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Preparation and application of porcine epidemic diarrhea virus IgA antibody ELISA kit

A porcine epidemic diarrhea and kit technology, applied in the field of immunoassay, can solve the problems of milk quality inspection, milk difficulty, and inability to directly evaluate the immune effect of piglets

Pending Publication Date: 2020-04-07
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic commercial products are only the PEDV IgA antibody ELISA detection kit produced by South Korea Anjie, but this kit can only detect the milk of sows. The special problem is that it is difficult for sows to collect milk during lactation, and the quality of milk is not good. need inspection
Since it cannot be used to test samples such as blood, it can only indirectly and vaguely evaluate the immune effect of piglets, and cannot directly evaluate the immune effect of individual piglets; nor can it evaluate the clinical PEDV infection situation, and epidemiological monitoring cannot be carried out

Method used

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  • Preparation and application of porcine epidemic diarrhea virus IgA antibody ELISA kit
  • Preparation and application of porcine epidemic diarrhea virus IgA antibody ELISA kit
  • Preparation and application of porcine epidemic diarrhea virus IgA antibody ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation and Identification of Porcine Epidemic Diarrhea Virus Antigen

[0050] 1.1 Selection of strains for antigen

[0051] Select representative strains of two types of porcine epidemic diarrhea virus classic strain and variant strain for culture and propagation, such as: the classic strain is CV777 strain, and the variant strains are HN1301 strain, HN1302 strain, HN1303 strain and their cultures.

[0052] 1.2 Antigen preparation method

[0053] The culture prepared in Example 1.1 was first centrifuged (5000-15000 rpm for 5-30 minutes) and then inactivated. The inactivation method includes: formaldehyde method (abbreviated as M1), and the concentration range of formaldehyde is 0.05%-0.5% (v / v), standing at 25-37°C or shaking at 50-200 rpm, inactivation time 24-48 hours; inactivation of BEI (referred to as M2), BEA first 2 hours of cyclization treatment, virus inactivation Preferably the concentration range is 3mM~20mM, 25~37°C shaking table 50~200 rpm...

Embodiment 2

[0059] The selection of embodiment 2 porcine epidemic diarrhea virus IgA antibody ELISA kit coating antibody

[0060] Monoclonal antibody 8A3A10, PEDV-McAB1, PEDV-McAB2 are respectively used as the antibody for coating, and the kit is prepared according to the method in Example 3.1 with the amount of 100ng / well of the antibody, and 40 parts of positive milk (wherein containing colostrum 20 ), 20 positive serums, 40 negative milks (including 20 colostrums), and 20 negative serums (all were identified by IPMA to confirm whether the IgA antibody was positive), and the coincidence rate of the samples with the IPMA method was calculated and detected simultaneously 1 clinically positive breast milk sample P1 (IPMA titer 1:256), the results are shown in Table 2.

[0061] Table 2 Evaluation results of porcine epidemic diarrhea virus antibody for kit coating

[0062]

[0063] It shows that all three strains of monoclonal antibodies can be used in the preparation of PEDV IgA antibod...

Embodiment 3

[0065] Preparation and detection method of embodiment 3 porcine epidemic diarrhea virus IgA antibody ELISA kit

[0066] 3.1 Kit preparation

[0067] Coated plate: Dilute porcine epidemic diarrhea virus antibody with coating solution, add 100 μl / well, coat, coat at 2-8°C for 16-24 hours or at 37°C for 1-3 hours; wash with washing solution , add blocking solution, block for 16-24 hours at 2-8°C or block for 1-3 hours at 37°C; wash with washing solution, add porcine epidemic diarrhea virus antigen, capture at 37°C for 0.5-2 hours; wash, dry, seal the plate .

[0068] Sample diluent: PBS solution containing 15%-25% fetal bovine serum.

[0069] Enzyme-labeled goat anti-pig IgA antibody: Dilute the enzyme-labeled goat anti-pig IgA antibody 1:8000-1:30000 with sample diluent, and pack in aliquots.

[0070] Washing solution: PBS solution containing 0.5%-1% Tween 20.

[0071] Chromogenic solution: Take 14.7 g of disodium hydrogen phosphate, 9.3 g of citric acid, and 0.3 g of carbam...

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Abstract

The invention provides a porcine epidemic diarrhea virus IgA antibody ELISA detection kit. The kit comprises a supporting medium coated with a porcine epidemic diarrhea virus antibody-antigen compound, the porcine epidemic diarrhea virus antibody-antigen compound being a porcine epidemic diarrhea virus IgG monoclonal antibody-inactivated antigen compound, enzyme labeled anti-swine IgA second antibody, and a detection reagent, the positive control and the negative control used for detecting the reaction of the porcine IgA and the porcine epidemic diarrhea virus antibody-antigen complex antigen-antibody. The kit disclosed by the invention is high in sensitivity, and can be used for accurately detecting a plurality of target samples in milk, serum, saliva, anus swab and excrement samples.

Description

technical field [0001] The invention relates to the preparation and application of a porcine epidemic diarrhea virus IgA antibody ELISA kit, belonging to the field of immunoassays. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a highly contagious, viral intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDVirus, PEDV), characterized by watery diarrhea, vomiting, dehydration, and loss of appetite. and high mortality in suckling piglets. This disease is all susceptible to pigs of various age stages, and the sickness rate of suckling piglet, shelf pig, fattening pig reaches 100%, is the most serious with suckling piglet especially, and case fatality rate reaches 100%. [0003] In practice, serological testing is still a common method for clinical diagnosis and immune evaluation. At present, PEDV antibody detection technology mainly focuses on the detection of PEDV IgG antibody level in the blood, and...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/536G01N33/569
CPCG01N33/535G01N33/536G01N33/56983
Inventor 田克恭王治平
Owner LUOYANG PULIKE WANTAI BIOTECH
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