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A kind of elispot detection kit for detecting brucellosis and application thereof

A detection kit, a technology for brucellosis, applied in measurement devices, instruments, scientific instruments, etc., can solve the problem of inability to detect brucella markers, detect brucella infection, and interfere with the accuracy of results. To achieve the effect of truly reflecting the immune level in the body, objective test results, and improving specificity and sensitivity

Active Publication Date: 2021-06-01
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the active ingredient of the antigen used in the traditional test of Brucella is mainly lipopolysaccharide (LPS), it has cross-reaction with Gram-negative bacteria, especially Yersinia type O:9, which interferes with the accuracy of the results
In addition, in the early stage of infection or when the antibody titer is low, especially within 12 to 16 days of infection with Brucella, it is difficult to detect Brucella infection by detecting antibodies
In addition, Brucella is an intracellular parasite, and in the late stage of Brucella infection, the serum markers of Brucella often cannot be detected
[0005] All in all, there is no rapid, accurate and clinically effective detection method for the pathogenic detection of Brucella

Method used

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  • A kind of elispot detection kit for detecting brucellosis and application thereof
  • A kind of elispot detection kit for detecting brucellosis and application thereof
  • A kind of elispot detection kit for detecting brucellosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046]Example 1 Preparation of ELISPOT Detection Test Box

[0047]Specifically, the ELISPOT detection kit assembly steps are as follows: 96-well filter plate, IFN-γ capture antibody, biotin-labeled IFN-γ detect antibody, specific stimulant, specific inhibitor, negative control (10% 1640 Culture) and positive control (plant lectin PHA) package assemble into a kit.

[0048]Further, the ELISPOT detection kit is based on the need to assemble it: enzyme-like affinity (affinity-alkaline phosphatase combination), NBT / BCIP color, concentrated PBS buffer, closed liquid, cell culture One or more of the liquid, phosphate buffer or phosphate spoophes.

Embodiment 2

[0049]Example 2 Using the ELISPOT detection kit of Example 1 to detect human peripheral blood samples

[0050]The detection method of this embodiment includes the following steps:

[0051]2.1 Dilute IFN-γ capture antibodies to 15 μg / ml using a sterile pH 7.4 (0.22 μm filter filtration) (0.22 μm filter filtration).

[0052]2.2 Remove the Millipore 96-Well PVDF Elispot Plate (Cat: MSIPS4510, Millipore, 96-well filter board), 15 μl of 35% ethanol for 2 min per well.

[0053]2.3 Sterile water (0.22 μm filter filtration) Clean filter plate 5 times, each time 200 μL per hole, after the liquid is abandoned, the liquid is placed on the sterile paper.

[0054]2.4 Each hole is added to the drug-catching antibody solution, and incubate overnight at 4 to 8 ° C.

[0055]2.5 Removing the antibody package in the filter plate, sterile PBS washing the filter plate 5 times, 200 μL per well, after the liquid is abandoned, and then buckled with the sterile paper.

[0056]2.6 Closed Filter Board: 200 μL of 10% 1640 cultur...

Embodiment 3

[0066]Example 3 Using the ELISPOT detection kit of Example 1 to detect human outer peripheral blood samples

[0067]The detection method of this embodiment includes the following steps:

[0068]2.1 Dilute IFN-γ capture antibodies to 15 μg / ml using a sterile pH 7.4 (0.22 μm filter filtration) (0.22 μm filter filtration).

[0069]2.2 Remove the Millipore 96-Well PVDF Elispot Plate (Cat: MSIPS4510, Millipore, 96-well filter board), 15 μl of 35% ethanol for 2 min per well.

[0070]2.3 Sterile water (0.22 μm filter filtration) Clean filter plate 5 times, each time 200 μL per hole, after the liquid is abandoned, the liquid is placed on the sterile paper.

[0071]2.4 Each hole is added to the drug-catching antibody solution, and incubate overnight at 4 to 8 ° C.

[0072]2.5 Removing the antibody package in the filter plate, sterile PBS washing the filter plate 5 times, 200 μL per well, after the liquid is abandoned, and then buckled with the sterile paper.

[0073]2.6 Closed Filter Board: 200 μL of 10% 1640 ...

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Abstract

The invention discloses an ELISPOT detection kit for detecting brucellosis and an application thereof. An ELISPOT detection kit for detecting brucellosis, said ELISPOT detection kit comprising: support medium, capture antibody, detection antibody, negative control and positive control and specific stimulator, said ELISPOT detection kit Specific inhibitors are also included to detect the presence or absence of Brucella infection bidirectionally by specific stimulators and specific inhibitors. The ELISPOT detection kit of the present invention determines whether there is Brucella infection through positive and negative experiments, and the detection result is more objective and accurate, and can truly reflect the immune level in the body, so as to solve the accuracy rate existing in the current detection of Brucella infection Low, long cycle and other issues.

Description

Technical field[0001]The present invention relates to the field of biological detection, and more particularly to an ELISPOT detection kit for detecting brucellosis and its applications.Background technique[0002]Brucellosis (Brucellosis, abbreviated disease) is a serious threat to human and multi-animal life-healthy infectious diseases. Sick sheep, cattle and other fatstocks are the main infectious sources of neutral disease, and Bruquys can spread by damaged skin mucosa, digestive tract and respiratory tract. Acute cases are greatly manifested by fever, fatigue, sweating, muscles, joint pain, and liver, spleen, lymphoid soda. The chronic period is mostly manifested as joint damage. Cloth disease is a B-like infectious disease stipulated in my country's "Infectious Disease Prevention and Control". According to my country's CDC 2019 statistics, people have increased to 37,947 people, and the top 10 cases of two types of infectious diseases were 10. The epidemic trend of the diamonds ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56911G01N33/577
Inventor 王文敬李金峰任慧黎诚耀
Owner SOUTHERN MEDICAL UNIVERSITY
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