WO2017211214A1 - Procédé de préparation d'une bandelette réactive, à or colloïdal, de détection de salmonella, destinée aux aliments, faisant appel à un anticorps monoclonal spécifique d'un polysaccharide de noyau de salmonella - Google Patents

Procédé de préparation d'une bandelette réactive, à or colloïdal, de détection de salmonella, destinée aux aliments, faisant appel à un anticorps monoclonal spécifique d'un polysaccharide de noyau de salmonella Download PDF

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WO2017211214A1
WO2017211214A1 PCT/CN2017/086561 CN2017086561W WO2017211214A1 WO 2017211214 A1 WO2017211214 A1 WO 2017211214A1 CN 2017086561 W CN2017086561 W CN 2017086561W WO 2017211214 A1 WO2017211214 A1 WO 2017211214A1
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salmonella
solution
colloidal gold
monoclonal antibody
group
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Chinese (zh)
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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
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Jiangnan University
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Jiangnan University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)

Definitions

  • the invention relates to a preparation method of a colloidal gold test strip for detecting Salmonella in food based on the monoclonal antibody SQX6D8 of Salmonella core polysaccharide, belonging to the field of immunoassay.
  • Salmonella is a global foodborne pathogen. Biologically, Salmonella is a kind of Gram-negative bacteria with blunt ends. It is free of spores and generally has no capsule. The main antigens are O antigen, H antigen and Vi antigen. Animal foods such as poultry, eggs, and dairy products are susceptible to contamination with Salmonella. After the human body ingests the bacteria-containing food, it may cause acute gastroenteritis, typhoid fever, and children with low immunity, and even symptoms such as sepsis.
  • the methods for detecting Salmonella mainly include biochemical culture methods, immunological detection methods, and molecular detection methods.
  • the traditional biochemical culture method is the national standard method for detecting Salmonella. Although the authority is reliable, it usually takes 5-10 days to obtain the results, and the operation process is cumbersome and cannot meet the requirements of rapid detection.
  • the molecular detection method is based on Salmonella deoxyribonucleic acid (DNA).
  • PCR Polymerase chain reaction
  • PCR polymerase chain reaction
  • RT-PCR real-time fluorescent quantitative PCR
  • LAMP loop-mediated isothermal amplification
  • RT-PCR has the characteristics of quantitative detection of target DNA, stronger specificity, effective solution to PCR contamination problems, and high degree of automation.
  • the LAMP method is characterized by simplicity, speed, and specificity. This technology is no less than conventional PCR technology in terms of sensitivity, specificity and detection range. It does not rely on special instruments and equipment, and can be quickly detected in the field with high throughput, and the detection cost is much lower than real-time PCR. However, it has been reported in the literature that the LAMP method has a false negative problem when detecting Salmonella in cow's milk. This may be caused by the influence of the primer on the sample matrix. Similarly, conventional PCR and real-time fluorescent PCR are also faced with the problems of high detection cost and high technical requirements for operators.
  • the colloidal gold test strip method has the advantages of being simple, rapid, and low in cost for detecting pathogenic microorganisms. Colloidal gold test strips for the detection of Salmonella at the genus level have not been reported. The main difficulties are the preparation of monoclonal antibodies with high cross-linking, high affinity, and the acquisition of paired antibodies for double antibody sandwiches. In the prior patent we disclose the synthesis of cross-over immunogens (Patent Application No.: 201410314040.3) and the establishment of a cross-type double antibody sandwich ELISA method (201510183851.9). However, the method of establishing a colloidal gold test strip with a paired antibody faces the problem of non-coloration of the T-line.
  • the reasons may be as follows: (1) The affinity of the selected monoclonal antibody satisfies the ELISA but is insufficient to meet the requirements of the colloidal gold test strip; (2) The core polysaccharide is difficult to be exposed on the surface of the bacteria. In the case where the reaction time of the colloidal gold test strip method is very short (10 min), it is difficult to effectively capture the Salmonella in the sample by the antibody immobilized on the T line. Therefore, we used a newly prepared monoclonal antibody SQX6D8 with higher affinity, and synthesized heterologous LPS conjugate as a T-line coating, which was tested by competitive principle, which overcomes the difficulty of capturing Salmonella by T-line antibodies. problem.
  • the detection sensitivity of the 12 strains of Salmonella tested was 10 5 -5 ⁇ 10 6 CFU/mL, and the crossover was uniform.
  • other test bacteria such as E.coli, E.coli O157:H7, Enterobacter sakazakii, jejunum Campylobacter, Campylobacter coli, Vibrio parahaemolyticus, Staphylococcus aureus, Listeria monocytogenes did not cross-react.
  • the object of the present invention is to establish a colloidal gold test strip for detecting Salmonella at the genus level for rapid detection of high specificity and high accuracy of Salmonella in food.
  • the present invention establishes a method for detecting a colloidal gold test strip of Salmonella in a food based on the competition principle of a Salmonella core polysaccharide monoclonal antibody, and the method further comprises an optimization process.
  • the monoclonal antibody SQX6D8 was obtained by immunizing 8 weeks old BALB/c mice with EDC LPS-BSA artificial antigen as immunogen, and was obtained by fusion and screening by hybridoma technique, which has the characteristics of high affinity and good inhibition effect.
  • the T-line was originally synthesized by the NaIO 4 method, and the LPS-BSA conjugate was better than the homologous EDC method.
  • test line T line
  • Salmonella core polysaccharide-specific monoclonal antibody SQX6D8 SQX6D8 and the red gold nanometer.
  • the particles were coupled as a gold standard antibody.
  • the Salmonella in the sample first binds to the gold-labeled antibody, thereby inhibiting the binding of the gold-labeled antibody to the T-line coating.
  • the color intensity of the T-line is inversely proportional to the amount of Salmonella in the sample.
  • Salmonella-based core polysaccharide-specific monoclonal antibodies ensure that the method has crossover for both Salmonella and no cross-reactivity to the extracellular bacteria.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee
  • a preparation method for detecting colloidal gold test strips of Salmonella in food based on monoclonal antibody SQX6D8 of Salmonella core polysaccharide the specific steps are as follows:
  • activation take the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg dissolved in ultrapure water according to the reaction mass ratio NaIO 4 ..Ra-LPS 5..1 drop 47 ⁇ L concentration of 200mM / L NaIO 4 solution to the mutant mouse
  • Salmonella typhimurium lipopolysaccharide Ra-LPS solution react at 25 ° C for 2 h; then take 10 ⁇ L of 1 M / L ethylene glycol solution into the reaction solution, react at 25 ° C for 2 h;
  • the Salmonella includes 12 strains, followed by paratyphoid A (group A), Agungnashamen (group B), typhoid typhus (group B), paratyphoid B (group B), and Thompson sand gate (C1). Group), Blockley Sandmen (C2 Group), Kentucky Sandmen (C3 Group), Enteritis Sand Gate (Group D), Typhoid Shamen (Group D), Dublin Sand Gate (Group D), Duck Sand Gate (Group E), Arizona Sand Gate.
  • the Salmonella-specific colloidal gold test strip provided by the invention is different from the principle of the conventional macromolecular colloidal gold test strip double antibody sandwich method, but is tested by a competition method.
  • the monoclonal antibody specific for Salmonella core polysaccharide SQX6D8 has higher affinity and uniformity.
  • the synthesized LPS-BSA conjugate is used as a T-line coating to compete with Salmonella in the sample for SQX6D8-conjugated gold nanoparticles.
  • the colloidal gold test strip can detect Salmonella at the genus level and does not cross-react with other test bacteria.
  • the method has the characteristics of simple and rapid operation, good stability and low cost, and has the promotion and application value.
  • Biomaterial sample preservation The monoclonal cell strain SQX6D8 has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, referred to as CGMCC. Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Deposit Date: January 20, 2016, deposit number CGMCC No. 12019.
  • Figure 1 is a schematic diagram of the principle of Salmonella-specific colloidal gold test strips.
  • FIG 2 shows Salmonella-specific colloidal gold test strips for detection of mutant lipopolysaccharide (RaLPS).
  • activation take the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg dissolved in ultrapure water, according to the reaction mass ratio NaIO 4 ..Ra-LPS 5..1 drop 47 ⁇ L concentration of 200mM / L NaIO 4 solution to the mutant
  • the Salmonella typhimurium lipopolysaccharide Ra-LPS solution was reacted at 25 ° C for 2 h; then 10 ⁇ L of 1 M / L ethylene glycol solution was taken into the reaction solution, and reacted at 25 ° C for 2 h;
  • Example 2 uses the colloidal gold test strip to detect Salmonella Ra LPS:
  • Ra LPS (1 mg/mL) was diluted with 0.01 M phosphate buffer to 100 ng/mL, 50 ng/mL, 25 ng/mL, 10 ng/mL and 5 ng/mL, and 0.01 M phosphate buffer as a control. Then, the Ra LPS was detected by a wet method, and 7 ⁇ L of the gold-labeled antibody prepared in the step (4) of Example 1 and 47 ⁇ L were taken. The suspension (0.1% Tween, 0.2% sucrose, 1% BSA in 0.01 M phosphate buffer) was placed in a microplate and mixed with a pipette.
  • Example 3 used the colloidal gold test strip to detect 12 strains of Salmonella:
  • the 12 strains were typhoid A (group A) CMCC 50093, Agunga Shamen (group B) CICC21586, paratyphoid B (group B) CMCC 50094, typhoid fever sand (group B) ATCC 13311, Thompson Shamen (C1 group) CICC21480, Brockley Shamen (C2 group) CICC 21489, Kentucky Shamen (C3 group) CICC 21488, Enteritis sand gate (Group D) ATCC13076, Salmonella typhi (D group) CMCC 50071, Dublin Shamen (Group D) CICC 21497, Yashamen (Group E) CICC 21498, Arizona Sand Gate ATCC 13314.
  • the specific detection process is: the test Salmonella pure culture is diluted with 0.01 M phosphate buffer to 10 7 CFU/mL, 10 6 CFU/Ml, 10 5 CFU/mL and 10 4 CFU/mL, with diluent As a blank control, the other processes are the same as in the second embodiment.
  • the specific test results are shown in Figure 3.
  • the other bacteria tested in 8 strains were: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157, common Escherichia coli, Enterobacter cloacae, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli.
  • the test concentration was 5 ⁇ 10 8 CFU/mL, and the detection process was the same as in Example 2, and the results are shown in FIG. 4 .

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Abstract

L'invention concerne le domaine des essais immunologiques et fournit un procédé de préparation d'une bandelette réactive, à or colloïdal, de détection de Salmonella, destinée aux aliments, faisant appel à un anticorps monoclonal spécifique d'un polysaccharide de noyau de Salmonella SQX6D8. Un conjugué de lipopolysaccharide de Salmonella typhimurium (LPS) synthétisé au moyen d'un procédé à base de périodate de sodium avec une albumine bovine (BSA) est utilisé en tant qu'antigène d'enveloppe d'une ligne d'essai (ligne T) de la bandelette réactive à or colloïdal. L'anticorps monoclonal SQX6D8 de polysaccharide de noyau de Salmonella est utilisé en tant qu'anticorps marqué à l'or. Contrairement au principe sandwich employé dans des bandelettes réactives classiques, à or colloïdal, de détection de bactéries pathogènes, le procédé selon la présente invention de bandelette réactive, à or colloïdal, de détection de Salmonella emploie un concept de compétition indirecte pour l'exécution d'un essai; en outre, un anticorps monoclonal spécifique d'un polysaccharide de noyau de Salmonella est utilisé pour garantir que le procédé est à réaction croisée avec une bactérie du genre de Salmonella et n'est pas à réaction croisée avec une bactérie qui n'est pas du genre de Salmonella, fournissant ainsi un moyen analytique à un essai rapide, pratique, simple, complet, de détection de Salmonella dans des aliments.
PCT/CN2017/086561 2016-06-07 2017-05-31 Procédé de préparation d'une bandelette réactive, à or colloïdal, de détection de salmonella, destinée aux aliments, faisant appel à un anticorps monoclonal spécifique d'un polysaccharide de noyau de salmonella Ceased WO2017211214A1 (fr)

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CN201610396856.4A CN106093409A (zh) 2016-06-07 2016-06-07 基于沙门氏菌核心多糖单克隆抗体的检测食品中沙门氏菌属的胶体金试纸条的制备方法
CN201610396856.4 2016-06-07

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