Kit of rapid detection of nitrofurazone by using one-step enzyme-linked immunosorbent assay technique
A nitrofurazone and enzyme-linked immunosorbent technology, which is used in measuring devices, material analysis, instruments and other directions by observing the impact on chemical indicators, can solve the problems of long detection time, can not meet the promotion and application of on-site sampling inspection, etc., and meets the requirements of instruments and equipment Low, no radioisotope contamination, time-saving effect
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Embodiment 1
[0028] Example 1 Synthesis of nitrofurazone metabolite immunogen and enzyme-labeled antigen
[0029] Friedel-Crafts alkylation reaction activates nitrofurazone drug metabolites to prepare immunogen: use 4-NP derivatization, use alkylating agent to react with nitrofurazone drug metabolites, introduce a carboxyl group with a 6-carbon spacer arm into the derivative; apply carbon 2 The imine method (EDC) was coupled with hemocyanin (KLH), and the immunogen was synthesized, purified by dialysis, and stored at -20°C for future use.
[0030] Friedel-Crafts acylation reaction activates nitrofurazone drug metabolites to prepare enzyme-labeled antigen: use 4-NP derivatization, use succinic anhydride as an acylating agent, activate nitrofurazone drug metabolites, and introduce a carboxyl group with 4 carbon atoms; use mixed anhydride method and Enzyme-labeled antigen was prepared by HRP reaction; purified by dialysis and stored at -20°C for later use.
example 2
[0031] Preparation of example 2 nitrofurazone metabolite polyclonal antibody
[0032] Six healthy male New Zealand white rabbits weighing 2-2.5 kg were selected. The immunogen nitrofurazone metabolite-KLH conjugate and an equal amount of Freund's complete adjuvant were mixed into a water-in-oil emulsion by syringe pumping method, and the amount of 1 mg / kg body weight was used for the first immunization, and multiple points were taken subcutaneously on the back. injection. Immunization was boosted once every two weeks, Freund's incomplete adjuvant was used instead of complete Freund's adjuvant, and the dosage and method were the same as the first immunization. From the third immunization, 10 days after each immunization, 1m1 of blood was collected from the ear vein to detect the antibody titer. When the antibody titer no longer increased, the last (seventh) immunization was carried out without adjuvant. Thigh intramuscular injection, carotid artery bloodletting after 7 days, ...
example 3
[0033] The mensuration of example 3 nitrofurazone metabolite antiserum potency
[0034] Measured by direct ELISA method. The ELISA plate was coated with anti-nitrofurazone metabolite antiserum, washed, blocked, and the negative serum of the same dilution was used as a control, and double-diluted nitrofurazone metabolite horseradish peroxidase was sequentially added to wells 1 to 12 in the first row Markers, all 100uL / well, incubate at 22-23°C for 10 minutes; then add horseradish peroxidase (ARP) substrate 100uL / well, after 10 minutes of incubation, stop the reaction and measure OD 450value. OD of antiserum 450 The value is the same as the negative serum OD of the same dilution 450 The titer of the nitrofuracilin metabolite antiserum was defined as the maximum dilution at which the ratio of values was greater than 2. The titer of the antiserum to the metabolite of nitrofurazone was determined to be 1000.
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