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Method for detecting interaction of antibody and polypeptides by adopting capillary electrophoresis

A technology of capillary electrophoresis and antibody detection, which is applied in the field of biological analysis, can solve the problems of inability to analyze multivalent interactions between antibodies and polypeptides, is not suitable for antibody-polypeptide interactions, and cannot provide resolution, etc., and achieves easy operation and short time required , the effect of reducing the error

Inactive Publication Date: 2013-03-27
CHANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, SPR and ELISA usually need to immobilize antibody molecules, which brings certain difficulties in operation, and is not suitable for detecting antibody-polypeptide interactions in solution.
HPSEC chromatography is suitable for separating proteins, free peptides and protein-peptide complexes in solution, but it cannot provide higher resolution
Therefore, most of the current analytical methods can only measure the binding process of a single polypeptide and antibody, but cannot analyze the multivalent interaction between antibody and polypeptide

Method used

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  • Method for detecting interaction of antibody and polypeptides by adopting capillary electrophoresis
  • Method for detecting interaction of antibody and polypeptides by adopting capillary electrophoresis
  • Method for detecting interaction of antibody and polypeptides by adopting capillary electrophoresis

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The instrument and operating conditions used in the example are as follows:

[0035] The capillary electrophoresis used is a self-built capillary electrophoresis system based on a fluorescence microscope, and the high-voltage power supply is produced by the Shanghai Institute of Nuclear Physics; the fluorescence spectrometer is Ocean Optics QE65000; the microscope interface is self-made, and the fluorescence from the standard port of the microscope is introduced into the spectrometer through an optical fiber. Voltage injection was used, the injection voltage was 12 KV, the injection time was 10 s, the electrophoresis voltage was 18 KV, and the electrophoresis was 10 min.

[0036] figure 2 For fluorescence detection of M2 and FLAG by capillary electrophoresis 2 -Cy5 interaction. The experimental results show that M2 and FLAG 2 Electrophoretic spectrum of -Cy5 mixture and FLAG 2 The electrophoresis spectra of -Cy5 are basically the same. This is due to M2 with FLAG ...

Embodiment 2

[0038] The experimental conditions are the same as in Example 1, except that M2 and FLAG are detected by capillary electrophoresis 1 -FAM interactions such as image 3 shown. The experimental results show that two electrophoretic peaks appeared at 506 seconds (P3) and 910 seconds (P4). According to the peak time, P3 is M2? (FLAG 1 -FAM) 1 The electrophoretic peak of the complex, P4 is M2? (FLAG 1 -FAM) 2 Electrophoretic peaks of the complex. Integrate the electrophoretic peaks, S P3 / S Total 81.3%, S P4 / S Total It is 9.3%, so the binding process of M2 and polypeptide can be used according to this method, and the content of the complex generated by the reaction can be calculated.

Embodiment 3

[0040] The experimental conditions are the same as in Example 1, except that the detection of M2 and different concentrations of FLAG by capillary electrophoresis 1 -Cy5 interaction such as Figure 4 shown. The experimental results showed that M2 with different concentrations of FLAG 1 The electrophoresis pattern of -Cy5 is different after mixing. Therefore, the binding process of M2 to different concentrations of polypeptides can be detected by capillary electrophoresis. The concentration of the M2 polypeptide complex can be judged according to the electrophoresis peak, and the dissociation constant between M2 and the polypeptide can be obtained according to formula (1) (2). M2:FLAG 1 When -Cy5 is 1:1, it is calculated K D.1 0.01 μM; M2:FLAG 1 When -Cy5 is 1:2, it is calculated K D.2 0.35 μM.

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Abstract

The invention relates to a method for detecting interaction of an antibody and polypeptides by adopting capillary electrophoresis, belonging to the field of bioanalysis. The method comprises the steps of firstly, mixing the antibody with polypeptides of different proportions, then carrying out capillary electrophoresis detection, simultaneously detecting an appearance time and a fluorescence intensity of an antibody polypeptide compound and a fluorescence polypeptide, integrating an electrophoresis peak of the antibody polypeptide compound, converting an integral area into a compound concentration, and calculating a dissociation constant (KD) of the antibody and the polypeptide according to a formula. The method can also be used for detecting a competition process of the interaction of two exogenous polypeptides and the antibody. Experiments prove that the method has the advantages of simpleness in operation, high detection sensitivity, and short required time. The method is a novel high-sensitivity technology for detecting the interaction of the polypeptides and the antibody through improvement of the traditional technology of detecting the interaction of the polypeptides and the antibody and combination with the advantages of high sensitivity and the like of fluorescence detection.

Description

technical field [0001] The invention relates to the field of biological analysis, in particular to a method for detecting the interaction between an antibody and a polypeptide by capillary electrophoresis. Background technique [0002] Multivalent binding interactions play a very important role in the study of interactions between biomolecules in solution. For example, an IgG antibody has two antigen polypeptide binding sites, which can bind two antigens, which is a kind of bivalent binding. However, chromatographically resolving 1:1 and 1:2 IgG antibody-peptide complexes in solution is very challenging because differences in molecular weight, surface shape, and hydrophobicity are very close between the two. Therefore, it is very difficult to achieve the separation of two antibody-peptide complexes in different ratios. The FLAG tag polypeptide is a polypeptide fragment composed of 8 hydrophilic amino acids fused to the recombinant protein, which tends to be located on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
Inventor 王建浩王车礼邱琳蒋鹏举夏江
Owner CHANGZHOU UNIV
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