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ELISPOT detection kit for detecting bovine brucellosis

A detection kit, the technology of Brucella bovis, applied in the field of diagnosis of bovine brucellosis, the field of ELISPOT detection kits for detecting bovine brucellosis, can solve the problem of unsatisfactory specificity and the inability to meet the requirements of bovine Brucella Specific detection of bacterial infection, can not meet the early detection of Brucella bovine infection and other problems, to eliminate the interference of false positives, good specificity, strong affinity

Active Publication Date: 2016-07-13
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But above-mentioned detection method still can not satisfy the real needs of the specific detection of this area to the bovine Brucella infection;
[0006] Although ELISA is currently recognized as a relatively high specificity and sensitivity diagnostic method for brucellosis, it is based on humoral immune detection and its specificity is not ideal

Method used

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  • ELISPOT detection kit for detecting bovine brucellosis
  • ELISPOT detection kit for detecting bovine brucellosis
  • ELISPOT detection kit for detecting bovine brucellosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0019] As an embodiment of the present invention, the first bovine interferon-gamma monoclonal antibody is bovine interferon-gamma monoclonal antibody 2G5, and the bovine interferon-gamma monoclonal antibody 2G5 is obtained from a hybridoma with a deposit number of CCTCCNO: C2012107 The cell line 2G5 or its passage cell line is secreted; and the second bovine interferon-gamma monoclonal antibody is bovine interferon-gamma monoclonal antibody 5E11, and the bovine interferon-gamma monoclonal antibody 5E11 has a preservation number of CCTCCNO: C2012108 The hybridoma cell line 5E11 or its subcultured cell line is secreted and produced.

[0020] Hybridoma cell line 2G5 and hybridoma cell line 5E11 are disclosed in patent CN102965343B.

[0021] As an embodiment of the present invention, the supporting medium is a microporous membrane filter plate. More preferably, the supporting medium is a PVDF film-coated microwell culture plate. Wherein, preferably, there is a PVDF membrane on ...

Embodiment 1

[0047] Embodiment 1 The preparation of the bovine brucellosis ELISPOT detection kit based on Br-PPD of the present invention

[0048] The ELISPOT kit assembly steps are as follows:

[0049] 1) Purification of bovine gamma interferon monoclonal antibody 2G5

[0050] The hybridoma cell line secreting bovine gamma interferon monoclonal antibody 2G5 was injected into the peritoneal cavity of mice. After ascites was produced, the ascites was collected, and the collected 2G5 ascites was purified by ProteinG affinity chromatography.

[0051] 2) Preparation of biotin-labeled bovine interferon-γ monoclonal antibody (referred to as Bio-5E11)

[0052] The hybridoma cell line secreting bovine gamma interferon monoclonal antibody 5E11 was injected into the peritoneal cavity of mice. After ascites was produced, the ascites was collected, and the collected 5E11 ascites was purified by ProteinG affinity chromatography.

[0053] Label the purified 5E11 monoclonal antibody with standard bioti...

Embodiment 2

[0064] Embodiment 2 detects bovine peripheral blood sample based on the ELISPOT detection kit of non-specific stimulant PWM

[0065] 1. Experimental materials

[0066] The bovine brucellosis ELISPOT detection kit prepared in Example 1 was used.

[0067] 2. Anticoagulation incubation and detection

[0068] ① Sterilely take 1mL of bovine blood and add it into blood collection tubes containing sodium heparin, and mix it upside down after blood collection to obtain anticoagulant blood;

[0069] ②Add the following reagents to the above-mentioned coated 96-well filter plate: each anticoagulant sample includes 2 test wells, 50 μL of cell culture solution to the negative control well, 50 μL of PWM diluted in the cell culture solution to the positive control well (final Concentration is 5 μg / mL), 50 μL bovine anticoagulant blood was added to each well, and the 96-well filter plate was placed at 37 ° C, 5% CO 2 Cultivate in the incubator for 24-48 hours;

[0070] ③Take out the 96-we...

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Abstract

The invention provides an ELISPOT detection kit for detecting bovine brucellosis.The ELISPOT detection kit comprises a supporting medium, a capture antibody, a detection antibody, a negative control, a positive control and a specificity irritant, wherein the capture antibody is the first bovine gamma interferon monoclonal antibody, the detection antibody is the second marked bovine gamma interferon monoclonal antibody, and the specificity irritant is brucellin Br-PPD.A micropore filter membrane is arranged on a contact face of the supporting medium and the reagent.The capture antibody coats the supporting medium or the capture antibody and the supporting medium are placed respectively.The capture antibody and the detection antibody can be subjected to the antigen antibody combination effect together with bovine gamma interferon.According to the detection kit, the adopted monoclonal antibody excreted by hybrid tumor cell strains has the advantages of being high in active valence, good in specificity and strong in natural antigen affinity.Compared with a brucellosis antibody ELISA detection kit, the ELISPOT can achieve early detection of bovine brucellosis infection, false positive interference brought by a cross reaction is eliminated, and the good specificity is shown.

Description

technical field [0001] The invention relates to a detection kit, in particular to an ELISPOT detection kit for detecting bovine brucellosis and its application in the field of bovine brucellosis diagnosis. Background technique [0002] Bovine brucellosis (hereinafter referred to as "bull brucellosis") is a zoonotic chronic infectious disease caused by Brucella. After infection, livestock will have symptoms such as infertility and abortion, and bulls will have arthritis and orchitis. The ability to breed has declined significantly, seriously threatening the health of the herd, and causing great harm to animal husbandry production and human health. [0003] Brucella is a facultative intracellular parasite that induces a cellular immune response after infection. The cellular immune response can produce a series of cytokines, which play an important role in the body's defense against infection. When the body is infected with Brucella, specific immunogenic proteins can activate...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/569
CPCG01N33/56911G01N33/577G01N33/6866G01N2333/57
Inventor 焦新安陈祥徐正中孟闯刘佳莹潘志明孙林
Owner YANGZHOU UNIV
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