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O antigen affinity medium and preparation method and application thereof

A technology of O antigen and medium, applied in the field of immunoassay, can solve the problem of indistinguishable antibodies, achieve high operability and technical value, increase yield, high affinity and specificity

Active Publication Date: 2019-08-27
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are unable to distinguish antibodies of different specificities

Method used

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  • O antigen affinity medium and preparation method and application thereof
  • O antigen affinity medium and preparation method and application thereof
  • O antigen affinity medium and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Extraction and Purification of O Antigen

[0062] The specific steps for the preparation of the O antigen ligand are:

[0063] (1) Acid hydrolysis: Take a certain volume of inactivated Salmonella pullorum standard type antigen solution for centrifugation, collect the bacteria, take 20g of wet bacteria collected after centrifugation, add 0.8% to 1.0% (w / v) sodium chloride solution to resuspend the cells, mix well and add 0.8% to 1% (v / v) acetic acid to make the final pH of the solution 3.5 to 4.7, boil at 100°C for 5 to 8 hours. End with 28% NH 4 OH for neutralization, resulting in a final pH of 6-7. Finally, the supernatant was obtained by centrifugation, and the specific conditions were 10000 rpm for 40 min.

[0064] (2) Concentration and exchange of liquid: Use a 15mL, 30kDa ultrafiltration tube to replace the solvent, sequentially replace the liquid with 20 times the volume of 1M NaCl and 10 times the volume of water, and finally concentrate to about 11...

Embodiment 2

[0072] The identification of embodiment 2 O antigen

[0073] The purified O-antigen was detected by gel permeation chromatography (Gel Permeation Chromatography, GPC), and the distribution range of its molecular weight was obtained. The selected chromatographic column is TSKgel GMPWXL, using an evaporative light scattering detector (ELSD) detector, and the detection conditions are set as follows: the concentration of the sample to be determined is 2 mg / mL, the sample volume is 100 μL, the mobile phase is H2O, and the flow rate is 0.6 mL / min , the duration is 30min, see the result figure 2 . Dextran standards with different molecular weights, including 500K, 40K, 5K, and 1K, were detected separately, and the molecular weight range of the extracted O antigen was judged by comparing the retention time in the chromatograms of the standard and the sample to be tested.

[0074] Depend on figure 2 It can be seen that the extracted O antigen is mainly divided into two size ranges...

Embodiment 3

[0075] Activation of embodiment 3 ultra-macroporous medium and O grafting

[0076] First, epichlorohydrin was used to epoxy-activate the ultra-macroporous polystyrene medium. Measure 7 mL (sediment volume) of super-macroporous medium stored in 20% (v / v) ethanol solution and place it in a G3 sintered glass funnel, wash repeatedly with excess distilled water to remove ethanol, and then vacuum dry. Weigh 7 g of the suction-filtered medium, wash it with 20%, 50% and 70% dimethyl sulfoxide aqueous solution in sequence, and vacuum-dry the cleaning solution after each washing. Weigh 5g of DMSO-treated spheres, place them in a 50mL conical flask, and then add epichlorohydrin (ECH), sodium hydroxide, and ultrapure water in sequence to make the total volume of the solution 20mL. The concentrations are 8-12% (w / v) and 0.6-1.0M, respectively. The suspension containing the medium is placed on a constant temperature air bath shaker and shaken at 170 r / min for 2-3 hours, and the reaction t...

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Abstract

The invention provides an O antigen affinity medium and a preparation method and application thereof. The O antigen affinity medium comprises an ultra-macroporous medium and an O antigen of Salmonella, wherein the O antigen affinity medium is chemically immobilized on the surface of the ultra-macroporous medium. The preparation process of the O antigen affinity matrix provided by the invention issimple, the purification operation is simple, and the efficiency is high; an antibody prepared by the affinity medium and the purification method has high specificity for the O antigen, and can be used for the application of assisting the diagnosis of Salmonella and the like.

Description

technical field [0001] The invention belongs to the field of immune analysis, and relates to an affinity chromatography medium and its preparation method and application, in particular to an O antigen affinity medium, its preparation method and application. Background technique [0002] Pullorum, chicken typhoid, paratyphoid, piglet paratyphoid, etc. are animal diseases caused by Salmonella, resulting in significant economic losses in the poultry industry. Salmonella is a Gram-negative bacterium and an important pathogen of bacterial food poisoning. Some Salmonella are zoonotic, and even cause infection and death, which has attracted great attention. [0003] The most important and fundamental method in the prevention and control of poultry diseases such as pullorum and typhoid fever is to eliminate sick poultry, purify and eradicate them, and keep them closed. There are many types of poultry diseases and they appear in clusters. Therefore, the key is to quickly and accurat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/255B01J20/286B01J20/30B01D15/38C07K16/12G01N33/543G01N33/569
CPCB01D15/3809B01J20/286B01J20/3085C07K14/255C07K16/1235G01N33/54306G01N33/56916
Inventor 张松平苏志国杨延丽尤星力
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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