Ketamine hybridoma cell strain and preparation method and application thereof
A hybridoma cell line, anti-ketamine technology, applied in biochemical equipment and methods, chemical instruments and methods, material testing products and other directions, can solve problems such as inability to meet, sensitivity limit of ketamine monoclonal antibody, etc., and achieve the effect of high sensitivity
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[0049] Step (1). Preparation of ketamine artificial antigen:
[0050] 1-1. Preparation and detection of ketamine hapten: using ketamine hydrochloride as a raw material, after freeing, it undergoes a substitution reaction with ethyl bromoacetate to introduce an ester group to obtain an ester of ketamine; hydrolyze under alkaline conditions to obtain a carboxyl-containing ketamine hapten :
[0051] Dissolve 200 mg of ketamine hydrochloride in 10 ml of deionized water, add ammonia water to adjust the pH to 10, add 90 ml of dichloromethane for extraction 3 times, collect the organic phase, dry with anhydrous magnesium sulfate, filter, and evaporate the solvent under reduced pressure to obtain free ketamine ; Free state ketamine is detected by thin-layer chromatography: the chromatographic liquid is petroleum ether: ethyl acetate=1:1, product R f =0.3~0.4.
[0052] Dissolve free ketamine in 10 ml of acetone, add 121 μl of ethyl bromoacetate and 300 mg of potassium carbonate, ref...
Embodiment 2
[0077] Example 2 Performance test of anti-ketamine monoclonal antibody
[0078] 2.1 Subtype identification of anti-ketamine monoclonal antibodies
[0079] The anti-ketamine monoclonal antibody prepared in Example 1 was subjected to isotype identification with reference to Monoclonal antibody Isotyping Kits (pierce #37501). The anti-ketamine monoclonal antibody belongs to the IgG1 subtype, and the light chain is a kappa chain.
[0080] 2.2 Anti-ketamine monoclonal antibody reactivity test
[0081] Indirect ELISA method to detect the anti-ketamine monoclonal antibody prepared in Example 1: Dilute the ketamine artificial antigen (KET-BSA) to 1ug / mL with pH9.6 carbonate buffer with a concentration of 0.05M, and then put it on a 96-well microtiter plate Add 100ul diluted ketamine artificial antigen to each well, coat overnight at 4°C; then wash the plate 3 times with PBS buffer containing 0.05% tween20; then add 100ul anti-ketamine monoclonal antibody solution with a concentration...
Embodiment 3
[0084] Example 3 Establishment and evaluation of ketamine colloidal gold test strip method
[0085] 1. Preparation of Colloidal Gold
[0086] First, prepare 20ml of 0.1g / L chloroauric acid solution, heat it to boiling with a constant temperature electromagnetic stirrer, add 0.4ml of 1% trisodium citrate solution, continue to heat and stir for 10min, cool to room temperature and add distilled water to the original volume to obtain colloidal gold solution;
[0087] 2. Colloidal Gold Labeling of Antibody Proteins
[0088] Use 0.1mol / L K 2 CO 3 Adjust the pH value of the colloidal gold solution to 9.0, and add 0.5 mg of anti-ketamine monoclonal antibody protein dropwise; after continuing to stir for 3 minutes, add dropwise the BSA solution with a mass-to-volume ratio of 5% until the mass-to-volume ratio of the BSA solution is 1%, and continue to stir for 10 minutes. An antibody solution is obtained; wherein the anti-ketamine monoclonal antibody protein is the antibody protein ...
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