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Preparation method for pathogenic yersinia enterocolitica test strips

A technology of enterocolitis and Yersinia, which is applied in the field of biomedicine, can solve the problems of poor specificity, tediousness, time-consuming detection methods, etc., and achieve the effect of rapid typing detection and sensitive typing detection

Inactive Publication Date: 2015-12-30
HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above situation, in order to solve the defects of the prior art, the object of the present invention is to provide a method for preparing a test strip for pathogenic Yersinia enterocolitica, which can effectively solve the problems of time-consuming, cumbersome and poor specificity of the existing detection methods. question

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  • Preparation method for pathogenic yersinia enterocolitica test strips

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Embodiment 1

[0023] In concrete implementation, the present invention is realized by the following steps:

[0024] (1) Preparation of purified monoclonal antibodies:

[0025] Take 1 part of mouse ascites and 3 parts of acetate buffer with pH 4.4, add 100 μl of n-octanoic acid dropwise for each ml of ascites, mix well, place at room temperature at 18-25°C for 30 minutes, and centrifuge at 15,000 rpm at 4°C For 20 minutes, discard the precipitate, adjust the pH value of the supernatant to 7.2 with NaOH, then add ammonium sulfate, the amount of ammonium sulfate added is 0.277g per ml of supernatant, stir at room temperature at 18-25°C for 30 minutes, 15000 rpm Separate and centrifuge at 4°C for 20 minutes, discard the supernatant, and dissolve the precipitate with a pH7.2 PBS solution that is 1 / 2 times the volume of the mouse ascites, and then put it into a pH7.2 PBS buffer as a dialysate Medium, 48 hours of dialysis at 4°C, changing the dialysate every 9-10 hours to produce purified monoclo...

Embodiment 2

[0035] In concrete implementation, the present invention is realized by the following steps:

[0036] (1) Preparation of purified monoclonal antibodies:

[0037] Take 1 part of mouse ascites and 3 parts of acetate buffer with pH 4.4, add 100 μl of n-octanoic acid dropwise for each ml of ascites, mix well, place at room temperature at 18-25°C for 25 minutes, and centrifuge at 15,000 rpm at 4°C After 15 minutes, discard the precipitate, adjust the pH value of the supernatant to 7.2 with NaOH, then add ammonium sulfate, the amount of ammonium sulfate added is 0.277g per ml of supernatant, stir at room temperature at 18-25°C for 25 minutes, 15000 rpm Separate and centrifuge at 4°C for 15 minutes, discard the supernatant, and dissolve the precipitate with 1 / 2 volume of PBS solution with pH 7.2 as the ascites of the mouse, and then put it into the PBS buffer with pH 7.2 as the dialysate , dialysis at 4°C for 45 hours, changing the dialysate every 9-10 hours to make purified monoclo...

Embodiment 3

[0047] In concrete implementation, the present invention is realized by the following steps:

[0048] (1) Preparation of purified monoclonal antibodies:

[0049] Take 1 part of mouse ascites and 3 parts of acetate buffer with pH 4.4, add 100 μl of n-octanoic acid dropwise for each ml of ascites, mix well, place at room temperature at 18-25°C for 35 minutes, and centrifuge at 15,000 rpm at 4°C After 25 minutes, discard the precipitate, adjust the pH value of the supernatant to 7.2 with NaOH, then add ammonium sulfate, the amount of ammonium sulfate added is 0.277g per ml of supernatant, stir at room temperature at 18-25°C for 35 minutes, 15000 rpm Separate and centrifuge at 4°C for 25 minutes, discard the supernatant, and dissolve the precipitate with 1 / 2 volume of PBS solution with pH 7.2 as the ascites of the mouse, and then put it into the PBS buffer with pH 7.2 as the dialysate , dialysis at 4°C for 50 hours, changing the dialysate every 9-10 hours to make purified monoclo...

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Abstract

The invention relates to a preparation method for pathogenic yersinia enterocolitica test strips, effectively solving the problems of high time consumption, complexity and poor specificity of existing detection methods. The preparation method includes adding n-caprylic acid to mouse ascites and acetate buffer solution, mixing evenly and centrifuging the mixture, regulating the pH (potential of hydrogen) value of supernatant, adding ammonium sulfate, performing centrifugation, dissolving sediments by PBS solution, and performing dialysis to obtain monoclonal antibodies; activating red, blue and yellow latex particles with carboxyl groups by carbodiimide respectively to obtain suspended activated latex particles; dialyzing to-be-marked antibodies, adding the monoclonal antibodies, dropping the mixture into the activated latex particles, rinsing with NaH2PO4 to enable sediments to be suspended in bovine serum albumin buffer solution, adsorbing markers by glass fibers, drying the markers, and curing anti-mouse IgG (immunoglobulin G) antibodies on fiber films to form quality control areas. The preparation method has the advantages that the advantages of immunoreaction specificity, chromatography rapidity and intuition of the colored latex particles serving as tracers are combined, and accordingly, simplicity, convenience, specificity and sensitiveness are achieved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing a test strip for pathogenic Yersinia enterocolitica. Background technique [0002] Yersinia enterocolitica is a pathogenic bacterium that causes severe enterocolitis in humans. The bacterium widely exists in nature such as lakes, soil and vegetation, as well as poultry, livestock and other animals. Pigs, cattle, dogs, cats, chickens, etc. are storage hosts. Humans are mainly infected through contaminated food. The clinical manifestations are gastroenteritis type, appendicitis and mesenteric lymphadenitis type, erythema nodosum and arthritis type, and sepsis type . Known Yersinia enterocolitica has 54 serotypes, and the serotypes O:3, O:9, and O:8 are closely related to humans. In China, serotypes O: 3 and O: 9 are the main pathogenic strains to humans, and serotype O: 8 is the main pathogenic strain in the United States, Canada and other countries. Due to the i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56916G01N33/577G01N2333/24
Inventor 杨书豪李珊珊周伏忠王玉金宁萌杜迅刁文涛刘丽
Owner HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY
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