Immune affinity chromatography purification method for specific egg yolk immunoglobulins

A purification method and specific technology, which is applied in the field of immunoaffinity purification of Listeria monocytogenes-specific yolk antibody, can solve the problems of few purification methods and inability to separate and obtain specific IgY, so as to improve binding efficiency, Effect of improving purification efficiency

Inactive Publication Date: 2010-09-01
OCEAN UNIV OF CHINA
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Problems solved by technology

[0003] At present, the methods for the preparation and purification of yolk antibodies are mainly salting-out method, organic solvent precipitation method, ultrafiltration method, and affinity purification method. Most of these methods are aimed at the extraction of IgY, and there are few methods for IgY purification. It is an affinity purification method, and it can only be purified to obtain IgY, but cannot be separated to obtain specific IgY

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  • Immune affinity chromatography purification method for specific egg yolk immunoglobulins
  • Immune affinity chromatography purification method for specific egg yolk immunoglobulins

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Embodiment 1

[0019] Example 1: Purification of Listeria monocytogenes-specific yolk antibody

[0020] 1. Preparation of Listeria monocytogenes cell antigen

[0021] Listeria monocytogenes was inoculated in tryptone soybean yeast extract broth (TSB-YE) medium, cultured at 37°C for 24 hours, centrifuged at 10,000r / min for 30 minutes to collect the bacteria, and prepared a bacterial suspension with physiological saline. Treat in boiling water bath for 10 minutes, wash with physiological saline three times, centrifuge at 4000r / min for 10 minutes each time, store at 4°C for future use.

[0022] 2. Amino activation of Listeria monocytogenes bacterial antigen

[0023] Dissolve 2mL of divinyl sulfone in 4mL of dimethylformamide, and mix well with 34mL of 0.5M pH1.0 carbonate buffer, and prepare the bacterial antigen (5×10 11 cfu) was resuspended in the above mixture, incubated at 21°C for 60 minutes, centrifuged at 4000r / min for 10 minutes to collect the bacteria and washed three times with doub...

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Abstract

The invention discloses an immune affinity chromatography purification method aiming at specific egg yolk immunoglobulins (IgY) against listeria monocytogenes. The method comprises the following steps of: combining listeria monocytogenes bacteria cells serving as specific adsorption materials and Sepharose4B filler subjected to amino activation and cyanogen bromide activation to form a solid phase carrier; during purification, combining the specific IgY antibody to a chromatographic column through specific adsorption with the listeria monocytogenes bacteria cells combined on the chromatographic column, and making non-specific antibody outflow along with eluent; after no non-specific antibody outflows, dissociating the antibody and the antigen combined on the chromatographic column by using dissociating buffer solution; and detecting the adsorption and dissociating conditions of the specific antibody by using a method for measuring ultraviolet absorption, and detecting the purification effect of the antibody by using an indirect enzyme-linked immunosorbent assay (ELISA). The method has simple operation, and can be applied to effective separation and purification of the specific IgY.

Description

technical field [0001] The invention relates to a method for immunoaffinity purification of specific egg yolk antibody, in particular to an immunoaffinity purification method for specific egg yolk antibody of Listeria monocytogenes. Background technique [0002] Egg yolk immunoglobulins (IgY) is the only immunoglobulin present in poultry egg yolk, which is formed by the transfer of immunoglobulin to egg yolk by the hen during laying. The biggest feature of IgY that is different from IgG is that it not only has antigen binding ability, but also has a significant inhibitory effect on the growth of its antigenic bacteria. With the continuous deepening of research, there have been a large number of research reports on the application of IgY to passive immune protection and preventive control of animal pathogens. [0003] At present, the methods for the preparation and purification of yolk antibodies are mainly salting-out method, organic solvent precipitation method, ultrafiltr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02C07K1/22
Inventor 林洪隋建新曹立民
Owner OCEAN UNIV OF CHINA
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