Establishing method and application of pasteurella multocida indirect haemagglutination assay

A Pasteurella and hemagglutination test technology, which is applied in biological testing, material testing products, measuring devices, etc., can solve the problems of increasing vaccine production costs, time-consuming and labor-intensive, etc., so as to save the link of strong poisoning and attacking poisons, and the operation is simple. , the effect of low cost

Active Publication Date: 2014-01-15
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two methods all have problems such as time-consuming and labor-intensive, increased vaccine production costs, and the risk of strong poison and loose poison.

Method used

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  • Establishing method and application of pasteurella multocida indirect haemagglutination assay
  • Establishing method and application of pasteurella multocida indirect haemagglutination assay
  • Establishing method and application of pasteurella multocida indirect haemagglutination assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Establishment of primary Pasteurella multocida indirect hemagglutination test

[0033] 1. Material

[0034] (1) Medium: Martin agar, modified Martin broth

[0035] (2) Strains: C51-2 (Pasteurella multocida, strain deposit number CVCC499), BS039 (Bronchibacterium bronchiseptica, strain deposit number HNAU0220), CMCC44149 (Escherichia coli, strain deposit number CVCC20725) , All purchased from the China Veterinary Drug Inspection Institute.

[0036] (3) Experimental animals: 1.5-2.0kg healthy and susceptible rabbits (purchased from Henan Kangda Experimental Animal Co., Ltd.).

[0037] (4) Reagent

[0038] Sodium dihydrogen phosphate (Tianjin Kaitong Chemical Reagent Co., Ltd., analytical grade, 20110308); potassium dihydrogen phosphate (Chengdu Kelon Chemical Reagent Factory, analytical grade, 20100125); disodium hydrogen phosphate (Chengdu Kelon Chemical Reagent Factory, analytical grade, 20091031); glutaraldehyde (batch number 060318, Green bird imported subpackage, S...

Embodiment 2

[0063] Example 2: Determination of Capsular Polysaccharide in Pasteurella multocida Capsular Antigen

[0064] 1. Material

[0065] Anhydrous glucose (sigma); phenol (imported sub-package); concentrated sulfuric acid (Luoyang Reagent Chemical Factory, 091102); 100℃ water bath, 50ml volumetric flask, 25ml volumetric flask.

[0066] 2. Method

[0067] (2) Drawing of standard curve

[0068] ① Preparation of reference solution

[0069] Take an appropriate amount of anhydrous glucose reference substance dried to constant weight at 105°C, accurately weigh it, add water to dissolve it to make a 0.5mg / ml solution, and shake it well.

[0070] ② Standard curve drawing

[0071] Precisely measure 1, 2, 3, 5, and 10ml of the above reference solution, put them in a 50ml measuring flask, and dilute each to the mark with water, and shake well. Precisely measure 2ml of the above solution and place it in a 25ml measuring flask, precisely add 1ml of 5% phenol solution, 5ml of concentrated sulfuric acid, shak...

Embodiment 3

[0082] Example 3: The sensitivity and reproducibility of the Pasteurella multocida indirect hemagglutination test

[0083] The three batches of capsular antigens R1-Ag, R2-Ag, and R3-Ag were diluted 2, 4, 8, 10, 16, 20, and 25 times, respectively, and then sensitized the aldehyde-treated sheep red blood cells to perform various self-made serums. Indirect blood coagulation test. The results showed that when the polysaccharide content in the capsular antigen was 100-200 μg / ml, the indirect hemagglutination titer of Pm serum was the highest (see Table 4 for details), and the Bb serum, E.coli serum and negative serum were all negative, indicating that the The method has good specificity and reproducibility.

[0084] Table 4: Sensitivity and repeatability test results of indirect hemagglutination test method

[0085]

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Abstract

The invention relates to an establishing method for pasteurella multocida indirect haemagglutination assay and application of the pasteurella multocida indirect haemagglutination assay in pasteurella multocida vaccine potency testing. According to the method, the stable pasteurella multocida indirect haemagglutination assay with good sensitivity and repeatability is established on the basis of polysaccharide content in the capsular antigen of pasteurella multocida through screening the optimal dilution of antigens in indirect hemagglutination test; the assay is used to detect the indirect hemagglutination titer of the serum of vaccinated animals and explore a parallel relationship between the indirect hemagglutination titer of the serum and a virulent virus attack protection rate after immunization and is applied to potency testing of pasteurella multocida vaccines.

Description

Technical field [0001] The invention belongs to the technical field of veterinary biological product inspection, and relates to a method for establishing a Pasteurella multocida indirect hemagglutination test method and its application in the efficacy test of a Pasteurella multocida vaccine. Background technique [0002] Pasteurella multocida (Pm) is a kind of zoonotic pathogen that can cause a variety of livestock and poultry, pets, wild animals and human infections. Studies have reported that the pathogen is widespread all over the world, has no host specificity, and can spread horizontally and vertically through the respiratory tract through mutual contact; Davies et al. reported in 2004 that the bacteria can even spread between different hosts. In my country, the main pathogen causing Pasteurellosis in livestock and poultry is the capsular serotype A Pm, followed by the capsular serotype D Pm. The main pathogen causing the bovine pasteurellosis is the capsular serotype B Pm, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/5091G01N33/86G01N2800/52
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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