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Labelled complex and preparation method therefor, and kit, use and detection system thereof

A complex and detection kit technology, applied in the field of detection systems, can solve problems such as high false positives, impact on immunological activity, and complexity

Active Publication Date: 2018-10-23
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 1) Most of the methods for labeling antigens and antibodies are direct labeling methods at present, but some antigens are directly labeled with the detected signal substances, and their immunological activity will be affected, thereby affecting the stability and sensitivity of the markers;
[0013] 2) The sensitivity of labeling some antigens can be improved by indirect labeling of small molecules or tagged proteins, but it is necessary to find suitable small molecules or tagged proteins and corresponding identifiable ligands, which will increase the reagent components and make it complicated ;
[0014] 3) Through the expression form of fusion protein-antigen chimeric antigen, the recombinant antigen has better solubility, and the labeling performance has also been improved, but there are certain deficiencies in the use of fusion proteins, for example, inclusion bodies often exist in prokaryotic expression systems , there will be a high false positive phenomenon after antigen labeling

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  • Labelled complex and preparation method therefor, and kit, use and detection system thereof
  • Labelled complex and preparation method therefor, and kit, use and detection system thereof
  • Labelled complex and preparation method therefor, and kit, use and detection system thereof

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preparation example Construction

[0062] According to another aspect of the present invention, a method for preparing the above-mentioned labeling complex is provided. The preparation method comprises the following steps: performing a coupling reaction on a labeled protein with a functional group on the surface and an antigen to obtain an intermediate product of the labeling complex; performing a coupling reaction on the signal generating substance and the intermediate product of the labeling complex, and separating, purifying and removing Unbound material yields labeled complexes.

[0063] Among them, the functional groups on the surface of the labeled protein include but are not limited to carboxyl (-COOH), amino (-NH 2 ), hydroxyl group (-OH), mercapto group (-SH), aldehyde group (-CHO), sugar group (-C=O) and imidazole group, functional group and free amino group, carboxyl group, mercapto group, aldehyde group or Glycosyl coupling (since the present invention has defined that the functional groups on the ...

Embodiment approach

[0064] According to a typical embodiment of the present invention, the specific steps of the preparation method are as follows: A. Select an appropriate method according to the coupling functional group to perform a coupling reaction between the labeled protein and the antigen; B. Remove unbound substances to obtain the intermediate product of the labeled complex ; C. Selecting an appropriate method according to the coupling functional group to perform a coupling reaction between the signal generating substance and the intermediate product of the labeling complex; D. Removing unbound substances to obtain the final labeling complex.

[0065] Select an appropriate method for the signal-generating substance according to the coupling functional group to perform a coupling reaction with the intermediate product of the labeling complex. The methods used for the coupling reaction include but are not limited to the mixed anhydride method, carbodiimide method, glutaraldehyde method, glut...

Embodiment 1

[0133] 1) Weigh 5 mg of HRP (M=44000) and dissolve it in 1 mL of distilled water (5 mg / mL).

[0134] 2) Add 0.5mL freshly prepared 0.06M NaIO to the above solution 4 (M=213.89) solution, mix well, and stand at 4°C in the dark for 30 minutes.

[0135] 3) Add 0.5mL 0.16M ethylene glycol (remove excess NaIO 4 ), mix well, and stand at room temperature for 30min.

[0136] 4) 1mg HIV-1 recombinant antigen (2mg / mL) was dialyzed with 0.05M pH 9.5 carbonate buffer at 4°C for 2h, then immediately added NaIO4 Oxidized HRP 1mg, stirred gently at room temperature for 2h in the dark.

[0137] 5) Add 50 μL of newly prepared 2mg / mL NaBH 4 (M=37.83) solution, after mixing, let stand at 4°C for 2h.

[0138] 6) Put the above solution into a dialysis bag, and dialyze against 0.01M pH7.4PBS at 4°C overnight.

[0139] 7) Choose a dialysis bag with a cut-off of 3500, measure the appropriate size, tie one end tightly after wetting, and test for leaks with purified water 3 times (it is better if...

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Abstract

Disclosed are a labelled complex, a preparation method therefor, a kit comprising the labelled complex and the use thereof, and a detection system comprising the kit. The labelled complex comprises: an antigen; a labelled protein coupled with the antigen to form an intermediate of the labelled complex; and a signal product coupled with the intermediate of the labelled complex to form the labelledcomplex.

Description

technical field [0001] The present invention relates to the field of in vitro diagnostic reagents, in particular to a labeling compound, its preparation method, a kit containing it and its application, and a detection system including the kit. Background technique [0002] At present, clinical immunoassay methods mainly include enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunocolloidal gold technique (GICT), radioimmunoprecipitation assay (RIPA), chemiluminescence assay (CLIA), etc. . ELISA and CLIA are suitable for clinical large-scale primary screening, but due to the instability or insufficient performance of reagent components, false positives and false negatives may occur in the test results. Therefore, it is necessary to further improve the sensitivity and stability of the reagent. [0003] ELISA is currently a commonly used detection technology in clinical blood screening. However, compared with the advantages of fully automatic operation and high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/576G01N33/531
CPCG01N33/531G01N33/56983G01N33/56988G01N33/5761G01N33/5767G01N33/5768G01N21/76G01N2333/02G01N2333/16G01N2333/20G01N2333/025G01N33/535G01N2469/20G01N33/532
Inventor 饶微方中刚袁锦云王燕梅李婷华王小莉
Owner SHENZHEN NEW INDS BIOMEDICAL ENG