Method for efficiently preparing Enterobacter sakazakii polyclonal antibody

A polyclonal antibody, Enterobacter sakazakii technology, applied in the field of immunoassay, can solve the problems of complex preparation process, easy contamination, low titer and purity, etc.

Inactive Publication Date: 2016-06-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the monoclonal antibody has high specificity, the preparation process is complicated and easily contaminated; the preparation process of polyclonal antibody is simple, but the traditional subcutaneous multi-point injection immunization method takes a long time, and the titer and purity are low
At present, there are no reports of patents and articles related to the preparation of polyclonal antibodies against Enterobacter sakazakii by the immunization technique in the present invention

Method used

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  • Method for efficiently preparing Enterobacter sakazakii polyclonal antibody
  • Method for efficiently preparing Enterobacter sakazakii polyclonal antibody
  • Method for efficiently preparing Enterobacter sakazakii polyclonal antibody

Examples

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example 1

[0018] (1) Antigen preparation

[0019] Enterobacter sakazakii ( Enterobacters akazakii ) Streak beef extract peptone (CM0002) solid medium, culture at 37°C for 24h to activate the strain. Wash the bacterial lawn with sterile water and wash it three times, and adjust the bacterial concentration to 1×10 by turbidimetric method. 7 cfu / ml; Formalin was inactivated at 4°C for 24h, spread on the plate and incubated at 37°C for 24h, if there were no viable bacteria, centrifuged at 12000rpm at 4°C for 10min to remove formalin, and the precipitate was obtained by resuspending with an equal volume of sterile water Enterobacter sakazakii particulate antigen.

[0020] (2) Preparation of antiserum

[0021] After one week of adaptive feeding of New Zealand white rabbits, 5-10 mL of blood was collected from the ear vein as negative serum. New Zealand rabbits were immunized with the inactivated Enterobacter sakazakii granular antigen, the specific procedure was as follows: basic immuniz...

example 2

[0028] (1) Antigen preparation

[0029] Enterobacter sakazakii ( Enterobacters akazakii ) Streak beef extract peptone (CM0002) medium, culture at 37°C for 24h to activate the strain. Wash the bacterial lawn with sterile water and wash it three times, and adjust the bacterial concentration to 1×10 by turbidimetric method. 8 cfu / ml; Formalin was inactivated at 4°C for 24h, spread on the plate and incubated at 37°C for 24h, if there were no viable bacteria, centrifuged at 12000rpm at 4°C for 10min to remove formalin, and the precipitate was obtained by resuspending with an equal volume of sterile water Enterobacter sakazakii particulate antigen.

[0030] (2) Preparation of antiserum

[0031] After one week of adaptive feeding of New Zealand white rabbits, 5-10 mL of blood was collected from the ear vein as negative serum. New Zealand rabbits were immunized with the inactivated Enterobacter sakazakii granular antigen, the specific procedure was as follows: basic immunization,...

example 3

[0038] (1) Antigen preparation

[0039] Enterobacter sakazakii ( Enterobacters akazakii ) Streak beef extract peptone (CM0002) medium, culture at 37°C for 24h to activate the strain. Wash the bacterial lawn with sterile water and wash it three times, and adjust the bacterial concentration to 1×10 by turbidimetric method. 9 cfu / ml; Formalin was inactivated at 4°C for 24h, spread on the plate and incubated at 37°C for 24h, if there were no viable bacteria, centrifuged at 12000rpm at 4°C for 10min to remove formalin, and the precipitate was obtained by resuspending with an equal volume of sterile water Enterobacter sakazakii particulate antigen.

[0040] (2) Preparation of antiserum

[0041] After one week of adaptive feeding of New Zealand white rabbits, 5-10 mL of blood was collected from the ear vein as negative serum. Use the inactivated Enterobacter sakazakii granular antigen to immunize New Zealand rabbits, the specific procedure is as follows: basic immunization, 0.7m...

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Abstract

The invention belongs to the technical field of immunoassay, and concretely relates to a method for efficiently preparing an Enterobacter sakazakii polyclonal antibody. The method comprises the following steps: immunizing New Zealand white rabbits with Enterobacter sakazakii granular antigens through adopting a subcutaneous multi-point injection and ear vein venous injection combination technology, and further purifying antiserum by a Protein A affinity chromatography column to rapidly obtain the high-tilter Enterobacter sakazakii polyclonal antibody. Compared with traditional immunization methods, the method disclosed in the invention has the advantages of shortening of the immunization time by 7-14d, and great increase of the antibody tilter. A West-blotting and IFAT combination technology is used to detect the specificity of the antibody, and a result shows that the prepared antibody has very high specificity to Enterobacter sakazakii. The method provided by the invention is simple and efficient, provides technical support for preparation of the Enterobacter sakazakii polyclonal antibody, and is helpful for developing the immunodetection technology of the Enterobacter sakazakii.

Description

technical field [0001] The invention belongs to the technical field of immune analysis. Specifically, it relates to a method for efficiently preparing polyclonal antibodies against Enterobacter sakazakii. Background technique [0002] Enterobacter sakazakii ( E. sakazakii ) belongs to facultative anaerobic Gram-negative bacilli, sporadic and outbreak cases of neonatal meningitis, sepsis and necrotizing enterocolitis caused by it have been reported in the world, and the fatal cases are as high as 40% to 80% %. Infections are mainly hospitalized newborn babies, especially those born prematurely or with other medical conditions. Enterobacter sakazakii ( E. sakazakii ) as a hygienic indicator that must be detected in infant formula milk powder. Traditional detection methods have the disadvantages of cumbersome detection steps and long detection cycle. Therefore, the establishment of a rapid detection method for Enterobacter sakazakii is the key to the prevention and control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/06C07K1/22
Inventor 汪东风朱英莲徐莹
Owner OCEAN UNIV OF CHINA
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