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Monoclonal antibody composition for quantitative detection of Coxiella burnetii I-phase strains

A technology of Coxiella basilica and composition, which is applied in the field of monoclonal antibody composition, can solve problems such as time-consuming and labor-intensive operation, difficulty in separating pathogens, complicated result judgment and infection risk assessment, etc. The result is a stable effect

Active Publication Date: 2020-01-17
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological detection methods are highly specialized, have high biosafety requirements, and high technical requirements. Patients with acute onset often cannot detect antibodies, and it is very difficult to separate pathogens from blood; nucleic acid detection needs to extract genomes in advance, which is time-consuming and labor-intensive. Professional equipment is required, and it needs to be operated in a professional laboratory. At the same time, the method of nucleic acid detection cannot determine whether the bacteria are alive, which will easily lead to the complexity of result judgment and infection risk assessment

Method used

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  • Monoclonal antibody composition for quantitative detection of Coxiella burnetii I-phase strains
  • Monoclonal antibody composition for quantitative detection of Coxiella burnetii I-phase strains
  • Monoclonal antibody composition for quantitative detection of Coxiella burnetii I-phase strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, preparation of anti-Cox body monoclonal antibody

[0046] 1) Immunization of mice

[0047] Using the inactivated antigen of whole bacterium of Coxiella spp. Xinqiao strain as the immunogen, female 8-week-old BALB / c mice were immunized by subcutaneous injection, and spleen lymphocytes were obtained for hybridoma fusion experiments.

[0048] Purified inactivated Coxiella beinii whole antigen was diluted with PBS pH 7.4. Five 8-week-old female BALB / c mice with similar body weight were selected. For the first immunization, 20 μg of the whole bacterial antigen was emulsified and mixed with 100 μl of Freund’s complete adjuvant and injected subcutaneously; the second subcutaneous immunization and the third subcutaneous immunization were performed every two weeks, and the adjuvant was changed to Freund’s for the second and third immunizations. Incomplete adjuvant; booster immunization was carried out 3 days before fusion, without adjuvant, intraperitoneal inject...

Embodiment 2

[0080] Embodiment 2. Specific detection (ELISA method) of anti-Cox body monoclonal antibody

[0081] Rickettsia spotted heat (Th1 epitope peptides induce protective immunity against Rickettsia rickettsii infection in C3H / HeN mice. Wang P, Xiong X, JiaoJ, Yang X, Jiang Y, Wen B, Gong W. Vaccine. 2017Dec 18; 35( 51):7204-7212), Escherichia coli, Salmonella type 5, Listeria, Brucella, Bacillus anthracis, and Legionella were preserved or cultured in this laboratory, see literature (Zhao Y, Wang H, Zhang P, Sun C, Wang X, Wang X, Yang R, Wang C, Zhou L. 2016. Rapid multiplex detection of 10foodborne pathogens with an up-converting phosphortechnology-based 10-channel lateral flow assay. Scientific reports 6:21342; HaoM, Zhang P, Li B, Liu X, Zhao Y, Tan H, Sun C, Wang X, Wang X, Qiu H, Wang D, Diao B, Jing H, Yang R, Kan B, Zhou L. 2017. Development and evaluation of an up -converting phosphor technology-based lateral flow assay for the rapid,simultaneous detection of Vibrio choler...

Embodiment 3

[0085] Example 3. Sensitivity detection (ELISA method) of anti-Cox coxella monoclonal antibody

[0086] Dilute the inactivated Coxiella beinerii whole bacterial antigen to 2x10 9 For copy number / ml, add 100 μl / well to the ELISA plate, each of 6 duplicate wells, coat overnight at 4°C, wash with PBST for 5 min×5 times, and pat the liquid in the wells clean. Add blocking solution at 200 μl / well and incubate at 37°C for 2 hours, wash with PBST for 5 minutes×5 times, and clean the liquid in the well. Take the monoclonal antibody (mAb 10) for doubling dilution from 250 μg / ml, add 100 μl / well, act at 37°C for 1 hour, wash 5 minutes×5 times; add 0.1% PBST to 100 μl / well to dilute the spicy Root peroxidase (HRP)-labeled goat anti-mouse antibody, incubated at 37°C for 1 hour, washed with PBST for 5 minutes×5 times; added TMB substrate chromogenic solution at 100 μl / well, reacted at room temperature for 15 minutes in the dark; added at 50 μl / well Observe the results of the stop solutio...

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Abstract

The invention provides a monoclonal antibody for quantitative detection of Coxiella burnetii I-phase strains. The monoclonal antibody performs detection in combination with an up-converting phosphor technology; and compared with a traditional Coxiella burnetii detection method, the monoclonal antibody has the advantages that detection is accurate, quantitative and free of background interference,the detection result is stable, and detection is convenient and rapid to perform.

Description

technical field [0001] The invention relates to a monoclonal antibody composition for quantitatively detecting phase I strains of Coxiella bezieri. Background technique [0002] Coxiella burnetii is the causative bacterium of Q fever (Qfever), an important zoonotic disease, with Gram-negative staining, obligate intracellular parasitism, small short rod-shaped or small club-shaped . There is a phase transition during the growth process of Coxiella basidioides. Phase I strains are usually virulent strains isolated from Q fever patients or infected animals. After tens or even hundreds of generations of artificial passage in the laboratory, phase I strains Part of the antigen loss of LPS, the formation of attenuated strains, that is, phase II strains. Phase I strains of Coxiella bezii contain phase I and phase II antigens, which can induce animals to produce phase I and phase II antibodies, while II strains mainly contain phase II antigens and can only induce phase II antibodi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569C07K16/12C12N15/13
CPCG01N33/577G01N33/56911C07K16/1203G01N2333/195C07K2317/565Y02A50/30
Inventor 熊小路张平平杨瑞馥焦俊赵勇王津周冬生
Owner ACADEMY OF MILITARY MEDICAL SCI
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