Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method

Abstract

The invention discloses a kit for detecting pig pseudorabies virus antibodies and a block enzyme-linked immuno sorbent assay (ELISA) method. The kit for detecting pig pseudorabies virus antibodies comprises pig pseudorabies virus monoclonal antibodies which are labelled by horseradish peroxidase, wherein the pig pseudorabies virus monoclonal antibodies are monoclonal antibodies obtained by pig pseudorabies viruses as immunogens and the pig pseudorabies viruses are pseudorabies virus strain Ea. The kit for detecting pig pseudorabies virus antibodies also comprises an enzyme label plate, a sample diluent, negative and positive contrasts, a coloured solution, a washing solution, and a stopping solution. The block ELISA method comprises the following steps of 1, taking out a detection plate pre-coated with virus antigens from the kit for detecting pig pseudorabies virus antibodies, adding diluted blood serum needing to be detected into the detection plate pre-coated with the virus antigens, and simultaneously, setting negative and positive contrast apertures, 2, shaking up the diluted blood serum in the negative and the positive contrast apertures, shaking off a solution in the negative and the positive contrast apertures, and washing the detection plate by the washing solution, and 3, adding the pig pseudorabies virus monoclonal antibodies labelled by horseradish peroxidase into the negative and the positive contrast apertures, washing, adding the colored solution into the negative and the positive contrast apertures to carry out room-temperature coloration in the dark, adding the stopping solution into the negative and the positive contrast apertures, and determining OD630nm values of the negative and the positive contrast apertures by an ELISA apparatus. The block ELISA method has the advantages of good singularity, high sensitivity, short detection time, and high accuracy because of utilization of an S / N ratio method in result determination.

Description

technical field

[0001] The invention relates to animal epidemic detection, in particular to an ELISA kit for detecting porcine pseudorabies virus antibody, and also relates to a blocking ELISA detection method for porcine pseudorabies virus antibody. It is suitable for detecting the antibody level of pseudorabies virus in pig serum. Background technique

[0002] Pseudorabies (PR) is an important infectious disease caused by pseudorabies virus (Pseudorabies Virus, PrV), including domestic animals and various wild animals, with symptoms of fever, itching and encephalomyelitis. The disease has caused huge economic losses to the pig industry, and pigs are the natural host and storage of the disease. Prevention, control and ultimate eradication of this disease is a difficult task that our country is currently facing. In the prevention and control of pseudorabies, it is very important to monitor and detect the level of pseudorabies virus antibody in pig serum. In pigs that have...

Examples

Embodiment 1

[0024] Example 1: Preparation of antigen-coated plates:

[0025] Virus culture and purification: BHK cells were cultured at 37°C with DMEM containing 10% calf serum. After the cells grew into a monolayer, discard the supernatant, inoculate 1 / 50 of the original volume of virus, and add maintenance solution (containing 3% volume ratio of calf serum DMEM), the liquid can just cover the cell layer, after 1 hour of adsorption at 37°C, add the maintenance solution to the original volume of the cells when culturing, and continue culturing in a 37°C incubator until more than 80% of the cells have lesions At that time, the cell bottle was placed in a -20°C refrigerator, and the virus liquid was collected after repeated freezing and thawing 3 times. The collected virus liquid was centrifuged at 5000r / min at 4°C for 30min to remove the precipitate. The supernatant was centrifuged at 27000r / min at 4°C for 1 hour, and the pellet was resuspended in 2ml of pH7.2 0.01mol / L PBS. Prepare 20% ...

Embodiment 2

[0027] Embodiment 2: Preparation and labeling of anti-pseudorabies virus monoclonal antibody:

[0028] Preparation steps of anti-pseudorabies virus monoclonal antibody:

[0029] 1) Preparation of feeder cells: One day before fusion, a 7-week-old female BALB / c mouse was killed by cervical dislocation, sterilized with 75% volume ratio alcohol for 5 minutes, dried and moved into an ultra-clean workbench, fixed in a UV-treated On a sterilized foam board, use sterilized ophthalmic scissors and curved tweezers to cut the skin (without damaging the peritoneum), and blunt dissection to fully expose the peritoneum. Use a 10mL disposable syringe to fill up the HAT-containing selective medium and inject it into the abdominal cavity of the mouse. The right hand fixes the syringe, and the left hand uses tweezers to grasp the alcohol cotton and gently rub the mouse abdomen. Then use the syringe to draw out the medium in the abdominal cavity and inject the sterile In the plate, add the abov...

Embodiment 3

[0046] Embodiment 3: kit assembly:

[0047] The kit contains 2 pieces of 96-well ELISA detection plates, 1 bottle of enzyme-labeled anti-pseudorabies virus monoclonal antibody (20mL / bottle), 2 tubes of negative and positive controls (1.5mL / tube), 1 bottle of sample diluent (50mL / bottle) bottle), 1 bottle of 20-fold concentrated washing solution (30mL / bottle), 1 bottle each of chromogenic solution A, chromogenic solution B, and stop solution (10mL / bottle), and 1 copy of instructions.

[0048] 20 times concentrated washing solution: 160g sodium chloride, 10ml Tween-20 dissolved in 1000ml distilled water.

[0049] Blocking solution: Phosphate buffered saline containing 5mg / mL BSA.

[0050] Sample diluent: DMEM medium containing 25 mg / mL gelatin.

[0051] Stop solution: 0.25% hydrofluoric acid by volume.

[0052] Preparation of positive and negative controls:

[0053] Add penicillin and streptomycin to the porcine pseudorabies virus standard positive serum obtained by screenin...

technical field

[0001] The invention relates to animal epidemic detection, in particular to an ELISA kit for detecting porcine pseudorabies virus antibody, and also relates to a blocking ELISA detection method for porcine pseudorabies virus antibody. It is suitable for detecting the antibody level of pseudorabies virus in pig serum. Background technique

[0002] Pseudorabies (PR) is an important infectious disease caused by pseudorabies virus (Pseudorabies Virus, PrV), including domestic animals and various wild animals, with symptoms of fever, itching and encephalomyelitis. The disease has caused huge economic losses to the pig industry, and pigs are the natural host and storage of the disease. Prevention, control and ultimate eradication of this disease is a difficult task that our country is currently facing. In the prevention and control of pseudorabies, it is very important to monitor and detect the level of pseudorabies virus antibody in pig serum. In pigs that have...

Examples

Embodiment 1

[0024] Example 1: Preparation of antigen-coated plates:

[0025] Virus culture and purification: BHK cells were cultured at 37°C with DMEM containing 10% calf serum. After the cells grew into a monolayer, discard the supernatant, inoculate 1 / 50 of the original volume of virus, and add maintenance solution (containing 3% volume ratio of calf serum DMEM), the liquid can just cover the cell layer, after 1 hour of adsorption at 37°C, add the maintenance solution to the original volume of the cells when culturing, and continue culturing in a 37°C incubator until more than 80% of the cells have lesions At that time, the cell bottle was placed in a -20°C refrigerator, and the virus liquid was collected after repeated freezing and thawing 3 times. The collected virus liquid was centrifuged at 5000r / min at 4°C for 30min to remove the precipitate. The supernatant was centrifuged at 27000r / min at 4°C for 1 hour, and the pellet was resuspended in 2ml of pH7.2 0.01mol / L PBS. Prepare 20% ...

Embodiment 2

[0027] Embodiment 2: Preparation and labeling of anti-pseudorabies virus monoclonal antibody:

[0028] Preparation steps of anti-pseudorabies virus monoclonal antibody:

[0029] 1) Preparation of feeder cells: One day before fusion, a 7-week-old female BALB / c mouse was killed by cervical dislocation, sterilized with 75% volume ratio alcohol for 5 minutes, dried and moved into an ultra-clean workbench, fixed in a UV-treated On a sterilized foam board, use sterilized ophthalmic scissors and curved tweezers to cut the skin (without damaging the peritoneum), and blunt dissection to fully expose the peritoneum. Use a 10mL disposable syringe to fill up the HAT-containing selective medium and inject it into the abdominal cavity of the mouse. The right hand fixes the syringe, and the left hand uses tweezers to grasp the alcohol cotton and gently rub the mouse abdomen. Then use the syringe to draw out the medium in the abdominal cavity and inject the sterile In the plate, add the abov...

Embodiment 3

[0046] Embodiment 3: kit assembly:

[0047] The kit contains 2 pieces of 96-well ELISA detection plates, 1 bottle of enzyme-labeled anti-pseudorabies virus monoclonal antibody (20mL / bottle), 2 tubes of negative and positive controls (1.5mL / tube), 1 bottle of sample diluent (50mL / bottle) bottle), 1 bottle of 20-fold concentrated washing solution (30mL / bottle), 1 bottle each of chromogenic solution A, chromogenic solution B, and stop solution (10mL / bottle), and 1 copy of instructions.

[0048] 20 times concentrated washing solution: 160g sodium chloride, 10ml Tween-20 dissolved in 1000ml distilled water.

[0049] Blocking solution: Phosphate buffered saline containing 5mg / mL BSA.

[0050] Sample diluent: DMEM medium containing 25 mg / mL gelatin.

[0051] Stop solution: 0.25% hydrofluoric acid by volume.

[0052] Preparation of positive and negative controls:

[0053] Add penicillin and streptomycin to the porcine pseudorabies virus standard positive serum obtained by screenin...

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