Fluorescence polarization immunoassay method for detecting carbaryl

A fluorescence polarization and immunoassay technology, applied in the field of immunoassay technology and pesticide residue detection, can solve the problems of complex operation and long time consumption, and achieve the effect of simple operation

Active Publication Date: 2017-06-20
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0005] The present invention aims at the disadvantages of complex operation and long time consumption in the existing carbaryl immunoassay technology, and establishes a fluorescence polarization immunoassay method for detecting carbaryl. The whole detection process only needs one-step competitive reaction, and the operation is simple and convenient. fast

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  • Fluorescence polarization immunoassay method for detecting carbaryl
  • Fluorescence polarization immunoassay method for detecting carbaryl
  • Fluorescence polarization immunoassay method for detecting carbaryl

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preparation example Construction

[0046] 5. Preparation, purification, subtype and identification of anti-carbaryl monoclonal antibody

[0047] The obtained anti-carbaryl monoclonal antibody hybridoma cell line Jnw1D2 was injected into BALB / c mice treated with incomplete Freund's adjuvant in advance, the ascites of the mice was collected, and the antibody was purified by octanoic acid-ammonium sulfate method, specifically The operation is: filter mouse ascites with double-layer filter paper, centrifuge at 12,000 r / min at 4°C for more than 15 minutes, absorb the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, slowly add n-octanoic acid while stirring, The volume of n-octanoic acid required per milliliter of ascitic fluid is 30-35 μL, mixed at room temperature for 30-60 min, and allowed to stand at 4 °C for more than 2 h. Centrifuge at 12000r / min at 4°C for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / ...

example 1

[0050] Example 1, the preparation of fluorescent markers

[0051] Step 1: Preparation of carbaryl hapten synthesis intermediate (1-naphthyloxy-4-nitrobenzene carbonate)

[0052] Install an electric stirrer in a 1L four-necked bottle, configure a thermometer and a constant pressure dropping funnel, add 240mL of dichloromethane, 33mL of triethylamine, and 31.7g of methylnaphthol at room temperature, stir and dissolve, then drop to 0°C with a low-temperature reaction bath ; 40.2g phenyl p-nitrochloroformate was dissolved in 60mL of dichloromethane solution, slowly added dropwise to the above solution, white smoke was produced, and the color change of the solution became darker along with the addition. After 1h dropwise addition, Insulation reaction for 3 hours, TLC monitors the complete reaction of the raw materials (developing agent: dichloromethane: petroleum ether = 1:3); add 360mL of 3% hydrochloric acid, stir for about 30min, separate the liquids, combine the organic phases,...

example 2

[0061] Example 2, Screening of the best fluorescent markers

[0062] Step 1: First, set the working concentration of each fluorescent marker to the concentration (5nM) of the corresponding fluorescent marker when the fluorescence intensity is 10 times the background fluorescence intensity of the borate buffer solution. According to 1 / 125, 1 / 250, 1 / 500, 1 / 1000, 1 / 2000, 1 / 4000, 1 / 8000, 1 / 16000 and 1 / 32000 dilution, draw the antibody binding curve to obtain the maximum value of the signal intensity change δmP (δmP=mP max -mP min ), where the signal change value of CNH-EDF is the largest. The experimental results are shown in Table 2:

[0063] Table 2 The signal intensity of three fluorescent markers combined with antibodies

[0064]

[0065] Step 2: First, the working concentration of each fluorescent marker is set to the concentration (5nM) of the corresponding fluorescent marker when the fluorescence intensity is 10 times the background fluorescence intensity of the BB b...

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Abstract

The invention relates to a fluorescence polarization immunoassay method for detecting carbaryl. The method comprises the steps of mixing a sample to be tested, a fluorescent marker solution and a carbaryl monoclonal antibody solution; incubating for carrying out competing reaction; determining a fluorescence polarization value of an obtained system; calculating the concentration of the carbaryl in the sample to be tested according to a fluorescence polarization value-carbaryl concentration standard curve in a carbaryl standard sample. According to the carbaryl fluorescence polarization immunoassay (FPIA) method provided by the invention, only sample adding is required, the separation and washing operation are not needed, so that a detection result can be obtained within ten minutes. The carbaryl residue detection limitation requirement can be met.

Description

technical field [0001] The invention belongs to the field of immunoassay technology and pesticide residue detection, and in particular relates to a fluorescence polarization immunoassay method for detecting carbaryl. Background technique [0002] Carbaryl, also known as carbaryl and carbaryl, is a carbamate insecticide. It has the characteristics of high efficiency, long duration, and high selectivity. It is a broad-spectrum pesticide with a large amount of use in China. It is widely used in fruits, In the production of crops such as vegetables, cotton and tea. Carbaryl is a moderately toxic pesticide with contact and stomach poisoning effects. It remains in water, soil, fruit, grain and other media for a long time, which can easily cause excessive residues in agricultural products and safety consumption problems. and endocrine system, threatening the sustainable development of the agricultural industry. Annex II in the fourth meeting of the EU Chemical Review Committee in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/533
CPCG01N33/533G01N33/577G01N21/6445G01N33/542G01N33/582G01N2021/6439
Inventor 李培武李慧杨青青唐晓倩张奇张文张兆威丁小霞
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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