CN111830289B - Method for directly imaging biotinylated antibody-IgE immune complex by atomic force microscopy - Google Patents
Method for directly imaging biotinylated antibody-IgE immune complex by atomic force microscopy Download PDFInfo
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- CN111830289B CN111830289B CN202010719926.1A CN202010719926A CN111830289B CN 111830289 B CN111830289 B CN 111830289B CN 202010719926 A CN202010719926 A CN 202010719926A CN 111830289 B CN111830289 B CN 111830289B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
- G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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Abstract
The invention relates to the technical field of direct imaging of antigen-antibody immune complexes, in particular to a method for directly imaging biotinylated antibody-IgE immune complexes by using atomic force microscopy. The invention creatively discovers that the morphology of the biotinylated antibody-IgE immune complex can be analyzed by utilizing atomic force microscopy, and the biotinylated antibody and the IgE in a single immune complex can be distinguished through the morphology. Compared with the traditional method, the method provided by the invention is simple, the sample consumption is less, and the binding condition of the biotinylated antibody and the IgE can be visually judged on a nanoscale. Therefore, the morphological analysis is helpful for researching the antigen-antibody interaction from the molecular level, and provides a new way for the research and application of vaccines and targeted therapy. In addition, successful imaging of biotinylated antibody-IgE immune complexes also enables direct imaging of other immune complexes.
Description
Technical Field
The invention relates to the technical field of direct imaging of antigen-antibody immune complexes, in particular to a method for directly imaging biotinylated antibody-IgE immune complexes by using Atomic Force Microscopy (AFM).
Background
The antigen-antibody combination research has important significance in the aspects of medical diagnosis, vaccine development, disease mechanism analysis, targeted therapy and the like. In recent years, the primary method of detecting antigen-antibody binding has been immunoassay, but this method uses a number of biochemical reagents in the detection process. The wide use of biochemical reagents not only requires cumbersome processes, but also causes serious environmental pollution.
AFM is a multifunctional biomolecule research technique with particularly high spatial resolution and extremely low invasiveness, and has attracted more and more attention in recent years. However, no method for imaging biotinylated antibody-IgE immune complexes using AFM has been reported. In fact, the sensitivity of conventional immunological techniques is not high enough to be far from the sensitivity of AFM. For the detection of IgE concentration, the ELISA method has a minimum detectable IgE concentration of 0.24ng ML -1 . In the invention, single-molecule detection of IgE can be realized by AFM. In addition, the traditional immunological method needs much more samples than AFM, and the practical application of the immunological method is limited because samples such as blood, cerebrospinal fluid and the like of infants are not easily obtained. More importantly, the fatal disadvantage of immunological techniques is the inability to provide visual images of antigen-antibody binding. The present invention can directly see the image of antigen-antibody binding by AFM.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for directly imaging a biotinylated antibody-IgE immune complex by using atomic force microscopy.
In order to achieve the purpose, the technical scheme of the invention is as follows: the biotinylated antibody used in the present invention is an IgG antibody linked to biotin, and can specifically bind to IgE. The invention discovers that the biotinylated antibody has one 'small tail' more than IgE, so that whether the biotinylated antibody is combined with the IgE can be directly judged according to the morphology.
1. A method for directly imaging biotinylated antibody-IgE immune complexes by AFM comprises the following steps:
a) Dropwise adding MgCl onto the surface of freshly cracked mica 2 ·6H 2 O solution and incubation;
b) To MgCl 2 ·6H 2 Dripping IgE solution on the mica surface after the incubation of the O solution for continuous incubation;
c) Exposing the mica surface covered with the IgE solution to a biotinylated antibody solution;
d) The mica surface that had been exposed to the biotinylated antibody solution was topographically tested using atomic force microscopy.
Preferably, the incubation time in the step A is 0.5 min-2 min.
Preferably, the incubation time in the step B is 1 min-10 min.
Preferably, the exposure time in the step C is 50min to 70min.
Preferably, the step D of performing the morphology test specifically comprises the following steps:
a1 Washing and drying the mica surface exposed in the biotinylated antibody solution;
a2 The surface of the blow-dried and cleaned mica was subjected to morphological analysis using atomic force microscopy in tapping mode.
Drawings
FIG. 1 is an AFM image of biotinylated antibody-IgE immune complexes prepared according to the present invention.
FIG. 2 is an AFM image of biotinylated antibodies made according to the invention.
FIG. 3 shows an AFM of IgE produced by the present invention.
Detailed Description
In order to facilitate an understanding of the present invention, the present invention is described in further detail below with reference to examples and the accompanying drawings, but embodiments of the present invention are not limited thereto and are provided for the purpose of making the disclosure of the present invention more thorough and complete.
Example 1
Firstly MgCl is dripped on the surface of freshly cracked mica 2 ·6H 2 O solution and incubation for 1min; then to MgCl 2 ·6H 2 Dripping IgE solution on the surface of the mica incubated by the O solution and continuing incubation for 5min; the mica surface covered with IgE solution was then exposed to biotinylated antibody solution for 60min; finally, the mica surface exposed in the biotinylated antibody solution was subjected to a topography test using atomic force microscopy.
Example 2
This example is the same as example 1 except that NaCl is used instead of MgCl 2 ·6H 2 And O, and incubating for 1min.
Example 3
This example is the same as example 1 except that KCl is used instead of MgCl 2 ·6H 2 And O, and incubating for 1min.
Example 4
This example is the same as example 1 except that NiCl was used 2 ·6H 2 O in place of MgCl 2 ·6H 2 And O, and incubating for 1min.
Example 5
This example is the same as example 1 except for the following features 2 ·6H 2 The incubation time of O on the mica surface was 0.5min.
Example 6
This example is the same as example 1 except for the following features 2 ·6H 2 The incubation time of O on the mica surface is 2min.
Example 7
This example is the same as example 1 for the IgE solution in MgCl, except for the following features 2 ·6H 2 The mica surface after the O solution incubation is continuously incubated for 1min.
Example 8
This example is the same as example 1 for the IgE solution in MgCl, except for the following features 2 ·6H 2 The mica surface after incubation in O solution is incubated for 10min.
Example 9
This example is the same as example 1 except that the mica surface covered with the IgE solution was exposed to the biotinylated antibody solution for 50min.
Example 10
This example is identical to example 1 except that the mica surface covered with the IgE solution was exposed to the biotinylated antibody solution for a period of 70min.
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several modifications and variations can be made, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A method for direct imaging of biotinylated antibody-IgE immune complexes using atomic force microscopy, comprising the steps of:
a) Dropwise adding MgCl on the surface of freshly cracked mica 2 ·6H 2 O solution and incubation;
b) To MgCl 2 ·6H 2 1ng mL of mica surface after O solution incubation is dripped -1 IgE solution of (a), and continuing incubation;
c) Exposing the mica surface covered with the IgE solution to a biotinylated antibody solution;
d) Carrying out morphology test on the surface of the mica exposed in the biotinylated antibody solution by using atomic force microscopy;
the incubation time in the step A is 0.5 min-2 min;
the incubation time in the step B is 1 min-10 min;
the exposure time in the step C is 50-70 min;
the step D of carrying out the morphology test specifically comprises the following steps:
d1 Washing and drying the mica surface exposed in the biotinylated antibody solution;
d2 Single protein molecular morphology analysis was performed on the blow-dried cleaned mica surface using atomic force microscopy in tapping mode.
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| CN107328939A (en) * | 2017-08-23 | 2017-11-07 | 武汉大学 | A kind of enzyme-linked immunosorbent assay for measuring spent based on biotinylated antigen or antibody inorganic salts hybridized nanometer |
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| DE10126798A1 (en) * | 2001-06-01 | 2002-12-19 | Nanotype Gmbh | Highly parallel determination of analytes, useful e.g. for diagnostic detection of proteins, by immobilization then contacting with surface carrying specific probes |
| EP2276500A4 (en) * | 2008-03-13 | 2015-03-04 | Univ Yale | Reactivation of axon growth and recovery in chronic spinal cord injury |
| CN102183679B (en) * | 2011-03-16 | 2012-12-19 | 中国科学院合肥物质科学研究院 | In-situ quasi synchronous detection method for detecting physicochemical properties of micro and nano structures |
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| CN105699699A (en) * | 2016-04-07 | 2016-06-22 | 中国科学院上海应用物理研究所 | Sample preparation method employing AFM (Atomic Force Microscope) for single antibody molecule imaging |
| CN107328939A (en) * | 2017-08-23 | 2017-11-07 | 武汉大学 | A kind of enzyme-linked immunosorbent assay for measuring spent based on biotinylated antigen or antibody inorganic salts hybridized nanometer |
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