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Preparation method of colloidal gold test paper strip for detecting salmonella in food based on salmonella core polysaccharide monoclonal antibody

A monoclonal antibody, colloidal gold test paper technology, applied in the field of immunoassays, can solve the problems of difficult exposure of core polysaccharides, difficult to effectively capture antibodies, and insufficient satisfaction, and achieve the effects of high affinity, low cost, and cross uniformity.

Inactive Publication Date: 2016-11-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when using paired antibodies to establish a colloidal gold test strip method, there is a problem that the T line does not develop color. The reasons may be: (1) the affinity of the screened monoclonal antibody meets the ELISA but is not enough to meet the requirements of the colloidal gold test strip; (2) The core polysaccharide is difficult to expose on the surface of the bacteria. In the case of a short reaction time (10 min) of the colloidal gold test strip method, it is difficult for the antibody immobilized on the T line to effectively capture Salmonella in the sample

Method used

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  • Preparation method of colloidal gold test paper strip for detecting salmonella in food based on salmonella core polysaccharide monoclonal antibody
  • Preparation method of colloidal gold test paper strip for detecting salmonella in food based on salmonella core polysaccharide monoclonal antibody
  • Preparation method of colloidal gold test paper strip for detecting salmonella in food based on salmonella core polysaccharide monoclonal antibody

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Experimental program
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Embodiment 1

[0030] The concrete steps of this colloidal gold test strip development are:

[0031](1) Preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8:

[0032] Using the immunogen synthesis method disclosed in Patent Application No.: 201410314040.3, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide ( NHS) method to synthesize mutant Salmonella lipopolysaccharide (Ra-LPS) and keyhole limpet hemocyanin (KLH) complete antigens, and as immunogens to immunize mice, through routine cell fusion and Salmonella cells with LPS and different O antigens as package The positive cells were screened by the original, and finally the genus-specific monoclonal antibody SQX6D8 with high affinity and uniform crossover to Salmonella was prepared.

[0033] (2) Synthesis of T-line-coated original LPS-BSA conjugate: Synthesize the conjugate using the synthesis method disclosed in Patent Application No.: 2014103140403, and the specific steps are as...

Embodiment 2

[0039] Embodiment 2 adopts this colloidal gold test strip to detect Salmonella Ra LPS:

[0040] First, Ra LPS (1mg / mL) was diluted with 0.01M phosphate buffer to 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL and 5 ng / mL, blank 0.01M phosphoric acid Salt buffer as a control;

[0041] Then use the wet method to detect Ra LPS, take 7 μL of the gold-labeled antibody prepared in step (4) of Example 1 and 47 μL of the resuspension (0.1% Tween, 0.2% sucrose, 1% BSA in 0.01M phosphate buffer ) into the microplate and mix with a pipette. Add 150 μL of different concentrations of Ra LPS to different microwell plates, mix well with a pipette gun, and react at room temperature for 5 minutes; insert the prepared colloidal gold test strip into the microwell plate, and react at room temperature for 10 minutes to read. Interpretation basis: blank sample, quality control line and test line have color at the same time, and the test line has a darker color, positive sample. The quality control lin...

Embodiment 3

[0042] Embodiment 3 Adopt this colloidal gold test strip to detect 12 strains of Salmonella:

[0043] The 12 strains were Paratyphoid A (Group A) CMCC 50093, Salmonella Agona (Group B) CICC 21586, Paratyphoid B (Group B) CMCC 50094, Salmonella Typhimurium (Group B) ATCC 13311, Tompu Salmonella genus (group C1) CICC21480, Salmonella Brockley (group C2) CICC 21489, Salmonella Kentucky (group C3) CICC 21488, Salmonella enteritidis (group D) ATCC13076, Salmonella typhi (group D) CMCC 50071, Salmonella Dublin (group D) ) CICC 21497, Duck Salmonella (Group E) CICC 21498, Arizona Salmonella ATCC 13314.

[0044] The specific detection process is as follows: the pure culture of Salmonella tested is diluted to 10% with 0.01M phosphate buffer solution. 7 CFU / mL, 10 6 CFU / Ml, 10 5 CFU / mL and 10 4 CFU / mL, the diluent was used as a blank control, and other processes were the same as in Example 2. Specific test results such as image 3 shown.

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Abstract

The invention provides a preparation method of a colloidal gold test paper strip for detecting salmonella in food based on salmonella core polysaccharide monoclonal antibody SQX6D8, and belongs to the field of immunological analysis. According to the method, a Salmonella typhimurium lipopolysaccharide (LPS) and bovine serum albumin (BSA) conjugate synthesized through a sodium periodate method is used as the coating antigen of the test line (T line) of the colloidal gold test paper strip, and salmonella core polysaccharide monoclonal antibody SQX6D8 is used as the gold-labeled antibody. According to the present invention, the principle of the method is different from the principle of the conventional pathogenic bacteria colloidal gold test paper strip sandwich method, the indirect competition principle is used to detect, it is ensured that the cross-reaction is provided for the intragenus salmonella while no cross-reaction is provided for the extragenus salmonella through the salmonella core polysaccharide specific monoclonal antibody, and the analysis means is provided for the complete, convenient and rapid detection of the salmonella in the food.

Description

technical field [0001] The invention relates to a preparation method of a colloidal gold test strip for detecting Salmonella in food based on the Salmonella core polysaccharide monoclonal antibody SQX6D8, belonging to the field of immune analysis. Background technique [0002] Salmonella is a global foodborne pathogen. Biologically, Salmonella is a kind of Gram-negative bacteria with blunt ends, no spores, and generally no capsule. The main antigens are O antigen, H antigen, and Vi antigen. Animal foods such as poultry, eggs, and dairy products are easily contaminated with Salmonella. After the human body ingests food containing bacteria, it will cause acute gastroenteritis, typhoid fever, children with low immunity and even sepsis and other symptoms. [0003] There are more than 2,000 serotypes of Salmonella, and the common serotypes in clinical practice are mainly Salmonella enteritidis, Salmonella typhimurium, and Salmonella paratyphi A. The application of well-regulat...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/531G01N33/577G01N33/56916G01N2333/255
Inventor 匡华王文彬胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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