Colloidal gold detection test strip for detecting Brucella antigen by sandwich method

A technology of Brucella and detection test paper, applied in the field of immune detection, can solve the problems of insufficient capture ability, inability to detect susceptible animals, low sensitivity, etc., and achieve the effect of eliminating interference

Inactive Publication Date: 2021-07-09
CHINA INST OF VETERINARY DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, only the complete Brucella particle antigen is captured by the bp26 monoclonal antibody, which obviously has the problem of low sensitivity due to insufficient capture ability
Zhang Mingzhou et al. (Application No. 201310033074.0) of Hangzhou Dean Technology Co., Ltd. used Brucella bovis antibody-labeled colloidal gold to detect only Brucella antigens in bovine blood samples, and cannot detect other susceptible animals. limitations
Hangzhou Aotai Biotechnology Co., Ltd. Chen Jinshu et al. (application number 201911398504.2) used Brucella monoclonal antibody and polyclonal antibody sandwich method to detect Brucella. Like the other methods mentioned above, the sensitivity and specificity of the method are not high

Method used

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  • Colloidal gold detection test strip for detecting Brucella antigen by sandwich method
  • Colloidal gold detection test strip for detecting Brucella antigen by sandwich method
  • Colloidal gold detection test strip for detecting Brucella antigen by sandwich method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1—Optimized selection of detection line coating antibody

[0051] 1. Streak inoculation of Brucella suis S2 into TSA medium, incubate at 37°C for 72 hours, wash the culture with normal saline, count the viable bacteria and calculate the concentration of the bacteria solution, and inactivate them in a water bath at 80°C for 2 hours. According to the result of live bacteria count, use 0.01M PBS to dilute the Brucella solution to 1×10 8 CFU / mL, 5×10 7 CFU / mL, 1×10 7 CFU / mL, 5×10 6 CFU / mL, 1×10 6 CFU / mL, 5×10 5 CFU / mL for use.

[0052]2. Escherichia coli O157 and Yersinia enterocolitica O9 were each inoculated into a suitable growth medium, cultured at a suitable temperature, harvested, and inactivated after counting the number of viable bacteria to calculate the concentration of the bacteria liquid, and using 0.01M according to the result of the count of viable bacteria Dilute each bacterial solution with PBS to 1×10 8 CFU / mL for use.

[0053] 3. Prepare 8 ...

Embodiment 2

[0060] Embodiment 2——Utilize 4 monoclonal antibody sandwich methods that bind to different epitopes to detect the detection test strip structure of Brucella antigen

[0061] Test strips for detecting Brucella antigens using the sandwich method, such as figure 1 As shown, the test strip is composed of a PVC bottom plate (7) and a sample pad (1), a gold standard pad (2), a nitrocellulose membrane (3) and an absorbent pad (4) pasted on the bottom plate in sequence. The pad (2) is coated with colloidal gold-labeled Brucella monoclonal antibody 4D7, the detection line (5) is coated with the mixture of Brucella monoclonal antibody 2B5, 5F3, and 6C2, and the quality control line (6) is coated with goat anti- Mouse IgG antibody.

Embodiment 3

[0062] Embodiment 3——The preparation of the detection test strip that sandwich method detects Brucella antigen

[0063] 1. Purification and quantification of LPS from different strains of Brucella suis S2, Brucella melis M5, Brucella bovis A19, Escherichia coli O157, Yersinia enterocolitica O9 and so on.

[0064] (1) Bacterial suspension preparation and inactivation will identify qualified Brucella suis S2, Brucella melis M5, Brucella bovis A19, Escherichia coli O157, Yersinia enterocolitica O9 Inoculate appropriate medium respectively, and after culturing at 37°C for 72 hours, add 10ml of physiological saline containing 0.5% phenol to each bottle to soak the surface of the bacteria for more than 10min, wash the culture, inactivate at 80°C for 2h, and conduct an inactivation test.

[0065] (2) Rough extraction of LPS Centrifuge the bacterial suspension that passed the inactivation test at 10,000 g for 20 min to collect the precipitate. Weigh and calculate the wet weight of th...

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Abstract

The invention relates to a colloidal gold detection test strip for detecting a Brucella antigen by a sandwich method. The detection test strip is composed of a Brucella antigen detection test strip and a plastic suction head, and the detection test strip is composed of a PVC base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad. A four-strain Brucella monoclonal antibody sandwich method combined with different antigen epitopes is adopted, a colloidal gold labeled Brucella monoclonal antibody 4D7 is coated on a gold label pad, a mixed solution of Brucella monoclonal antibodies 2B5, 5F3 and 6C2 is coated on a detection line, and a goat anti-mouse IgG antibody is coated on a quality control line. The test strip for detecting the Brucella antigen can be used for rapidly detecting bovine breed, sheep breed and swine breed Brucella on site and effectively eliminating interference of enterocolitis yersinia O9 and escherichia coli O157 on Brucella antigen detection. The test strip is high in sensitivity and sensitivity, good in specificity, simple and convenient to use, applicable to detection of samples such as clinical tissues, environments and milk, and also applicable to identification of purely cultured Brucella.

Description

technical field [0001] The invention relates to a method for diagnosing animal brucellosis—a colloidal gold detection test strip for detecting Brucella antigen by a sandwich method, which belongs to the technical field of immune detection. Background technique [0002] Brucellosis (referred to as "brucellosis") is a chronic multi-organ damage zoonotic infectious disease caused by Brucella. WHO regards Brucellosis as "the most widespread human and animal disease in the world." It is a common disease, but it is also one of the seven important infectious diseases that are most easily overlooked by people." Brucellosis is mainly contracted through direct or indirect contact with infected animals or animal products. After human beings suffer from brucellosis, it will cause fever, muscle and joint pain, etc., and severe cases will completely lose their ability to work; livestock infected with brucellosis will cause abortion and infertility, causing huge economic losses to the bre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/58G01N33/558G01N33/532
CPCG01N33/56911G01N33/558G01N33/577G01N33/587G01N33/532G01N2333/23G01N2469/10
Inventor 蒋卉彭小薇丁家波沈青春范学政张莹辉秦玉明许冠龙
Owner CHINA INST OF VETERINARY DRUG CONTROL
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