Time resolved fluoroimmunoassay kit for detecting chlorpromazine and detecting method thereof
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A chlorpromazine and kit technology, applied in the field of time-resolved fluorescence immunoassay, can solve the problems of complex operation, inability to meet urgent needs, low sensitivity and the like
Inactive Publication Date: 2011-11-09
FOOD INSPECTION CENT OF CIQ SHENZHEN
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However, these methods are expensive in equipment, complex in operation, low in sensitivity, and not suitable for t...
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Embodiment 1
[0011] Prepare the kit and test the urine sample as follows:
[0012] (1) Preparation of Eu3+-goat anti-mouse antibody:
[0013] Take 1-2ml of 5g / L goat anti-mouse antibody dissolved in 50mmol / LPBS pH7.0, and switch the buffer conditions through a PD-10 column. The eluent is 50mmol / LNa2CO3-NaHCO3pH8.5 buffer containing 0.155mol / LNaCl. The protein peaks were collected, quantified by UV absorption analysis (1.46A280-0.74A260), and the goat anti-mouse antibody was diluted to 2g / L with the above eluent. Take 500-1000 μl of diluted goat anti-mouse antibody and add it to a vial containing 0.2-0.4 mg of Eu3+-N2-[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu3+-DTTA), at 30°C The reaction was stirred magnetically for 20 hours. The reaction solution was chromatographed on a Sepharose CL-6B column (1×40cm) equilibrated with 80mmol / L Tris-HCl pH7.8 buffer, and the protein peak was collected by A280 monitoring, and diluted for use.
[0014] (2) Preparation of solid-phase a...
Embodiment 2
[0041] Prepare the kit and test pork samples as follows:
[0042] (1) Preparation of the kit is the same as (1)-(5) of Example 1.
[0043] (2) The specific detection steps are as follows:
[0044] Take 5.00 g of the prepared pork sample, crush it with a homogenizer, add 10 mL of ethyl acetate and 1.0 mL of 1.0 mol / L NaOH solution in sequence, homogenize at 12000 r / min for 1 min, let it stand for 0.5 h, then centrifuge at 3000 r / min for 5 min, and take 5mL of the supernatant was adjusted to pH 7.0 with 1.0mol / L HCl for later use.
[0045] Take the CPZ-OVA strip, add 50 μl of CPZ standard or processed samples to their respective microwells, each standard and sample must use a new tip, add 50 μl of CPZ antibody diluted with buffer 1:5000, and pipette Do not touch the tip of the tube with the liquid put into the hole, shake at 37°C for 1 hour, wash with washing solution four times, add 100 μl of Eu3+-goat anti-mouse antibody diluted 1:400 in buffer, shake at 37°C for 45 minutes,...
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Abstract
The invention provides a kit for detecting chlorpromazine (CPZ) and a detecting method thereof, belonging to the technical field of time resolved fluoroimmunoassay (TRFIA) and used for detecting the content of CPZ in meat, fish and other animal derived foods. The kit prepared by the method is used for detecting CPZ by using TRFIA, and the detection is based on a labeled immunoreaction. A microporous plate is coated with CPZ-OVA (ovalbumin), a CPZ standard or sample is added, and a CPZ antibody is added; free CPZ competes for the CPZ antibody with the CPZ-OVA on the microporous plate, the CPZ antibody which is not connected is washed off and removed, Eu3+- antiMIgG is added, and the Eu3+- antiMIgG which is not connected is washed off and removed after the labeled immunoreaction; after an enhancing solution is added, the fluorescence intensity cps of the enhancing solution is detected by using a time-resolved fluorescence spectrofluorometer, the fluorescence intensity is in inverse proportion to the concentration of CPZ in the sample; and the content of CPZ in the detected sample can be determined in comparison with a standard curve. The kit for detecting CPZ provided by the invention has the advantages of simple structure, convenience in use, low cost and high sensitivity.
Description
technical field [0001] A kit for detecting chlorpromazine (CPZ) and a detection method thereof belong to the technical field of time-resolved fluorescence immunoassay (TRFIA), and are used for detecting the content of chlorpromazine in samples such as urine, tissue, and feed. Background technique [0002] Chlorpromazine is a representative drug of phenothiazines. This drug is mainly metabolized in the liver and easily produces drug residues. Residual chlorpromazine and some active metabolites of the original drug can cause leukopenia and agranulocytosis, resulting in Human liver and kidney lesions can also cause eye complications. [0003] At present, the determination methods of chlorpromazine and its metabolites in food of animal origin are mainly physical and chemical detection methods. For example, GC-MS is used to determine the residual amount of chlorpromazine in pig liver. Chlorpromazine residues in feed were determined by phase chromatography with a detection limit ...
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