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Immune microsphere in use for detecting SARS antigen, preparation method and application

A technology of immune microspheres and antigens, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of false positive experimental errors, unstable immune microspheres, and inconvenient enzyme-linked immunosorbent assays, etc.

Inactive Publication Date: 2005-08-31
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the existing enzyme-linked immunoassay for detecting SARS antigens which is not easy and fast enough; and the immune microspheres of the immune microsphere method are very unstable and easily cause false positive experimental errors, thereby providing a method that is both It can be quickly and easily detected, and is very stable for the detection of SARS antigen immune microspheres

Method used

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  • Immune microsphere in use for detecting SARS antigen, preparation method and application
  • Immune microsphere in use for detecting SARS antigen, preparation method and application
  • Immune microsphere in use for detecting SARS antigen, preparation method and application

Examples

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Effect test

Embodiment 1

[0139] Preparation of immune microspheres for detecting SARS antigen coupled with antibodies produced by immunizing rabbits with SARS coronavirus S protein

[0140] 1. Preparation of SARS coronavirus S protein antigen

[0141] Use the RT-PCR method to amplify the gene fragment of SARS coronavirus S, and then clone it into an expression vector such as prokaryotic expression vector pQE30 and yeast expression vector pPICZαA after the gene fragment is sequenced to confirm that the gene sequence is correct, and then express and protein purification.

[0142] 2. Identification of SARS coronavirus S protein antigen

[0143] After antigen purification, it must be identified by the following tests:

[0144] (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 280nmOdx1.45-260nmODx0.74 to calculate the protein content of the antigen...

Embodiment 2

[0156] Example 2: Preparation of immune microspheres coupled with SARS coronavirus N protein polyclonal antibody

[0157] 1. Preparation of SARS coronavirus N protein antigen:

[0158] Use the RT-PCR method to amplify the gene fragment of SARS coronavirus N, and after the gene fragment is sequenced to confirm that the gene sequence is correct, then it is cloned into an expression vector such as prokaryotic expression vector pQE30, yeast expression vector pPICZαA, and then expressed and protein purification.

[0159] 2. Identification of antigens:

[0160] After antigen purification, it must be identified by the following tests:

[0161] (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 280nmOdx1.45-260nmODx0.74 to calculate the protein content of the antigen.

[0162] (2) Determination of protein purity: Take 20 microl...

Embodiment 3

[0173] Example 3: Preparation of immune microspheres coupled with SARS coronavirus M protein polyclonal antibody

[0174] 1. Preparation of SARS coronavirus M protein antigen:

[0175] Use the RT-PCR method to amplify the gene fragment of SARS coronavirus M, and then clone it into an expression vector such as prokaryotic expression vector pQE30 and yeast expression vector pPICZαA after the gene fragment is sequenced to confirm that the gene sequence is correct, and then express and protein purification.

[0176] 2. Antigen identification:

[0177] After antigen purification, it must be identified by the following tests:

[0178] (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 280nmOdx1.45-260nmODx0.74 to calculate the protein content of the antigen.

[0179] (2) Determination of protein purity: Take 20 microliters of...

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Abstract

The present invention relates to a kind of immune microsphere in use for detecting SARS antigen, 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 antigen, a preparation method and application thereof. 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 for detecting SARS antigen in scientific research and clinical practice is enzyme-linked immunosorbent assay (ELISA). Use enzyme-linked immunosorbent assay (ELISA) to detect antigen, that is, the antibody is coated on the enzyme 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 sample to be tested is incubated. Wash after 1 hour, add enzyme-labeled antibody, incubate, wash several times, and then add color-developing substrate. Alt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/546
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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