Streptomycin hapten and its preparation method and application
A streptomycin and hapten technology, applied in the field of hapten and its preparation, can solve the problems of high cost, expensive equipment, complicated operation and the like, and achieve the effects of convenient use, low detection cost and simple synthesis method
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Embodiment 1
[0018] Example 1: Synthesis and identification of streptomycin hapten
[0019] The mixed solution of 0.58g streptomycin and 0.07g ethylenediamine in 50ml methanol is reacted at room temperature for 5-10 hours, the solvent is evaporated, and the streptomycin hapten is obtained quantitatively.
[0020] Take the above-mentioned product and measure by hydrogen nuclear magnetic resonance spectroscopy, such as figure 2 As shown, the newly added peaks at 1.6, 2.3, and 3.4 ppm (mg / L) in the spectrum are the signal peaks of the aminoethyl fragment, and the peaks around 7.7 ppm are the CH signal peaks on the enamine double bond, indicating the synthesis of hapten success.
Embodiment 2
[0021] Example 2: Streptomycin antigen
[0022] The streptomycin hapten is coupled with the carrier protein to obtain the streptomycin antigen.
[0023] 1. Preparation of immunogen-synthesis of streptomycin hapten-bovine serum albumin conjugate
[0024] Take 20 mg of streptomycin hapten and dissolve it in 2 ml of water to obtain solution I; take 0.5 ml of 3% glutaraldehyde and add dropwise to solution I, stir at room temperature for 24 hours to obtain solution II; take 50 mg of bovine serum albumin (BSA) Dissolve with 4ml of water to obtain solution III; slowly add solution II to solution III, stir at room temperature and react overnight; add NaBH 4 30mg reduction; dialyzed with 0.02M PBS for three days and replaced the dialysate three times a day to obtain streptomycin immunogen.
[0025] 2. Preparation of the original coating-synthesis of streptomycin hapten-ovalbumin conjugate
[0026] Take 50mg ovalbumin (OVA) and dissolve in 4ml water to obtain solution I; take 50mg each of carbod...
Embodiment 3
[0029] Example 3: Streptomycin monoclonal antibody
[0030] 1. Preparation of Streptomycin Monoclonal Antibody
[0031] Animal immunization: Inject the immunogen into Balb / c mice at a dose of 150μg / mouse to produce polyclonal antibodies.
[0032] Cell fusion and cloning: After the mouse serum assay result is high, take its spleen cells and fuse with SP2 / 0 myeloma cells at a ratio of 8:1. Use indirect competitive ELISA to measure the cell supernatant and screen positive wells. Use the limiting dilution method to clone the positive wells until a hybridoma cell line secreting monoclonal antibodies is obtained.
[0033] Cell cryopreservation and resuscitation: The monoclonal hybridoma cell line of streptomycin is made into 5×10 with cryopreservation solution 7 Cells / ml cell suspension, long-term storage in liquid nitrogen. Take out the cryopreservation tube during resuscitation, immediately put it into a 37℃ water bath for rapid thawing, centrifuge to remove the cryopreserved liquid, and...
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