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Fluorescence analysis method for detecting LM (lincomycin) in shrimp meat

A lincomycin and antibody technology, applied in the field of lincomycin content detection, can solve the problems of complicated operation, low sensitivity, expensive instruments and equipment, etc.

Inactive Publication Date: 2019-05-14
ZHENJIANG XIANCHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are expensive in equipment, complex in operation, low in sensitivity, and are not suitable for the detection of large quantities of samples, and cannot meet the urgent needs of the domestic food safety testing market.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Follow the steps below to prepare the kit and test the shrimp samples:

[0009] (1) Eu 3＋ - Preparation of goat anti-mouse antibody:

[0010] 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 500-1000μl diluted goat anti-mouse antibody and add Eu containing 0.2-0.4mg 3＋ -N2-[p-isocyanic acid-benzyl]-diethylenetriaminetetraacetic acid (Eu 3 ＋ -DTTA) in a vial with magnetic stirring at 30°C 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 solution, and the protein peak was collected by A280 monitoring, and diluted for use.

[0011] (2) Preparation of...

Embodiment 2

[0036] Prepare the kit and test pork samples as follows:

[0037] (1) preparation kit is the same as (1)~(5) of embodiment 1;

[0038] (2) The specific detection steps are as follows:

[0039] Take 5.00 g of the prepared chicken 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.

[0040] Take the LM-OVA strip, add 50 μl of LM standard or processed samples to their respective microwells, each standard and sample must use a new tip, add 50 μl of LM 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 1:400 diluted Eu 3＋ - Goat anti-mouse antibody 100 μl, shake at 37°C for 45 minutes, wash 4 time...

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Abstract

The invention relates to a kit for detecting LM (lincomycin) and a detection method thereof, and belongs to the technical field of TRFIA (Time-Resolved Fluorescence Immunoassay). The kit prepared by the invention adopts TRFIA to detect the LM, and the basis of measurement is labeled immunoreaction. A micropore plate is coated with LM-OVA, an LM standard or sample is added, and then LM antibodies are added. The dissociative LM and the LM-OVA on the micropore plate compete for the LM antibodies, the LM antibodies which are not connected are removed by washing, Eu3+-goat-anti-mouse antibodies areadded, and after the labeled immunoreaction, the Eu3+-goat-anti-mouse antibodies which are not connected are removed by washing. After enhancement solution is added, fluorescence intensity cps of thesample is measured by a time-resolved fluorescence instrument, the fluorescence intensity is inversely proportional to LM concentration in the sample, and by comparing with a standard curve, contentof the LM in the detected sample can be determined. The LM detection kit provided by the invention is simple in structure, convenient to use, cheap and high in sensitivity, and operation time is greatly reduced; and the kit and the detection method can be used for detecting residual LM in aquatic products (fish and shrimp meat).

Description

technical field [0001] A kit for detecting lincomycin (LM) and a detection method thereof belong to the technical field of time-resolved fluorescence immunoassay (TRFIA), and are used for detecting the content of lincomycin in samples such as fish and shrimp. Background technique [0002] Lincomycin is a broad-spectrum antibiotic, commonly used in the treatment of various infectious diseases in animals, and has a strong inhibitory effect on a variety of pathogenic bacteria. Lincomycin has serious side effects, which can inhibit the hematopoietic function of human bone marrow and cause human diseases such as aplastic anemia. Therefore, lincomycin residues in animal foods pose a huge threat to human health. [0003] At present, the determination methods of lincomycin and its metabolites in food of animal origin are mainly physical and chemical detection methods. For example, chemical analysis is used to determine the residual amount of lincomycin in shrimp meat, and the detect...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 洪霞张淑雅秦悦
Owner ZHENJIANG XIANCHUANG BIOTECH CO LTD
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