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Immunization microsphere in use for detecting SARS antibody, preparation method and application

A technology of immune microspheres and antibodies, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of enzyme-linked immunoassays that are not simple enough, fast, false positive experiment errors, unstable immune microspheres, etc.

Inactive Publication Date: 2008-10-15
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the disadvantages that the existing enzyme-linked immunosorbent method is not simple and fast enough; and the immune microspheres of the immune microsphere method are very unstable, which is easy to cause false positive experimental errors, thereby providing a fast and simple method. Detection, and very stable immune microspheres for detecting SARS antibodies

Method used

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  • Immunization microsphere in use for detecting SARS antibody, preparation method and application
  • Immunization microsphere in use for detecting SARS antibody, preparation method and application
  • Immunization microsphere in use for detecting SARS antibody, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0136] Embodiment 1, the preparation of the immune microsphere coupled with the SARS coronavirus S protein as antigen

[0137] Preparation and identification of SARS coronavirus S protein antigen:

[0138]Preparation—cloning, expressing and purifying SARS coronavirus S protein as an antigen by gene recombination technology: using RT-PCR method to amplify the gene fragment of SARS coronavirus S, and after the gene fragment is sequenced to confirm that the gene sequence is correct, then the It is cloned into an expression vector such as prokaryotic expression vector pQE30, or yeast expression vector pPICZαA, and then expressed and purified.

[0139] Identification—After antigen purification, identification is performed by the following tests:

[0140] 1) Determination of antigenic protein content: Take 1 ml of antigen and measure its protein content with a spectrophotometer. After measuring the OD values ​​at 260nm and 280nm respectively, use the formula 280nm Odx1.45-260nmODx0...

Embodiment 2

[0150] Embodiment 2, the preparation of the immune microsphere coupled with the SARS coronavirus N protein as antigen

[0151] Preparation and identification of SARS coronavirus N protein antigen:

[0152] Preparation—Using genetic recombination technology to clone, express and purify the SARS coronavirus N protein as an antigen: use RT-PCR to amplify the gene fragment of SARS coronavirus N, and after the gene fragment is sequenced to confirm that the gene sequence is correct, the It is cloned into an expression vector such as prokaryotic expression vector pQE30, or yeast expression vector pPICZαA, and then expressed and purified.

[0153] Identification—After antigen purification, identification is performed by the following tests:

[0154] 1) Determination of antigenic protein content: Take 1 ml of antigen and measure its protein content with a spectrophotometer. After measuring the OD values ​​at 260nm and 280nm respectively, use the formula 280nm Odx1.45-260nmODx0.74 to c...

Embodiment 3

[0164] Embodiment 3, the preparation of the immune microsphere coupled with the SARS coronavirus M protein as antigen

[0165] Preparation and identification of SARS coronavirus M protein antigen:

[0166] Preparation—cloning, expressing and purifying SARS coronavirus M protein as an antigen by gene recombination technology: using RT-PCR method to amplify the gene fragment of SARS coronavirus M, and after the gene fragment is sequenced to confirm that the gene sequence is correct, then the It is cloned into an expression vector such as prokaryotic expression vector pQE30, or yeast expression vector pPICZαA, and then expressed and purified.

[0167] Identification—After antigen purification, identification is performed by the following tests:

[0168] 1) Determination of antigenic protein content: Take 1 ml of antigen and measure its protein content with a spectrophotometer. After measuring the OD values ​​at 260nm and 280nm respectively, use the formula 280nm Odx1.45-260nmODx...

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Abstract

The present invention relates to a kind of immunization microsphere in use for detecting SARS antibody, preparation method and application.This immune microsphere takes polystyrene microsphere which surface is modified with carboxyl as kernel,the carboxyl couples with the antibodyies which resists SARS coronavirus of S N M or E proteini through multi polylysine chemically.The immune microsphere can be used for detecting SARS antigen as detecing reagent.Comparing to the existing technology,the immune microsphere using for detecting SARS antigen possesses better sensitivity and specificity,higher accuracy,avoiding the disadvantage of false positive of PCR disgnosis kit.the process is fast.on the other hand,detection needs no instrument equipment,so this immune microsphere is suitable for the largeness base sanitary and anti-epidemic units to use.

Description

technical field [0001] The invention relates to an immune microsphere for detecting SARS antibody, its preparation method and application. technical background [0002] SARS (Severe Acute Respiratory Syndrome) coronavirus is a newly discovered coronavirus. The new disease caused by it has a fast onset and rapid spread. If it is not diagnosed and treated in time, the fatality rate is very high. [0003] At present, the commonly used detection method in scientific research and clinical practice is enzyme-linked immunoassay (ELISA). Use enzyme-linked immunosorbent assay (ELISA) to detect antibodies, that is, the antigen is coated on the enzyme-labeled plate, and calf serum or skim milk is added to block, and the serum to be tested is added and incubated for 1 hour. After repeated washing, the enzyme-labeled secondary antibody is added. , incubated, and washed several times before adding the chromogenic substrate. Although this method has good sensitivity and specificity, it t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/547G01N33/546G01N33/531
Inventor 陈佺杨建国朱玉山顾莹邢娟金海京王晓惠
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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