CN113834932A - Protein chip for detecting autoimmune antibody of diabetes, preparation and detection method thereof - Google Patents
Protein chip for detecting autoimmune antibody of diabetes, preparation and detection method thereof Download PDFInfo
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- CN113834932A CN113834932A CN202111147467.5A CN202111147467A CN113834932A CN 113834932 A CN113834932 A CN 113834932A CN 202111147467 A CN202111147467 A CN 202111147467A CN 113834932 A CN113834932 A CN 113834932A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Abstract
The invention relates to the technical field of protein chips and discloses a protein chip for detecting autoimmune antibodies of diabetes, which comprises a titration plate, a filter membrane and a glass slide, wherein the filter membrane is arranged on the titration plate, the protein is dripped on the filter membrane, the glass slide is arranged on the filter membrane and is adhered with the titration plate to form an antibody array, the chip contains reaction sites defined for various analyses, the flat chip can simultaneously detect a plurality of immobilized proteins in a mixture consisting of the protein, metabolites and other molecules through a ligand in a soluble stage, and redundant information can be generated by adopting a multiplex assay method. The most valuable function of an autoantibody chip is that it can generate a profile of autoantibodies. The result of the multiplexing chip is analyzed by computer spectrum recognition software, so that differential diagnosis of diseases is performed, the diagnosis accuracy can be improved, and the analysis flux and cost benefit can be improved.
Description
Technical Field
The invention relates to the technical field of protein chips, in particular to a protein chip for detecting autoimmune antibodies of diabetes, a preparation method and a detection method thereof.
Background
The protein chip is a high-throughput monitoring system, and the interaction between protein molecules is monitored through the interaction between target molecules and capture molecules. Capture molecules are usually pre-immobilized on the chip surface and are widely used as capture molecules due to the high specificity and strong binding property of antibodies to antigens. The research on protein chips is very critical to effectively fix antibodies on the chip surface, and particularly, the research on protein chips is very critical to enhance the sensitivity of protein chips in terms of the consistency of the fixed antibodies. The G protein is an antibody binding protein that specifically binds to antibody FC fragments and has therefore been widely used to immobilize different types of antibodies.
Protein chips are more difficult to develop than nucleic acid chips. This is because proteins differ greatly in molecular size, charge, shape, hydrophobicity, type and degree of post-translational formation of genes, and quaternary structure, and it is difficult for some proteins to retain specificity and activity after attachment to a solid support. Simultaneous detection of self-antigens, as with various immunoassay methods, requires a reasonably set, reproducible, tunable and quantitative antigen. There are problems in the measurement of autoantigens using the chip technology, including how to obtain the expression of functional proteins required for chip construction, development of a technique for attaching proteins to the surface of a carrier and maintaining their activities, and simultaneous achievement of high analytical sensitivity and large dynamic range by chemical and detection systems. Assay normalization, data interpretation and storage are also possible.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a protein chip for detecting autoimmune antibodies of diabetes, a preparation method and a detection method thereof.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: the protein chip for detecting the autoimmune antibody of the diabetes comprises a titration plate, a filter membrane and a glass slide, and is characterized in that: the filter membrane is placed on the titration plate, the protein is dripped on the filter membrane, the glass slide is placed on the filter membrane, and the glass slide is attached to the titration plate to form an antibody array;
antibody arrays are stained with fluorescent antibiosomes on the antibody array and encoded by a fluorescent pattern.
Preferably, the protein chip is a chip for detecting, by classifying each antibody array according to a distribution region set in advance by a digital signal processing technique, and the respective proteins in each antibody array are sequentially immobilized on various media carriers such as a slide glass.
Preferably, the fluorescence intensity of each array on the chip analyzes the relationship between proteins to determine the expression function of each gene.
Preferably, the specification of the titer plate and the glass slide is 18mm multiplied by 60mm, the thickness is 2mm to 3mm, and the reaction tanks are squares with the side length of 0.2mm to 18 mm.
Preferably, the first step: selection and preparation of antigen: self-preparing nuclear antigens and proteins;
the second step is that: selection and preparation of the slide and selection of the sample solution: preparing five different fluorescence-modified slides respectively;
the third step: the preparation of the protein chip and the optimization of the detection condition adopt an Lg (34) orthogonal method, a ScanArray4000 laser copolymerization scanner is used for imaging, QuantArray software is used for analyzing the fluorescence intensity, and the optimal condition is screened according to the result;
the fourth step: different probe molecules are fixed on the antibody array to form an antibody array protein chip system;
the fifth step: reacting the antibody array of the labeled probe with a sample;
and a sixth step: and (5) detecting a reaction result.
Preferably, the repeatability and stability test of the chip prepares a chip capable of simultaneously detecting 7 kinds of autoantibodies according to the above results, and detects the repeatability and stability of the chip, comprising the following detection steps:
1)1:2 diluting the serum to be detected, hybridizing the diluted serum with a chip in a hybridization box at 37 ℃ for 15min, and sealing the hybridization box with a sealant at 37 ℃ for 15 min;
2) PBS wash 3 times, 15s each;
3) adding Cy3 labeled goat anti-human IgG with the concentration of 33 mug/ml, and incubating in a hybridization box for 30 min;
4) washing 3 times, each time 15s, drying and scanning, and finishing the whole experiment within 1 hour.
Preferably, the peptides in the antigen are derived from heat shock proteins, tissue antigens, immune system components, structural antigens, hormones, enzymes, plasma proteins, synthetic oligonucleotides, and bacterial antigens, respectively.
(III) advantageous effects
Compared with the prior art, the invention provides a protein chip for detecting the autoimmune antibody of diabetes, a preparation method and a detection method thereof, and the protein chip has the following beneficial effects:
1. the diabetes autoimmune antibody detection protein chip and the preparation and detection method thereof adopt a two-dimensional microchip, the chip contains reaction sites defined for various analyses, and the flat chip can simultaneously detect a plurality of immobilized proteins in a mixture consisting of proteins, metabolites and other molecules through a ligand at a soluble stage.
2. According to the diabetes autoimmune antibody detection protein chip, the preparation method and the detection method thereof, each array antibody device is independently processed in an optimal mode, and then different array antibody devices are combined to prepare a final multiplexing array antibody reagent. A large number of analytes can be accommodated in a chip assay, and thus multiple internal controls can be used to ensure the desired performance of the assay system. All assay signals were normalized using a fluorescent internal standard. This standardization compensates for fluctuations in the illumination and detection systems, and also allows for the design of other internal assays in the autoimmune assay based on the corresponding clinical specimen type and sample loading volume.
3. The diabetes autoimmune antibody detection protein chip, the preparation method and the detection method thereof adopt a multiplex determination method to generate redundant information. The most valuable function of an autoantibody chip is that it can generate a profile of autoantibodies. The result of the multiplexing chip is analyzed by computer spectrum recognition software, so that differential diagnosis of diseases is performed, the diagnosis accuracy can be improved, and the analysis flux and cost benefit can be improved.
4. The diabetes autoimmune antibody detection protein chip, the preparation method and the detection method thereof provide important basis for the typing identification of immune-mediated diabetes patients by determining the existence or the content of the diabetes autoimmune antibody in serum, and can also be widely used for the prediction of the early clinical stage and the early onset stage of the type I diabetes in diabetes high risk groups, thereby having important clinical practical value.
5. The diabetes autoimmune antibody detection protein chip, the preparation method and the detection method thereof have stable quality control. The highly purified antigen protein ensures the stability of the antigen quality, and reduces the batch-to-batch difference, thereby effectively realizing the detection standardization and reducing the fluctuation of the detection result. Meanwhile, each chip is provided with an independent quality control detection microarray, so that the quality reliability of each chip is ensured.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The protein chip for detecting the autoimmune antibody of the diabetes comprises a titration plate, a filter membrane and a glass slide, wherein the filter membrane is arranged on the titration plate, the protein is dripped on the filter membrane, the glass slide is arranged on the filter membrane, and the glass slide is attached to the titration plate to form an antibody array; the protein chip classifies each antibody array according to a preset distribution area by a digital signal processing technology, various proteins in each antibody array are orderly fixed on various medium carriers such as a glass slide and the like to form a chip for detection, the fluorescence intensity of each array on the chip analyzes the interaction relationship between the proteins so as to achieve the purpose of measuring various gene expression functions, the titer plate and the glass slide have the specification of 18mm multiplied by 60mm, the thickness is 2mm to 3mm, and the reaction cells are squares with the side length of 0.2mm to 18 mm.
A protein chip for detecting autoimmune antibodies of diabetes and a preparation method thereof comprise the following steps: selection and preparation of antigen: the nuclear antigen and the protein are prepared by self, the antigens aiming at seven autoantibodies are selected according to the requirement of the current clinical detection, namely ANA, dsDNA, Ro-60/SSA, Ro52/SSA, La/SSB, Jo-1 and cardiolipin, the nuclear protein is extracted from the epithelial cell of the human laryngeal cancer as the nuclear antigen, the protein concentration of the nuclear antigen is measured, plasmid DNA is extracted from a prokaryotic expression vector pcDNAII as the antigen for detecting the anti-dsDNA antibody, and the antigen does not contain ssDNA through agarose electrophoresis verification. The self-prepared antigen can meet the requirement of antibody detection,
the second step is that: selection and preparation of the slide and selection of the sample solution: five different fluorescence-modified glass slides are respectively prepared, and the screened APES-modified glass slide can be used as a solid phase substrate to fix protein, nucleic acid and lipid simultaneously, so that the method is very suitable for the characteristic of antigen diversification and fixation of an autoimmune disease antibody detection chip; the screened PBST can be used as a sample solution, so that the antigen can be well fixed, the detection sensitivity can be improved, and the lowest detection amount can reach 50 pg. The cardiolipin spotting fluid was DMSO.
The third step: the preparation and detection conditions of the protein chip are optimized by an Lg (34) orthogonal method, a ScanArray4000 laser copolymerization scanner is used for imaging, QuantArray software is used for analyzing fluorescence intensity, the optimal conditions are screened according to results, and the preparation and detection conditions of the protein chip screened by an orthogonal design test are that the protein chip is incubated for 2 hours in a hybridization box at 37 ℃; washing with PBS; blocking with PBST containing 1% BSA and 2.5% sucrose for 30 min; reacting with a secondary antibody for 30min, and carrying out orthogonal design test to screen out the secondary antibody and serum under the reaction conditions of serum dilution 1: 2; the concentration of Cy 3-labeled goat anti-human IgG was 33 pug/ml; the serum reaction time was 15 min.
The fourth step: different probe molecules are fixed on an antibody array to form an antibody array protein chip system, the array antibody matrix of the labeled probe is reacted with a sample, the probe can be specifically combined with corresponding target molecules, and a reporter molecule with green report fluorescence is also specifically combined with the target molecules to quantify the reaction.
The fifth step: reacting the antibody array of the labeled probe with a sample;
and a sixth step: and (5) detecting a reaction result.
The repeatability and stability experiment of the chip capable of simultaneously detecting 7 kinds of autoantibodies is prepared according to the results, and the repeatability and stability of the chip are detected, which comprises the following detection steps:
1) diluting the serum to be detected, hybridizing the diluted serum with a chip at 37 ℃ for 15min in a hybridization box in a mode of 1:2, 1:3, 1:4 and 1:5, sealing the diluted serum with a sealant at 37 ℃ for 15min to form four detection solutions with different concentrations, selecting two specific reaction tanks, and attaching two additional quality control reaction tanks to the chip to detect the quality of the chip. The result judgment criteria are: if the ratio of the average fluorescence intensity of the protein spot sample points on each detection piece of the positive quality control detection microarray to the background fluorescence intensity is higher than 50: 1, and the ratio of the average fluorescence intensity of the protein spot sample points on each detection piece of the negative quality control detection microarray to the background fluorescence intensity is lower than 50: 1, the quality of the chip is good, and the detection result is effective;
2) PBS wash 3 times, 15s each;
3) adding Cy3 labeled goat anti-human IgG with the concentration of 33 mug/ml, and incubating in a hybridization box for 30 min;
4) washing 3 times, each time 15s, drying and scanning, and finishing the whole experiment within 1 hour.
The peptides in the antigen are respectively from heat shock protein, tissue antigen, immune system components, structural antigen, hormone, enzyme, plasma protein, synthetic oligonucleotide and bacterial antigen, and the antigen concentration and serum dilution result is nuclear antigen of 300 ug/ml; dsDNA,200 ug/ml; Ro-60/SSA,100 ug/ml; Ro-52/SSA,2004 g/ml; La/SSB,300 ug/ml; jo-1,200 ug/ml; cardiolipin, 2000 μ g/ml; serum was diluted 1: 2.
Through comparison of the point sample solution and the serum dilution, the serum dilution with the highest signal-to-noise ratio is 1:2, the screened TBST containing 0.1% Tween20 is used as the point sample solution, the antigen can be well fixed, the detection sensitivity can be improved, the concentration-gray value linear relation of HumanIgG and 12 antigens (the point sample solution is the TBST containing 0.1% Tween20, and the serum dilution is 1: 2) of the protein chip is better, R2 is greater than 0.97, the linear relation is better, and the protein chip can perform qualitative and semi-quantitative detection on corresponding antibodies in serum under the antigen concentration in the range selected by people.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The protein chip for detecting the autoimmune antibody of the diabetes comprises a titration plate, a filter membrane and a glass slide, and is characterized in that: the filter membrane is placed on the titration plate, the protein is dripped on the filter membrane, the glass slide is placed on the filter membrane, and the glass slide is attached to the titration plate to form an antibody array;
antibody arrays are stained with fluorescent antibiosomes on the antibody array and encoded by a fluorescent pattern.
2. The protein chip for detecting autoimmune antibodies in diabetes mellitus according to claim 1, wherein: the protein chip classifies antibody arrays according to a preset distribution region by a digital signal processing technology, and various proteins in each antibody array are orderly fixed on various medium carriers such as a glass slide to become a chip for detection.
3. The protein chip for detecting autoimmune antibodies in diabetes mellitus according to claim 1, wherein: the fluorescence intensity of each array on the chip analyzes the interaction relationship between the proteins, thereby achieving the purpose of determining the expression function of each gene.
4. The method for preparing the protein chip for detecting autoimmune antibodies in diabetes mellitus according to claim 1, wherein the protein chip comprises: the specification of the titer plate and the glass slide is 18mm multiplied by 60mm, the thickness is 2mm to 3mm, and the reaction tank is a square with the side length of 0.2mm to 18 mm.
5. The method for preparing the protein chip for detecting autoimmune antibodies in diabetes mellitus according to claim 1, wherein the protein chip comprises:
the first step is as follows: selection and preparation of antigen: self-preparing nuclear antigens and proteins;
the second step is that: selection and preparation of the slide and selection of the sample solution: preparing five different fluorescence-modified slides respectively;
the third step: the preparation of the protein chip and the optimization of the detection condition adopt an Lg (34) orthogonal method, a ScanArray4000 laser copolymerization scanner is used for imaging, QuantArray software is used for analyzing the fluorescence intensity, and the optimal condition is screened according to the result;
the fourth step: different probe molecules are fixed on the antibody array to form an antibody array protein chip system;
the fifth step: reacting the antibody array of the labeled probe with a sample;
and a sixth step: and (5) detecting a reaction result.
6. The method for detecting a protein chip for detecting autoimmune antibodies in diabetes according to claim 1, wherein: the repeatability and stability experiment of the chip capable of simultaneously detecting 7 kinds of autoantibodies is prepared according to the results, and the repeatability and stability of the chip are detected, which comprises the following detection steps:
1)1:2 diluting the serum to be detected, hybridizing the diluted serum with a chip in a hybridization box at 37 ℃ for 15min, and sealing the hybridization box with a sealant at 37 ℃ for 15 min;
2) PBS wash 3 times, 15s each;
3) adding Cy3 labeled goat anti-human IgG with the concentration of 33 mug/ml, and incubating in a hybridization box for 30 min;
4) washing 3 times, each time 15s, drying and scanning, and finishing the whole experiment within 1 hour.
7. The method of claim 5, wherein the protein chip for detecting autoimmune antibodies in diabetes mellitus comprises: the peptides in the antigen are derived from heat shock proteins, tissue antigens, immune system components, structural antigens, hormones, enzymes, plasma proteins, synthetic oligonucleotides, and bacterial antigens, respectively.
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| CN202111147467.5A CN113834932A (en) | 2021-09-29 | 2021-09-29 | Protein chip for detecting autoimmune antibody of diabetes, preparation and detection method thereof |
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