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Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae

A technology of monoclonal antibody and pleuropneumonia, which is applied in the direction of introducing foreign genetic material, measuring devices, anti-bacterial immunoglobulin, etc. through the use of carriers, which can solve the problems of accurate diagnosis of missed detection, poor cross-reaction, difficult coverage of serotypes, etc. , to achieve good repeatability, high antigen capture performance, and high sensitivity

Inactive Publication Date: 2011-01-19
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The sensitivity of the above-mentioned detection technology is very high, and it can be used for the specific diagnosis of the serotype of the disease. However, due to the large number of serotypes of the pathogen, and there is no good cross-reaction among the serotypes, this will inevitably lead to all APP pathogenic bacteria. The serotype is difficult to cover, and there are problems of missed detection and accurate diagnosis

Method used

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  • Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae
  • Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae
  • Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae

Examples

Experimental program
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Effect test

Embodiment 1

[0029] 1. Preparation of APP standard strain template DNA and negative control template for all serotypes

[0030]APP serotypes 1-12 standard strains, APP serotypes 1, 3, 5 and 7 clinical isolates and Haemophilus parasuis types 1-15, Salmonella choleraesuis, Proteus mirabilis, Bordetella bronchiseptica Bacteria, Erysipelas suis, Actinobacillus suis, and Pasteurella multocida from pigs were provided by the applicant's research laboratory, and the genomic DNA extraction of the above-mentioned bacteria was carried out according to the previously reported methods. Specifically: take 1 mL of the overnight bacterial culture and centrifuge at room temperature 8,000 r / min for 5 min, suspend the pellet in 1 mL PBS, add 6 μL of 50 mg / mL lysozyme, act at 37°C for 2 hours, add 50 μL proteinase K, and act at 50°C for 3 hours. h or overnight at 37°C. Add an equal volume of phenol: chloroform: isoamyl alcohol for extraction, precipitate with 0.6 times the volume of isopropanol for more than...

Embodiment 2

[0051] ELISA detection kit includes: ELISA plate coated with APP rApxⅣN protein monoclonal antibody, biotin-labeled monoclonal antibody (Biotin-McAb) for detection, washing solution, chromogenic solution, stop solution, positive control, negative control.

[0052] The method of use of the above kit is:

[0053] Ten samples of serum and diseased lung tissue samples from suspected APP-infected pigs from 3 pig farms in Jiangsu were used to isolate and identify bacteria in the diseased lung tissue, and the serum of pigs with different disease courses was detected by the double-antibody sandwich ELISA method established above.

[0054] Suspicious colonies were isolated from 6 out of 10 lung tissue samples (samples 2, 5, 6, 8, 9 and 10) of suspicious APP lesions after cultured on sheep blood agar plate and Gram staining. The results of biochemical identification of suspicious colonies were all positive for CAMP, positive for urease, and negative for mannitol fermentation, and were f...

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Abstract

The invention relates to the research field of biological diagnostic reagents, in particular to an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting APP (Actinobacillus Pleuropneumoniae). The kit comprises a washing solution, a colorimetric solution, a stopping solution, a positive control specimen, a negative control specimen and an ELISA plate coating a first antibody as well as a second antibody for detection, wherein the first antibody is a monoclonal protein antibody of the APP rApxIVN, and the second antibody is a biotin-labeled monoclonal protein antibody of the APP rApxIVN. The invention can be applied to detecting APP-infected pig serum and has favorable importance and sensitivity.

Description

technical field [0001] The invention relates to the field of development of biological diagnostic reagents, in particular to an ELISA kit for detecting early infection of porcine infectious pleuropneumonia and a use method thereof. Background technique [0002] Porcine infectious pleuropneumonia is a highly contagious disease of pigs caused by Actinobacillus pleuropneumoniae (APP). There are 15 pathogenic serotypes and 2 biotypes of APP reported so far, of which serotype 1 Types ~12 and 15 are biological type I, and serum types 13 to 14 are biological type II. The pathogenicity of biotype Ⅰ to pigs was stronger than that of biotype Ⅱ. Except for serotype 15, serotypes 1-12 of APP could cause severe disease and death in infected pigs. [0003] Since Pattison first reported porcine infectious pleuropneumonia in 1957, the disease has been widely prevalent in many countries around the world, and has become one of the internationally recognized important infectious diseases (fiv...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569C12N15/31C12N15/63C07K14/195C07K1/22C07K16/12
Inventor 朱国强张志妮王建业姚丰华朱军舒燕朱晓芳
Owner YANGZHOU UNIV
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