Immunoassay kit for detecting ethopabate, and preparation method and application thereof
A technology of ethopabate and methyl ethoxybenzoate, which is applied in the field of immunoassay kits and preparations for the detection of ethopabate, can solve problems affecting drug monitoring, etc., and improve fusion efficiency and effect , improve affinity and sensitivity, and enhance the effect of stimulation
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[0032] A preparation method of an immunoassay kit for detecting ethopabate, the steps are as follows:
[0033] Step 1, the preparation of ethopabate hapten
[0034] The ethopabate hapten refers to methyl 4-amino-2-ethoxybenzoate, which can combine with the corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate the animal body to produce antibodies alone. The synthesis method of the hapten can be prepared through the following two ways.
[0035] Route 1: Using p-aminosalicylic acid as a raw material, undergo two-step reactions of methyl esterification and ethylation to prepare methyl 4-amino-2-ethoxybenzoate. The specific steps are: first react p-aminosalicylic acid, methanol and concentrated sulfuric acid at 55°C-75°C for 5-24 hours to generate 4-amino-2-hydroxybenzoic acid methyl ester; then, take p-aminosalicylic acid Add methyl ester, diethyl sulfate, sodium carbonate and N,N-dimethylformamide into a microwave reactor, heat up to 50°C-100°C, ...
Embodiment 1
[0072] Example 1 A preparation method of an immunoassay kit for detecting ethopabate
[0073] Preparation of ethopabate hapten: using p-aminosalicylic acid as raw material, react 5g of p-aminosalicylic acid, 50mL of methanol and 200mL of concentrated sulfuric acid at 65°C for 16h to generate 4-amino-2-hydroxybenzoic acid Methyl ester: Take 2g of methyl p-aminosalicylate, 3g of diethyl sulfate, 10g of sodium carbonate and N,N-dimethylformamide into a microwave reactor, heat up to 70°C, microwave power is 5W, and react for 2h . Cool the reaction kettle to room temperature, take out the product, add water, extract twice with dichloromethane, combine the extracts, and distill under reduced pressure to obtain 4-amino-2-ethoxybenzoic acid methyl ester, namely ethopabate half antigen.
[0074] Preparation of ethopabate immunogen and coater: dissolve 20mg polylysine in 30mL 0.01mol / L pH 7.4 phosphate buffer, add 7mL 25% glutaraldehyde solution, react at room temperature 18h, after ...
Embodiment 2
[0086] Example 2 Ethoxylate immunoassay kit detects ethopabate in chicken
[0087] Weigh 5g of fresh chicken tissue sample, cut it into pieces, grind it, mix it with phosphate buffer solution of pH 7.4, add 50mmol / L hydrochloric acid solution, mix it, and extract it by ultrasonic wave for 1h. After cooling to room temperature, filter, take 10 mL of the filtrate, add 1.0 mL of 1mol / L sodium hydroxide solution, mix well, add 5.0 mL of 0.50 mol / L potassium dihydrogen phosphate solution, place at 4°C for 1 hour, filter, and take the filtrate Add 5.0 mL of C18 solid-phase extraction cartridge, elute the sample with 3.0 mL of methanol, evaporate the eluent to dryness in a water bath, dissolve it in 1.0 mL of distilled water and detect it.
[0088] During detection, take out the kit prepared in Example 1, take out the microplate as required after returning to room temperature, add 50 μ L / well of ethopabate standard solution or sample solution to be tested in the microplate, and then ...
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