ELISPOT detection kit for detecting brucellosis and application of ELISPOT detection kit

Abstract

The invention discloses an ELISPOT detection kit for detecting brucellosis and application of the ELISPOT detection kit. The ELISPOT detection kit comprises a support medium, a capture antibody, a detection antibody, a negative control, a positive control, a specific stimulant, and a specific inhibitor; whether brucella infection exists or not can be tested and determined through two-way detectionof the specific stimulant and the specific inhibitor. According to the ELISPOT detection kit, whether brucella infection exists or not is determined through positive and negative experiments, a detection result is objective and accurate, and an in-vivo immune level can be truly reflected; problem that existing brucella infection detection is low in accuracy rate and long in period.

Description

technical field

[0001] The invention relates to the field of biological detection, in particular to an ELISPOT detection kit for detecting brucellosis and an application thereof. Background technique

[0002] Brucellosis (brucellosis for short) is a zoonotic infectious disease that seriously threatens the life and health of humans and various animals. Sick sheep, cattle and other diseased animals are the main source of infection for brucellosis. Brucella can be transmitted through damaged skin and mucous membranes, digestive tract and respiratory tract. In the acute stage, the main manifestations are fever, fatigue, sweating, muscle and joint pain, and enlargement of liver, spleen and lymph nodes. Most cases in the chronic phase show joint damage. Brucellosis is a Class B infectious disease stipulated in my country's "Law on the Prevention and Control of Infectious Diseases". According to the 2019 statistical report of my country's CDC, the number of people with human bru...

Examples

Embodiment 1

[0046] The preparation of embodiment 1 ELISPOT detection kit

[0047] Specifically, the assembly steps of the ELISPOT detection kit of the present invention are as follows: 96-well filter plate, IFN-γ capture antibody, biotin-labeled IFN-γ detection antibody, specific stimulator, specific inhibitor, negative control (10% 1640 culture medium) and the positive control (phytohemagglutinin PHA) were packaged and assembled into kits respectively.

[0048] Further, the ELISPOT detection kit is assembled as required: enzyme-labeled avidin (avidin-alkaline phosphatase conjugate), NBT / BCIP chromogenic solution, concentrated PBS buffer, blocking solution, cell culture One or more of buffered saline, phosphate buffered saline or phosphate Tween buffered solution.

Embodiment 2

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

[0050] The detection method of the present embodiment comprises the following steps:

[0051] 2.1 Dilute the IFN-γ capture antibody to 15 μg / ml with sterile pH 7.4 calcium and magnesium ion-free 1×PBS (ie DPBS) (filtered with a 0.22 μm filter).

[0052] 2.2 Take out the Millipore 96-well PVDF ELISPOT plate (cat: MSIPS4510, Millipore, 96-well filter plate), and add 15 μl of 35% ethanol to each well for 2 minutes.

[0053] 2.3 Wash the filter plate 5 times with sterile water (filtered through a 0.22 μm filter), add 200 μl to each well each time, discard the liquid in the last pass, and gently dry it on sterile paper.

[0054] 2.4 Add 100 μl of the capture antibody solution diluted in step 2.1 to each well, and incubate overnight at 4-8°C.

[0055] 2.5 Remove the antibody coating solution in the filter plate, wash the filter plate 5 times with sterile PBS, 200 μl per w...

Embodiment 3

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

[0067] The detection method of the present embodiment comprises the following steps:

[0068] 2.1 Dilute the IFN-γ capture antibody to 15 μg / ml with sterile pH 7.4 calcium and magnesium ion-free 1×PBS (ie DPBS) (filtered with a 0.22 μm filter).

[0069] 2.2 Take out the Millipore 96-well PVDF ELISPOT plate (cat: MSIPS4510, Millipore, 96-well filter plate), and add 15 μl of 35% ethanol to each well for 2 minutes.

[0070] 2.3 Wash the filter plate 5 times with sterile water (filtered through a 0.22 μm filter), add 200 μl to each well each time, discard the liquid in the last pass, and gently dry it on sterile paper.

[0071] 2.4 Add 100 μl of the capture antibody solution diluted in step 2.1 to each well, and incubate overnight at 4-8°C.

[0072] 2.5 Remove the antibody coating solution in the filter plate, wash the filter plate 5 times with sterile PBS, 200 μl per w...

technical field

[0001] The invention relates to the field of biological detection, in particular to an ELISPOT detection kit for detecting brucellosis and an application thereof. Background technique

[0002] Brucellosis (brucellosis for short) is a zoonotic infectious disease that seriously threatens the life and health of humans and various animals. Sick sheep, cattle and other diseased animals are the main source of infection for brucellosis. Brucella can be transmitted through damaged skin and mucous membranes, digestive tract and respiratory tract. In the acute stage, the main manifestations are fever, fatigue, sweating, muscle and joint pain, and enlargement of liver, spleen and lymph nodes. Most cases in the chronic phase show joint damage. Brucellosis is a Class B infectious disease stipulated in my country's "Law on the Prevention and Control of Infectious Diseases". According to the 2019 statistical report of my country's CDC, the number of people with human bru...

Examples

Embodiment 1

[0046] The preparation of embodiment 1 ELISPOT detection kit

[0047] Specifically, the assembly steps of the ELISPOT detection kit of the present invention are as follows: 96-well filter plate, IFN-γ capture antibody, biotin-labeled IFN-γ detection antibody, specific stimulator, specific inhibitor, negative control (10% 1640 culture medium) and the positive control (phytohemagglutinin PHA) were packaged and assembled into kits respectively.

[0048] Further, the ELISPOT detection kit is assembled as required: enzyme-labeled avidin (avidin-alkaline phosphatase conjugate), NBT / BCIP chromogenic solution, concentrated PBS buffer, blocking solution, cell culture One or more of buffered saline, phosphate buffered saline or phosphate Tween buffered solution.

Embodiment 2

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

[0050] The detection method of the present embodiment comprises the following steps:

[0051] 2.1 Dilute the IFN-γ capture antibody to 15 μg / ml with sterile pH 7.4 calcium and magnesium ion-free 1×PBS (ie DPBS) (filtered with a 0.22 μm filter).

[0052] 2.2 Take out the Millipore 96-well PVDF ELISPOT plate (cat: MSIPS4510, Millipore, 96-well filter plate), and add 15 μl of 35% ethanol to each well for 2 minutes.

[0053] 2.3 Wash the filter plate 5 times with sterile water (filtered through a 0.22 μm filter), add 200 μl to each well each time, discard the liquid in the last pass, and gently dry it on sterile paper.

[0054] 2.4 Add 100 μl of the capture antibody solution diluted in step 2.1 to each well, and incubate overnight at 4-8°C.

[0055] 2.5 Remove the antibody coating solution in the filter plate, wash the filter plate 5 times with sterile PBS, 200 μl per w...

Embodiment 3

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

[0067] The detection method of the present embodiment comprises the following steps:

[0068] 2.1 Dilute the IFN-γ capture antibody to 15 μg / ml with sterile pH 7.4 calcium and magnesium ion-free 1×PBS (ie DPBS) (filtered with a 0.22 μm filter).

[0069] 2.2 Take out the Millipore 96-well PVDF ELISPOT plate (cat: MSIPS4510, Millipore, 96-well filter plate), and add 15 μl of 35% ethanol to each well for 2 minutes.

[0070] 2.3 Wash the filter plate 5 times with sterile water (filtered through a 0.22 μm filter), add 200 μl to each well each time, discard the liquid in the last pass, and gently dry it on sterile paper.

[0071] 2.4 Add 100 μl of the capture antibody solution diluted in step 2.1 to each well, and incubate overnight at 4-8°C.

[0072] 2.5 Remove the antibody coating solution in the filter plate, wash the filter plate 5 times with sterile PBS, 200 μl per w...

Images