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Anti-H7N9 subtype avian influenza virus monoclonal antibody epitope as well as screening method and application thereof

A monoclonal antibody, avian influenza virus technology, applied in the field of immune detection, can solve the problems of affecting binding, hindering the binding of the latter antibody to the antigen, unable to locate the antigen surface, etc., achieving the effect of simple method, rapid identification, and short screening period

Active Publication Date: 2017-12-08
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the result judges that there is competition between monoclonal antibodies, there may be the following factors: (1) the two antibodies recognize the same epitope; (2) the epitopes recognized by the two antibodies are close, and the former antibody binds to the antigen. , which hinders the binding of the latter antibody to the antigen; (3) Although there is a certain distance between the epitopes recognized by the two antibodies, their spatial conformation positions are similar. binding of an antibody
Therefore, when the competitive inhibition ELISA method is used to analyze the epitope recognized by the monoclonal antibody, the positive result can only indicate the above possibilities, and it cannot accurately locate the antigen table targeted by the monoclonal antibody to a specific antigen table. bit

Method used

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  • Anti-H7N9 subtype avian influenza virus monoclonal antibody epitope as well as screening method and application thereof
  • Anti-H7N9 subtype avian influenza virus monoclonal antibody epitope as well as screening method and application thereof
  • Anti-H7N9 subtype avian influenza virus monoclonal antibody epitope as well as screening method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0075] 1 method

[0076] 1. Preparation of 1H7N9 Subtype Avian Influenza Monoclonal Antibody

[0077] 1.1.1 Antigen preparation

[0078] 1.1.1.1 Virus amplification and inactivation

[0079] A / CHICKEN / JIANGSU / W1-8 / 2015 (H7N9 subtype avian influenza virus, isolated from cotton swab samples of chickens in the live poultry trading market by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University) was sterile PBS conducts 10 -4 Dilute and inoculate 10-day-old SPF chicken embryos, inoculate chicken embryos with 0.2 mL of the diluted virus solution through the allantoic cavity, incubate at 35°C, discard dead chicken embryos within 24 hours, and place dead chicken embryos at 4°C overnight after 24 hours of inoculation. The allantoic fluid was collected, formalin was slowly added dropwise to a final concentration of 0.1%, placed at 4°C, 90 rpm, and shaken for 24 hours to inactivate, and finally the inactivated virus fluid was...

Embodiment 2

[0102] (1) Dilute the wild-type H7N9 subtype avian influenza virus (A / CHICKEN / JIANGSU / W1-8 / 2015) to 10 with PBS with a concentration of 0.01M and pH=7.4 6 EID 50 / 0.5ml, then take 0.5ml of monoclonal antibody ascites and the diluted viral allantoic fluid and mix them with room temperature for 1 hour.

[0103] (2) Inoculate four 10-day-old SPF chicken embryos with the above-mentioned virus and antibody mixture, inoculate 0.1ml of each chicken embryo, and culture at 35°C for 72h.

[0104] (3) Collect the allantoic fluid after 72 hours and measure its HA titer. The specific method is as follows:

[0105] Ⅰ. Add 25 μl of PBS to each well of a 96-well hemagglutination plate.

[0106] Ⅱ. Add 25 μl of virus to the first row of wells of the 96-well hemagglutination plate, dilute to the 11th well from left to right, and discard 25 μl. Well 12 was a negative control.

[0107] Ⅲ. Add 25μl PBS to each well.

[0108] Ⅳ. Add 25 μl of 1% red blood cells to each well, and the final volum...

Embodiment 4

[0130] Preparation of colloidal gold test strips

[0131] 1. Antibody purification effect identification

[0132] The 1B6 and 1A2 monoclonal antibodies were purified by Protein G affinity chromatography, and the concentrations were 1.231mg / mL and 2.663mg / mL, respectively. The processed samples were subjected to SDS-PAGE at a concentration of 0.5 mg / mL to observe the results. Such as figure 1 There are mainly two bands of heavy chain and light chain of immunoglobulin, among which M: protein molecular marker; 1 represents 1B6; 2 represents 1A2, and the purification effect is better.

[0133] 2. Optimal amount of protein labeled with colloidal gold

[0134] According to the color of the finger tube, it was found that the concentration started to be stable at 40 μl / ml, so the optimal amount of labeled monoclonal antibody was determined to be 40 μl / ml.

[0135] 3. Determination of the dilution factor of the coating antibody

[0136] Dilute the coated monoclonal antibody to 0.6...

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Abstract

The invention provides an anti-H7N9 subtype avian influenza virus monoclonal antibody epitope as well as a screening method and application thereof, belonging to the technical field of immunodetection. The screening method comprises the following steps: mixing wild type H7N9 subtype avian influenza virus liquid with a corresponding monoclonal antibody with a neutralizing property for incubation, and inoculating the mixture to an SPF chick embryo, so as to obtain allantoic fluid with a positive hemagglutination titer; and carrying out gradient dilution on the positive allantoic fluid, mixing the positive allantoic fluid with the monoclonal antibody for incubation, inoculating the mixture to the SPF chick embryo, determining a hemagglutination inhibition titer of the monoclonal antibody by selecting the allantoic fluid with the positive hemagglutination titer as an antigen, when the determined hemagglutination inhibition titer is lower than the hemagglutination inhibition titer of a wild type virus by 8log2, determining the positive allantoic fluid as an escape mutant of the wild type H7N9 subtype avian influenza virus, measuring an HA gene sequence of the positive allantoic fluid, and determining the epitope recognized by the monoclonal antibody. By virtue of the method, the specific epitope can be clearly screened; the method is simple, accurate and short in screening period.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and in particular relates to an anti-H7N9 subtype avian influenza virus monoclonal antibody antigen epitope and a screening method and application thereof. Background technique [0002] The H7N9 subtype avian influenza virus is a type of influenza A. In order to effectively prevent H7N9 subtype avian influenza, effective detection means must be established, and antigen capture ELISA based on monoclonal antibodies, immune colloidal gold test strips, etc. are commonly used methods. To establish this type of detection method, multiple monoclonal antibodies targeting different epitopes are required to obtain a detection method with high sensitivity and specificity. [0003] At present, the methods for determining antigenic epitopes are mainly competitive inhibition ELISA methods. The screening mechanism of the competitive inhibition ELISA method is as follows: if the labeled antibody and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983G01N33/577
Inventor 彭大新孙志豪陈素娟秦涛刘秀梵
Owner YANGZHOU UNIV
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