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Method for preparing aspergillus fumigatus galactomannan polyclonal antibody

A galactomannan and polyclonal antibody technology, applied in the biological field, can solve the problems of unsatisfactory polyclonal antibody titer and low polyclonal antibody titer, achieving good specificity, high titer, and broad application prospects Effect

Inactive Publication Date: 2016-12-14
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The Chinese invention patent with application number CN201210011794.2 discloses a method for preparing a polyclonal antibody against Aspergillus fumigatus. In this invention, the polyclonal antibody against Aspergillus fumigatus is obtained by preliminary purification of serum by ammonium sulfate salting out method and affinity chromatography. However, This method only removes some impurities and obtains polyclonal antibodies with low titer
[0008] The Chinese patent of CN105435758A discloses an immunoaffinity chromatography column with astragaloside IV as a ligand and its application. In this invention, astragaloside IV is used as a ligand to bind to an antibody. The solid phase carrier agarose gel, cellulose , dextran gel, polyacrylamide gel, porous glass or polyacrylamide-agarose gel, the monoclonal antibody of astragaloside IV with higher purity was obtained, but the effect of the purified monoclonal antibody The price only reaches 1:7500
[0009] In summary, although the preparation method of Aspergillus fumigatus galactomannan polyclonal antibody is disclosed in the prior art, the potency of the polyclonal antibody of the product obtained by the above method cannot meet the application in clinical detection of Aspergillus fumigatus, There is an urgent need for a new method to prepare high-titer polyclonal antibodies against galactomannan antigens

Method used

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  • Method for preparing aspergillus fumigatus galactomannan polyclonal antibody

Examples

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Embodiment 1

[0040] Example 1 Preparation of polyclonal antibody against Aspergillus fumigatus galactomannan antigen

[0041] The used Aspergillus fumigatus galactomannan was prepared by the method in Example 4 of the patent CN104945527A.

[0042] 1. Immunization of animals

[0043] Mix equal volumes of Aspergillus fumigatus galactomannan antigen and Freund's complete adjuvant to an appropriate volume. After full emulsification, New Zealand big-eared rabbits were injected subcutaneously at multiple points, and the immune dose of each rabbit was controlled at 0.01-1mg. Ear blood was collected 3 days before immunization, and serum was separated as a negative control. After the initial immunization, immunize once every 2 weeks, and the method is the same as the first time.

[0044] 2. Obtaining polyclonal antibodies

[0045] 1) Titer determination: During the immunization process, blood was collected every few days to measure the titer once after immunization, and the number of immunizati...

Embodiment 2

[0066] The detection of embodiment 2 antibody

[0067] 1. SDS-PAGE electrophoresis detection

[0068] The antibody prepared in Example 1 was subjected to SDS-PAGE electrophoresis, and the resulting gel was stained with Coomassie brilliant blue. See the experimental results figure 1 (pAb lane is polyclonal antibody, M lane is protein marker). It can be seen from the figure that there are clear and obvious bands in the 25KD and 50KD molecular weight regions without impurity bands, indicating that the purity of the antibody is very high.

[0069] 2. Potency determination

[0070] Antibody titer by indirect ELISA The antibody obtained in Example 1 was tested for titer. The enzyme-labeled secondary antibody used was goat anti-rabbit IgG labeled with horseradish peroxidase, and the negative control was phosphate buffer solution. See the test results figure 2 . It can be seen from the results that the antibody titer is greater than 1:1×10 6 , indicating a high titer of the a...

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Abstract

The invention provides an aspergillus fumigatus galactomannan antigen resisting polyclonal antibody and a preparation method thereof. The preparation method of the polyclonal antibody includes: adopting aspergillus fumigatus galactomannan prepared by affinity chromatography to perform animal immunization, taking blood from immunized animals to obtain serum, and purifying the serum by a saturated ammonium sulfate salting-out method and immunoaffinity chromatography to obtain the polyclonal antibody. The polyclonal antibody prepared according to the method is high in titer and purity, is the first aspergillus fumigatus galactomannan antigen resisting polyclonal antibody prepared by immunoaffinity chromatography in China and has a promising application prospect in the field of medical and scientific research.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polyclonal antibody for the antigen of Aspergillus fumigatus galactomannan (Galactomannan, GM) and a preparation method thereof. Background technique [0002] Aspergillus is an important opportunistic pathogen that often infects immunocompromised or immunocompromised patients. Aspergillus fumigatus is the most common pathogen causing severe deep Aspergillus infection in immunosuppressed patients with a high case-fatality rate. [0003] In recent years, with the extensive abuse of antibiotics and the widespread use of immunosuppressants in clinical practice, the morbidity and mortality of invasive deep fungal infections are increasing. Among them, Aspergillus, especially Aspergillus fumigatus, has gradually become a clinically important pathogenic fungus. Invasive aspergillosis has a mortality rate as high as 70% to 90% in patients with blood diseases and hematopoietic stem cell t...

Claims

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Application Information

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IPC IPC(8): C07K16/14C07K16/06G01N33/569
CPCC07K16/14G01N33/56961G01N2800/26
Inventor 刘春龙翟栓柱张舟李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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