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ELIS pot detection kit for detecting bovine tuberculosis

A detection kit and antibody detection technology, applied in measurement devices, disease diagnosis, instruments, etc., can solve the specific effects of tuberculin intradermal allergy, complex components, inability to distinguish tuberculosis-positive infection, immune individuals and environmental classification. Mycobacterial infections, etc.

Active Publication Date: 2018-12-21
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of tuberculin pure protein derivatives (PPD), many proteins contained in pathogenic mycobacteria and environmental non-pathogenic mycobacteria can not be distinguished from tuberculosis-positive infection, immune individuals and environmental mycobacteria infection and so on, so that the specificity of tuberculin intradermal allergy is affected to a certain extent

Method used

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  • ELIS pot detection kit for detecting bovine tuberculosis
  • ELIS pot detection kit for detecting bovine tuberculosis
  • ELIS pot detection kit for detecting bovine tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 The acquisition of hybridoma cell lines and the preparation of monoclonal antibodies

[0119] Obtaining the hybridoma cell line with the preservation number CCTCC NO: C2017283 and the hybridoma cell line with the preservation number CCTCC NO: C2017282.

[0120] 1. Animal immunization

[0121] The specific immunization procedure is as follows: for the first immunization, 100 μg recombinant bovine interferon gamma purified protein fully emulsified with Freund’s complete adjuvant was injected subcutaneously at multiple points in the abdomen, and 100 μg recombinant bovine interferon gamma protein fully emulsified with Freund’s incomplete adjuvant was injected subcutaneously at multiple points 2 weeks later. The purified protein was used for the second immunization, and 100 μg of purified protein without adjuvant was injected intraperitoneally after two weeks for the third immunization. After 7 days, orbital blood was collected to determine the serum antibody titer...

Embodiment 2

[0228] Example 2 Assembly of bovine tuberculosis gamma-interferon ELISpot detection kit

[0229] The assembly steps of bovine tuberculosis gamma-interferon ELISpot kit are as follows:

[0230] (1) Preparation of biotin-labeled bovine interferon-γ monoclonal antibody (referred to as Bio-6F8):

[0231] The purified monoclonal antibody 6F8 was labeled with a standard biotin labeling method to obtain biotin-labeled bovine interferon-γ monoclonal antibody Bio-6F8. Dissolve 2-10 mg of monoclonal antibody 6F8 protein in 1 mL of phosphate buffer, and calculate the dissolved millimoles; equilibrate biotin to room temperature, add 2 mg of Sulfo-NHS-Biotin to 100 μL of ultrapure water, and add a certain concentration of Biotin; 30 minutes at room temperature, or 2 hours on ice; pre-wash the purification column with 30mL PBS, load the sample, add the same buffer as the amount to be collected, collect 0.5mL or 1mL in a separate tube, and measure the absorption at 280nm Value determinatio...

Embodiment 3

[0256] Example 3 Detection of Bovine Peripheral Blood Samples Based on ELISpot Detection Kit of Non-specific Stimulator PWM

[0257] 1. Experimental materials

[0258] The bovine tuberculosis γ-interferon ELISpot detection kit prepared in Example 2 was used.

[0259] 2. PBMC isolation, incubation and detection

[0260] ① Sterilely take 5mL of bovine blood and add it into the blood collection tube containing sodium heparin, and mix it upside down after blood collection to obtain anticoagulant blood;

[0261] ②Take a lymphocyte separation tube and equilibrate to room temperature, centrifuge at 800g for 3min, make the separation liquid fall under the sieve plate, open the tube cap, carefully superimpose the blood sample on the sieve plate with a dropper, centrifuge at 18-22°C at 800g 20min. After centrifugation, the liquid in the tube is divided into four layers, from top to bottom: yellow clear plasma layer, cloudy cell layer, colorless separation liquid layer, red blood cell...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an ELIS pot detection kit for detecting bovine tuberculosis and an application thereof in the field of bovine tuberculosis diagnosis. The detection kit comprises a support medium, a capture antibody and a detection antibody; the capture antibody can be produced by a hybridoma cell strain with accession number of CCTCC NO: C2017283 or a passage cell strain thereof; and the detection antibody is a gamma interferon monoclonal antibody different from the capture antibody. According to the ELIS pot detection kit for detecting bovine tuberculosis, compared with the bovine tuberculosis BOVIGAM ELISA detection kit and the bovine tuberculosis antibody detection reagent, more sensitive and high-specificity detection of mycobacterium tuberculosis infection can be realized, the false positive interference brought by the cross reaction is eliminated, and good prospects in application are shown.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an ELISpot detection kit for detecting bovine tuberculosis and its application in the field of bovine tuberculosis diagnosis. Background technique [0002] Bovine tuberculosis is a zoonotic chronic infectious disease caused by Mycobacterium bovis. It is characterized by tuberculous nodular granuloma and caseous, calcified necrotic lesions in tissues and organs, and mostly affects the lungs, breasts, and intestines. and lymph nodes etc. Seriously threaten the health of cattle, and cause great harm to animal husbandry production and human health. [0003] Mycobacterium tuberculosis is a facultative intracellular parasite, which can cause cellular immune response after infecting the body, and then produce a series of cytokines, which play an important role in the body's defense against infection. When the body is infected with Mycobacterium tuberculosis, specific immunogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/5695G01N33/577G01N2333/35G01N2800/26
Inventor 焦新安陈祥李昕徐正中刘佳莹孟闯孙林
Owner YANGZHOU UNIV
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