polyethylene glycol (PEG) 4000, and screen to obtain hybridoma cell lines that can stably secrete monoclonal antibodies .
[0039] A monoclonal hybridoma cell line of melamine was obtained through screening. The monoclonal hybridoma cell line of melamine can produce unlimited amount of melamine-specific antibody, which is specific to melamine, and the sensitivity reaches 0.05 μg / L.
[0040] Cell cryopreservation and recovery: the hybridoma cells were made into 1×10 cryopreservation medium 9 cells / ml for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 3...
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