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Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody

A technology of Cryptococcus neoformans and polyclonal antibodies, which is applied in antifungal/algae/lichen immunoglobulins, measuring devices, instruments, etc., can solve the problems of unguaranteed purity and specificity of antigens, poor immunogenicity, and poor properties of polyclonal antibodies. Stability and other issues

Active Publication Date: 2016-06-08
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method directly uses the GXM antigen as the immunogen. However, due to the special structure, poor immunogenicity, and similar structure of polysaccharides, many of them are haptens, it is difficult to obtain antibodies as easily as protein immunogens; in addition, directly using the antigen as the immunogen Originally, since the purity and specificity of the antigen cannot be guaranteed, the properties of the obtained polyclonal antibody are also not stable.

Method used

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  • Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody
  • Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody
  • Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody

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Embodiment 1

[0075] Embodiment 1: the preparation of the polyclonal antibody of the anti-Cryptococcus neoformans capsular polysaccharide GXM antigen (hereinafter referred to as GXM antigen) of preliminary purification

[0076] 1. The inactivated Cryptococcus neoformans (ATCC, strain number: 208821) was crushed by repeated freezing and thawing and ultrasonic crushing.

[0077] The specific method is:

[0078] (1) Formaldehyde solution was added to the liquid medium to make the final concentration of formaldehyde 3.7%, and placed at 4°C for 24 hours to inactivate the spores.

[0079] (2) Centrifuge the inactivated Cryptococcus neoformans at 4000 rpm for 10 min at 4°C, and remove the supernatant.

[0080] (3) Add liquid nitrogen and grind with a sterilized mortar for 3-5 times, add water, and use probe-type ultrasonic crushing for 30 minutes under ice bath.

[0081] (4) Carry out BCA protein quantification and phenol sulfate method sugar quantification.

[0082] 2. Immunized animals

[00...

Embodiment 2

[0090] Example 2: Preparation of Cryptococcus neoformans Capsular Polysaccharide GXM Immunoaffinity Chromatography Column

[0091] (1) Dissolve the GXM antigen in carbonate buffer, bind 4 mg of GXM antigen per milliliter of wet protein A microbeads, mix the GXM antigen and protein A microbeads to form a thin homogenate, and mix in a total of 10 mL of carbon dioxide Add about 2mL of protein A microbeads to salt buffer solution, incubate at room temperature for 1h, and shake gently to mix.

[0092](2) Wash the homogenate twice with 5-15 times the volume of 0.2mol / L sodium borate (pH8.0-9.5), centrifuge at 3000g for 2min each time, or centrifuge at 10000g for 30s to obtain GXM antigen-protein A microbeads mixture.

[0093] (3) Resuspend the mixture of GXM antigen-protein A microbeads with 5-15 times the volume of 0.2mol / L sodium borate (pH8.0-9.5), take a sample equivalent to 10mL of wet protein A microbeads, add enough Add an appropriate amount of dimethylpimelate (solid) to t...

Embodiment 3

[0096] Example 3: Immunoaffinity purification of polyclonal antibody against Cryptococcus neoformans capsular polysaccharide GXM antigen

[0097] (1) Wash the column with 10-25 times the column bed volume of pre-elution buffer.

[0098] (2) Load the initially purified polyclonal antibody against the capsular polysaccharide GXM antigen of Cryptococcus neoformans, and let the sample flow through the chromatographic column at a flow rate of about 1 mL / h per milliliter of column volume, and control the flow rate with a peristaltic pump.

[0099] (3) Wash the column with 10-25 times the column bed volume of binding buffer.

[0100] (4) Wash the column with 10-25 times the column bed volume of pre-elution buffer.

[0101] (5) Using the segmented elution method, continuously pass the elution buffer of 0.4-0.8 times the column bed volume through the chromatography column, and collect each component in separate tubes.

[0102] (6) Detect the antibody content of each tube, and combine...

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Abstract

The invention provides a polyclonal antibody of a GXM (glucuronoxylomannan) antigen of Cryptococcus neoformans. The polyclonal antibody is prepared through the following steps: inactivated Crytococcus Neoformans bacteria are taken as an immunogen immunized animal, separated and purified, and a primarily purified polyclonal antibody is obtained; the GXM antigen of Cryptococcus neoformans is cross-linked with an affinity chromatography substrate, and a GXM immunoaffinity chromatography column of Cryptococcus neoformans is obtained; after the primarily purified polyclonal antibody is purified with the immunoaffinity chromatography column, the polyclonal antibody resisting the GXM antigen of Cryptococcus neoformans is obtained. The polyclonal antibody prepared with the method has the characteristics of high titer and high purity and has broad application prospect in the fields of medical treatment and scientific research.

Description

technical field [0001] The present invention relates to an antibody and a preparation method thereof, in particular to a polyclonal antibody and a preparation method thereof for the Cryptococcus neoformans capsular polysaccharide glucuronoxyloma-nnan (GXM) antigen. Background technique [0002] Cryptococcus neoformans is one of the opportunistic pathogenic fungi that is very harmful to human health. It often infects immunocompromised or immunocompromised patients and causes cryptococcosis. Treatment is often delayed due to untimely detection or misdiagnosis , the fatality rate is high. [0003] At present, the detection methods of Cryptococcus neoformans in the world mainly include the following: Indian ink staining method, when ink staining is used, the background is colored but the bacteria itself is not colored, thus detecting the capsule of bacteria or fungi, this method has poor sensitivity , the positive detection rate is about 55%; blood culture or cerebrospinal flui...

Claims

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Application Information

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IPC IPC(8): C07K16/14G01N33/569
CPCC07K16/14
Inventor 彭洁史东东李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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