CN110618264A - Method for detecting anti-AQP 4 antibody based on quantum dot polystyrene microspheres - Google Patents

Abstract

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, which combines a new type of quantum dot nanoprobe with flow cytometry analysis technology for the detection of self-anti-AQP4 antibodies, breaking through the traditional method of using organic Fluorescent dyes are the bottleneck of detection tools. At the same time, the advantages of rapid detection, easy standardization and automation of flow cytometry analysis technology are used to make up for the shortcomings of the existing AQP4 antibody detection technology. Simple, rapid and reproducible detection of AQP4 antibodies.

Description

Method for detecting anti-AQP4 antibody based on quantum dot polystyrene microspheres

technical field

The invention relates to the fields of bioanalytical chemistry and nano-biotechnology, in particular to a method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres.

Background technique

Human autoantibodies are important serological markers of autoimmune diseases, and their rapid detection has important application value for the diagnosis, treatment and prognosis evaluation of autoimmune diseases. With the improvement of the sensitivity and specificity of autoantibody detection methods, the diagnostic criteria of related autoimmune diseases are also updated and adjusted accordingly.

Neuromyelitis optica (NMO) is an acute or subacute inflammatory demyelinating lesion involving the optic nerve and spinal cord simultaneously or sequentially. In 2004, Lennon et al first found NMO-IgG antibodies in the serum of NMO patients, that is, anti-aquaporin 4 (AQP4) antibodies. AQP4 is the main aquaporin in the central nervous system, located on the foot processes of astrocytes, and is the main target of NMO-IgG. AQP4 antibody enters the central nervous system through the permeable part of the blood-brain barrier, encounters astrocytes and causes cell-dependent cytotoxic responses, astrocyte foot processes are degraded by NMO-IgG and complement, and then activate giant cells Phages, eosinophils, and neutrophils produce a series of cytokines, oxygen free radicals, etc., which cause damage to blood vessels and parenchyma, and eventually lead to damage to white and gray matter including axons and oligodendrocytes. In 2015, serum anti-AQP4 antibody was included in the diagnostic criteria of NMO as a supporting condition.

At present, the commonly used detection methods for clinically detecting AQP4 mainly include the following two types: (1) Indirect immunofluorescence assay (IIF) based on mouse brain tissue sections with high expression of AQP4 protein. Although this method has high sensitivity, However, the operation is complex, the production of tissue slices is highly demanding, quality control is difficult, and it requires inspectors to have rich neurophysiological knowledge to accurately determine the test results. (2) A cell-based assay (CBA) based on cells successfully expressing AQP4 protein. This method has relatively high specificity. Low expression levels affect the interpretation of test results, and due to inconsistent transfection efficiencies, it is difficult to establish uniform quality control standards, making it difficult to achieve large-scale commercial production.

Therefore, in view of the deficiencies of the prior art, it is necessary to provide a method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres to overcome the deficiencies of the prior art.

Contents of the invention

The purpose of the present invention is to avoid the shortcomings of the prior art and provide a method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, which has the characteristics of simple and fast preparation process and repeatable detection of AQP4 antibodies.

The object of the present invention is achieved through the following technical measures.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres is provided, comprising the following steps:

(1) Preparation of quantum dot polystyrene QPs microspheres;

(2) obtaining the functional fragment target gene of AQP4 protein for expression;

(3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

(4) Flow cytometric detection by oil-soluble CdSe quantum dots with a fluorescence emission peak of 600nm to 650nm and FITC-labeled goat anti-human IgG red-green dual fluorescence localization system, and the detection results of the functionalized QPs microspheres to read.

Preferably, the above step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

(1.1) Disperse and suspend 1 mg to 10 mg of PS microspheres in 0.5 mL to 1 mL of a saturated n-butanol solution with a concentration of 99.99%, to prepare a PS microsphere suspension;

(1.2) Dissolve 0.1 mg to 1 mg of quantum dot QD in 50 μL to 100 μL of 99.99% saturated dichloromethane solvent and shake and mix to make QD-dichloromethane solution;

(1.3) Add the QD-dichloromethane solution dropwise to the PS microsphere suspension at a drop rate of 2.5 μL/sec to 3.5 μL/sec, and shake at room temperature for 4 to 5 hours;

(1.4) Heat the shaken mixture in a water bath at a temperature of 45°C to 55°C for 20 to 25 hours;

(1.5) The mixture after the water bath was centrifuged and washed with an ethanol solution with a volume fraction of 20% to 80%, and the precipitate was collected to obtain QPs microspheres.

Preferably, the above step (2) specifically includes:

(2.1) artificially synthesize the target gene of the functional fragment of AQP4 protein, and insert the synthesized target gene into the pET32a plasmid vector to obtain the recombinant plasmid of AQP4-pET32a;

(2.2) Transform the recombinant plasmid of AQP4-pET32a into BL21 strain, and add IPTG inducer to induce expression;

(2.3) centrifuging the protein product obtained after the induced expression, and taking the centrifuged centrifuged supernatant and precipitate respectively for identification by polyacrylamide gel electrophoresis;

(2.4) The protein product obtained after the induced expression was purified through a nickel column, and identified again by western-blotting.

Preferably, the above step (2.2) specifically includes the following steps:

Mix 0.1 μg to 0.5 μg AQP4-pET32a recombinant plasmid with 50 μL to 100 μL BL21 strain with a volume fraction of 50% and mix in ice bath for more than 30 minutes, then place the mixed bacterial solution in water at 40°C to 45°C for 85 seconds to 95 seconds, then put it in an ice bath at 0°C for 2 to 3 minutes, rotate the mixed bacterial solution at a speed of 220 rpm for more than 45 minutes at a temperature of 35°C to 40°C, and inoculate the bacterial solution containing For ampicillin-based agar plates, incubate the agar plates at 35°C to 40°C for more than 24 hours.

Preferably, the above step (2.3) specifically includes the following steps: pick positive clones, rotate the culture medium at a speed of 220 rpm under an environment of 35°C to 40°C, incubate until the culture medium is slightly turbid, and separate the monoclonal bacterial liquid There are six tubes with 1mL bacterial solution in each tube, grouped according to the induction time and IPTG dilution ratio, where the induction time is divided into 4 hours and 6 hours, and the IPTG dilution ratio is divided into three types: 0, 1:1000, and 1:100 , the specific grouping is as follows:

1) The induction time is 4 hours, and the IPTG dilution ratio is 0 as a group;

2) The induction time is 4 hours, and the IPTG dilution ratio is 1:1000 as a group;

3) The induction time is 4 hours, and the IPTG dilution ratio is 1:100 as a group;

4) The induction time is 6 hours, and the IPTG dilution ratio is 0 as a group;

5) The induction time is 6 hours, and the IPTG dilution ratio is 1:1000 as a group;

6) The induction time is 6 hours, and the IPTG dilution ratio is 1:100 as a group.

Preferably, above-mentioned step (3) specifically comprises the following steps:

(3.1) Pretreatment of QPs microspheres: Take 0.1mg to 1mg of QPs microspheres and add them to 0.5mL to 1mL of MES buffer with a pH value of 6.1 and a concentration of 50mmol/L for mixing, and place the obtained mixture in Rotate for more than 5 minutes at 18,000 rpm for centrifugation;

(3.2) Activation of QPs microspheres: Reset the centrifuged product to 100 μL to 500 μL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L, and add 5 μg/μL to 20 μg/μL of EDC reagent Mix with 5 μg/μL to 20 μg/μL NHS reagent, and shake the mixed product for more than 30 minutes at room temperature;

(3.3) Termination of activation: place the shaken mixed product in an environment of 18,000 rpm for more than 5 minutes, perform centrifugation, and add 0.5mL to 1mL of PBS reagent to the centrifuged product for washing twice to 3 times;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

(3.4) QPs microspheres coupled with AQP4 protein: mix AQP4 protein and QPs microspheres in 0.5mL to 1mL of PBS reagent, and place the mixed product at a temperature of 3°C to 45°C overnight or at room temperature Shake for 2 to 4 hours under certain conditions to complete the operation of coupling AQP4 protein with QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

(3.5) Sealing: add BSA reagent with a mass concentration of 3% to 5% to the mixed product after shaking, and shake the mixture for more than 30 minutes at room temperature;

(3.6) Termination of the reaction: place the product obtained by adding BSA reagent with a mass concentration of 3% to 5% after shaking and place it at a speed of 18,000 rpm for more than 5 minutes for centrifugation, and add 0.5mL to the centrifuged product Wash with 1 mL of PBS reagent for 2 to 3 times, and finally add 100 μL to 500 μL of PBST reagent to resuspend the functionalized QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

Specifically, the pH range of the PBST reagent is 7.2 to 7.4, and the concentration is 0.01 mol/L.

Preferably, the basis for the interpretation of the above results are:

(4.1) The size of the QPs microspheres is uniform and its red fluorescence intensity is high, and there is no doped fluorescence, so it has excellent performance as a detection probe.

(4.2) The present invention uses an indirect method to form an immune complex composed of functionalized QPs microspheres-anti-AQP4 antibody-FITC-labeled goat anti-human IgG, and helps to interpret the detection results by means of a red-green dual fluorescent positioning system .

The flow immunological method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention uses quantum dot (Quantum dot, QD) nanomaterials to have high quantum yield, high fluorescence intensity, wide excitation spectrum range and narrow emission spectrum, Due to the advantages of long fluorescence lifetime, the method of combining new quantum dot nanoprobes with flow cytometry (FlowCytometry/on AQP4-QPs) is used to detect self-anti-AQP4 antibodies, which breaks through the traditional method of using organic fluorescent dyes as the basis. The bottleneck of detection tools; at the same time, it also utilizes the advantages of rapid detection, easy standardization and automation of flow cytometry technology, which makes up for the shortcomings of existing AQP4 antibody detection technology. It has simple, rapid and reproducible detection of AQP4 antibody.

Description of drawings

The present invention will be further described by using the accompanying drawings, but the content in the accompanying drawings does not constitute any limitation to the present invention.

Fig. 1 is a molar excitation spectrum and a fluorescence emission spectrum diagram of QPs microspheres of a method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention.

Fig. 2 is a western-blotting electrophoresis identification diagram of IPTG-induced AQP4 protein expression in a method for detecting anti-AQP4 antibody based on quantum dot polystyrene microspheres of the present invention.

Fig. 3 is a schematic diagram of the results of detecting anti-AQP4 antibodies in serum samples by flow cytometry.

Detailed ways

The present invention will be further described in conjunction with the following examples.

Example 1.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, as shown in Figures 1 to 3, comprising the following steps:

(1) Preparation of quantum dot polystyrene QPs microspheres;

(2) obtaining the functional fragment target gene of AQP4 protein for expression;

(3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

(4) Flow cytometric detection by oil-soluble CdSe quantum dots with a fluorescence emission peak of 600nm to 650nm and FITC-labeled goat anti-human IgG red-green dual fluorescence localization system, and the detection results of the functionalized QPs microspheres to read.

Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

(1.1) Disperse and suspend 1 mg to 10 mg of PS microspheres in 0.5 mL to 1 mL of a saturated n-butanol solution with a concentration of 99.99%, to prepare a PS microsphere suspension;

(1.2) Dissolve 0.1 mg to 1 mg of quantum dot QD in 50 μL to 100 μL of 99.99% saturated dichloromethane solvent and shake and mix to make QD-dichloromethane solution;

(1.3) Add the QD-dichloromethane solution dropwise to the PS microsphere suspension at a drop rate of 2.5 μL/sec to 3.5 μL/sec, and shake at room temperature for 4 to 5 hours;

(1.4) Heat the shaken mixture in a water bath at a temperature of 45°C to 55°C for 20 to 25 hours;

(1.5) The mixture after the water bath was centrifuged and washed with an ethanol solution with a volume fraction of 20% to 80%, and the precipitate was collected to obtain QPs microspheres.

Step (2) specifically includes:

(2.1) artificially synthesize the target gene of the functional fragment of AQP4 protein, and insert the synthesized target gene into the pET32a plasmid vector to obtain the recombinant plasmid of AQP4-pET32a;

(2.2) Transform the recombinant plasmid of AQP4-pET32a into BL21 strain, and add IPTG inducer to induce expression;

(2.3) centrifuging the protein product obtained after the induced expression, and taking the centrifuged centrifuged supernatant and precipitate respectively for identification by polyacrylamide gel electrophoresis;

(2.4) The protein product obtained after the induced expression was purified through a nickel column, and identified again by western-blotting.

Wherein, step (2.2) specifically comprises the following steps:

Mix 0.1 μg to 0.5 μg AQP4-pET32a recombinant plasmid with 50 μL to 100 μL BL21 strain with a volume fraction of 50% and mix in ice bath for more than 30 minutes, then place the mixed bacterial solution in water at 40°C to 45°C for 85 seconds to 95 seconds, then put it in an ice bath at 0°C for 2 to 3 minutes, rotate the mixed bacterial solution at a speed of 220 rpm for more than 45 minutes at a temperature of 35°C to 40°C, and inoculate the bacterial solution containing For ampicillin-based agar plates, incubate the agar plates at 35°C to 40°C for more than 24 hours.

Wherein, the step (2.3) specifically includes the following steps: picking positive clones, specifically rotating the culture medium at a speed of 220 rpm under an environment of 35°C to 40°C, incubating until the culture medium is slightly turbid, and separating the monoclonal bacterial liquid There are six tubes with 1mL bacterial solution in each tube, grouped according to the induction time and IPTG dilution ratio, where the induction time is divided into 4 hours and 6 hours, and the IPTG dilution ratio is divided into three types: 0, 1:1000, and 1:100 , the specific grouping is as follows:

1) The induction time is 4 hours, and the IPTG dilution ratio is 0 as a group;

2) The induction time is 4 hours, and the IPTG dilution ratio is 1:1000 as a group;

3) The induction time is 4 hours, and the IPTG dilution ratio is 1:100 as a group;

4) The induction time is 6 hours, and the IPTG dilution ratio is 0 as a group;

5) The induction time is 6 hours, and the IPTG dilution ratio is 1:1000 as a group;

6) The induction time is 6 hours, and the IPTG dilution ratio is 1:100 as a group.

Wherein, step (3) specifically comprises the following steps:

(3.1) Pretreatment of QPs microspheres: Take 0.1mg to 1mg of QPs microspheres and add them to 0.5mL to 1mL of MES buffer with a pH value of 6.1 and a concentration of 50mmol/L for mixing, and place the obtained mixture in Rotate for more than 5 minutes at 18,000 rpm for centrifugation;

(3.2) Activation of QPs microspheres: Reset the centrifuged product to 100 μL to 500 μL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L, and add 5 μg/μL to 20 μg/μL of EDC reagent Mix with 5 μg/μL to 20 μg/μL NHS reagent, and shake the mixed product for more than 30 minutes at room temperature;

(3.3) Termination of activation: place the shaken mixed product in an environment of 18,000 rpm for more than 5 minutes, perform centrifugation, and add 0.5mL to 1mL of PBS reagent to the centrifuged product for washing twice to 3 times;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

(3.4) QPs microspheres coupled with AQP4 protein: mix AQP4 protein and QPs microspheres in 0.5mL to 1mL of PBS reagent, and place the mixed product at a temperature of 3°C to 45°C overnight or at room temperature Shake for 2 to 4 hours under certain conditions to complete the operation of coupling AQP4 protein with QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

(3.5) Sealing: add BSA reagent with a mass concentration of 3% to 5% to the mixed product after shaking, and shake the mixture for more than 30 minutes at room temperature;

(3.6) Termination of the reaction: place the product obtained by adding BSA reagent with a mass concentration of 3% to 5% after shaking and place it at a speed of 18,000 rpm for more than 5 minutes for centrifugation, and add 0.5mL to the centrifuged product Wash with 1 mL of PBS reagent for 2 to 3 times, and finally add 100 μL to 500 μL of PBST reagent to resuspend the functionalized QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2 to 7.4, and the concentration is 0.01mol/L;

Specifically, the pH range of the PBST reagent is 7.2 to 7.4, and the concentration is 0.01 mol/L.

The results are based on:

(4.1) The size of the QPs microspheres is uniform and its red fluorescence intensity is high, and there is no doped fluorescence, so it has excellent performance as a detection probe.

(4.2) The present invention uses an indirect method to form an immune complex composed of functionalized QPs microspheres-anti-AQP4 antibody-FITC-labeled goat anti-human IgG, and helps to interpret the detection results by means of a red-green dual fluorescent positioning system .

The invention adopts the technique of combining the novel quantum dot probe with the flow cytometry analysis technique, and breaks through the traditional bottleneck of using organic fluorescent dyes as detection tools. On the one hand, the use of QD nanomaterials as a detection tool overcomes the shortcomings of organic fluorescent dyes such as weak fluorescence intensity, short fluorescence lifetime and easy quenching. At the same time, the use of QPs microspheres as a detection carrier can effectively avoid the inconsistency of detection results caused by batch differences On the other hand, analyzing the molecular structure of AQP4 protein, the use of prokaryotic expression system can realize large-scale production, which is conducive to commercialization; finally, combined with fast and sensitive flow cytometry analysis technology, it can quickly and accurately realize the detection of single QPs-AQP4 microsphere probe The detection greatly improves the sensitivity and accuracy of the detection, and is beneficial to the automation and standardization of the detection of self-anti-AQP4 antibodies.

The present invention cites the common method of fluorescent microspheres to prepare chemical osmosis to improve the oil-soluble QD, and to increase the water solubility of the QD by wrapping PS microspheres on the surface of the QD. Therefore, the present invention uses n-butanol as the dispersion system and dichloromethane as the dispersion system. The swelling agent is easy to operate, and the prepared fluorescent microspheres have stable properties and high fluorescence intensity.

The invention constructs a stable AQP4 protein expression system, which overcomes the limitation of the method relying on tissues or cells with high protein expression as matrix. At the same time, the present invention uses EDC-NHS to couple quantum dot polystyrene microspheres QPs and AQP4 protein to generate stable functionalized QPs microspheres; the large specific surface area of QPs and the covalent combination of AQP4 protein can greatly improve the detection method sensitivity and detection range. In addition, the combination of QPs microsphere probes and flow cytometry makes the detection process easy to control and automate, effectively avoids various errors, and is helpful for clinical auxiliary diagnosis of the occurrence and development of NMO and prognosis assessment.

The flow immunological method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention uses quantum dot (Quantum dot, QD) nanomaterials to have high quantum yield, high fluorescence intensity, wide excitation spectrum range and narrow emission spectrum, Due to the advantages of long fluorescence lifetime, the method of combining new quantum dot nanoprobes with flow cytometry (FlowCytometry/on AQP4-QPs) is used to detect self-anti-AQP4 antibodies, which breaks through the traditional method of using organic fluorescent dyes as the basis. The bottleneck of detection tools; at the same time, it also utilizes the advantages of rapid detection, easy standardization and automation of flow cytometry technology, which makes up for the shortcomings of existing AQP4 antibody detection technology. Enables simple, rapid and reproducible detection of AQP4 antibodies.

Example 2.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, other features are the same as in Example 1, the difference is that the specific detection steps of the detection sample are as follows:

(1) Take 10 μL serum sample, dilute it with PBS solution according to the ratio of serum: PBS solution = 1:10, fully mix the diluted serum with 80 μg of QPs-AQP4 complex, and incubate at 37°C for one hour.

(2) Wash the incubated product 3 times with PBS solution, 5 minutes each time, and spin dry.

(3) Dilute FITC-labeled goat anti-human IgG with PBS solution according to the ratio of FITC-labeled goat anti-human IgG: PBS solution = 1:50, and add 100 μL of diluted FITC-labeled goat anti-human IgG to every 100 μL serum mixture sample , and incubate the mixture at 37°C for one hour in the dark.

(4) Wash the incubated functionalized QPs microspheres-anti-AQP4 antibody-goat anti-human IgG immune complex three times with PBS solution, and resuspend with 100 to 500 μL of PBS solution after washing.

(5) Flow detection.

It should be noted that the PBS solution used in this example has a pH range of 7.2 to 7.4, and a concentration of 0.01 mol/L.

In the method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention, the detection of anti-AQP4 antibodies meets the standards for clinical detection of anti-AQP4 antibodies, and has high sensitivity and strong specificity. Quality control overcomes the contradiction that the current clinical detection of anti-AQP4 antibodies cannot be compatible with high sensitivity, strong specificity and repeatability; in addition, the present invention makes it easier and more accurate to judge the detection results, and relatively reduces the requirements for inspectors. Stable in nature, easier to be automated, microquantified and high-throughput, providing a strong basis for clinical auxiliary diagnosis of NMO, thereby improving the quality of life of patients.

Example 3.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, comprising the following steps:

(1) Preparation of quantum dot polystyrene QPs microspheres;

(2) obtaining the functional fragment target gene of AQP4 protein for expression;

(3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

(4) The red-green dual fluorescence localization system of oil-soluble CdSe quantum dots with a fluorescence emission peak of 600nm and FITC-labeled goat anti-human IgG was used for flow cytometry detection, and the detection results of the functionalized QPs microspheres were interpreted.

Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

(1.1) Disperse and suspend 1 mg of PS microspheres in 0.5 mL of 99.99% saturated n-butanol solution to prepare PS microsphere suspension;

(1.2) Dissolve 0.1 mg of quantum dot QD in 50 μL of 99.99% saturated dichloromethane solvent and shake and mix to make QD-dichloromethane solution;

(1.3) Add the QD-dichloromethane solution dropwise to the PS microsphere suspension at a drop rate of 2.5 μL/sec, and shake at room temperature for 4 to 5 hours;

(1.4) Place the shaken mixture in a water bath at a temperature of 45°C for 20 hours;

(1.5) The mixture after the water bath was centrifuged and washed with an ethanol solution with a volume fraction of 20%, and the precipitate was collected to obtain QPs microspheres.

Step (2) specifically includes:

(2.1) artificially synthesize the target gene of the functional fragment of AQP4 protein, and insert the synthesized target gene into the pET32a plasmid vector to obtain the recombinant plasmid of AQP4-pET32a;

(2.2) Transform the recombinant plasmid of AQP4-pET32a into BL21 strain, and add IPTG inducer to induce expression;

(2.3) centrifuging the protein product obtained after the induced expression, and taking the centrifuged centrifuged supernatant and precipitate respectively for identification by polyacrylamide gel electrophoresis;

(2.4) The protein product obtained after the induced expression was purified through a nickel column, and identified again by western-blotting.

Wherein, step (2.2) specifically comprises the following steps:

Mix 0.1 μg of AQP4-pET32a recombinant plasmid with 50 μL of 50% volume fraction of BL21 strain, and ice-bath for more than 30 minutes, then place the mixed bacterial solution in water at 40°C for 85 seconds, and then place it at 0°C Put it in an ice bath for 2 minutes, and incubate the mixed bacterial solution at a speed of 220 rpm for more than 45 minutes at a temperature of 35°C, inoculate the bacterial solution on an agar plate containing ampicillin, and place the agar plate at 35°C Incubate in the environment for more than 24 hours.

Wherein, step (2.3) specifically includes the following steps: picking positive clones, specifically rotating the culture medium at a speed of 220 rpm at 35°C, incubating until the culture medium is slightly turbid, and dividing the monoclonal bacterial liquid into six tubes And put 1mL bacterial solution in each tube, and group them according to the induction time and IPTG dilution ratio. The induction time is divided into 4 hours and 6 hours, and the IPTG dilution ratio is divided into 0, 1:1000, and 1:100. details as following:

1) The induction time is 4 hours, and the IPTG dilution ratio is 0 as a group;

2) The induction time is 4 hours, and the IPTG dilution ratio is 1:1000 as a group;

3) The induction time is 4 hours, and the IPTG dilution ratio is 1:100 as a group;

4) The induction time is 6 hours, and the IPTG dilution ratio is 0 as a group;

5) The induction time is 6 hours, and the IPTG dilution ratio is 1:1000 as a group;

6) The induction time is 6 hours, and the IPTG dilution ratio is 1:100 as a group.

Wherein, step (3) specifically comprises the following steps:

(3.1) Pretreatment of QPs microspheres: Add 0.1 mg of QPs microspheres to 0.5 mL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L for mixing, and place the obtained mixture at 18,000 rpm Rotate for more than 5 minutes at a high speed environment, and perform centrifugation;

(3.2) Activation of QPs microspheres: Reset the centrifuged product into 100 μL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L, and add 5 μg/μL of EDC reagent and 5 μg/μL of Mix the NHS reagents, and shake the mixed product for more than 30 minutes at room temperature;

(3.3) Termination of activation: place the shaken mixed product in an environment of 18,000 rpm for more than 5 minutes, perform centrifugation, and add 0.5 mL of PBS reagent to the centrifuged product for washing 2 to 3 times Second-rate;

Specifically, the pH range of the PBS reagent is 7.2, and the concentration is 0.01mol/L;

(3.4) QPs microspheres coupled with AQP4 protein: mix AQP4 protein and QPs microspheres in 0.5 mL of PBS reagent, and place the mixed product at a temperature of 3°C to 45°C overnight or at room temperature Shake for 2 hours to complete the operation of coupling AQP4 protein with QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2, and the concentration is 0.01mol/L;

(3.5) Sealing: add BSA reagent with a mass concentration of 3% to the mixed product after shaking, and shake the mixture for more than 30 minutes at room temperature;

(3.6) Termination of the reaction: place the product obtained after adding 3% BSA reagent in mass concentration and shake it at a speed of 18,000 rpm for more than 5 minutes for centrifugation, and add 0.5 mL of PBS reagent to the centrifuged product Wash for 2 to 3 times, and finally add 100 μL of PBST reagent to resuspend the functionalized QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.2, and the concentration is 0.01mol/L;

Specifically, the pH range of the PBST reagent is 7.2, and the concentration is 0.01 mol/L.

The results are based on:

(4.1) The size of the QPs microspheres is uniform and its red fluorescence intensity is high, and there is no doped fluorescence, so it has excellent performance as a detection probe.

(4.2) The present invention uses an indirect method to form an immune complex composed of functionalized QPs microspheres-anti-AQP4 antibody-FITC-labeled goat anti-human IgG, and helps to interpret the detection results by means of a red-green dual fluorescent positioning system .

The invention adopts the technique of combining the novel quantum dot probe with the flow cytometry analysis technique, and breaks through the traditional bottleneck of using organic fluorescent dyes as detection tools. On the one hand, the use of QD nanomaterials as a detection tool overcomes the shortcomings of organic fluorescent dyes such as weak fluorescence intensity, short fluorescence lifetime and easy quenching. At the same time, the use of QPs microspheres as a detection carrier can effectively avoid the inconsistency of detection results caused by batch differences On the other hand, analyzing the molecular structure of AQP4 protein, the use of prokaryotic expression system can realize large-scale production, which is conducive to commercialization; finally, combined with fast and sensitive flow cytometry analysis technology, it can quickly and accurately realize the detection of single QPs-AQP4 microsphere probe The detection greatly improves the sensitivity and accuracy of the detection, and is beneficial to the automation and standardization of the detection of self-anti-AQP4 antibodies.

The present invention cites the common method of fluorescent microspheres to prepare chemical osmosis to improve the oil-soluble QD, and to increase the water solubility of the QD by wrapping PS microspheres on the surface of the QD. Therefore, the present invention uses n-butanol as the dispersion system and dichloromethane as the dispersion system. The swelling agent is easy to operate, and the prepared fluorescent microspheres have stable properties and high fluorescence intensity.

The invention constructs a stable AQP4 protein expression system, which overcomes the limitation of the method relying on tissues or cells with high protein expression as matrix. At the same time, the present invention uses EDC-NHS to couple quantum dot polystyrene microspheres QPs and AQP4 protein to generate stable functionalized QPs microspheres; the large specific surface area of QPs and the covalent combination of AQP4 protein can greatly improve the detection method sensitivity and detection range. In addition, the combination of QPs microsphere probes and flow cytometry makes the detection process easy to control and automate, effectively avoids various errors, and is helpful for clinical auxiliary diagnosis of the occurrence and development of NMO and prognosis assessment.

The flow immunological method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention uses quantum dot (Quantum dot, QD) nanomaterials to have high quantum yield, high fluorescence intensity, wide excitation spectrum range and narrow emission spectrum, Due to the advantages of long fluorescence lifetime, the method of combining new quantum dot nanoprobes with flow cytometry (FlowCytometry/on AQP4-QPs) is used to detect self-anti-AQP4 antibodies, which breaks through the traditional method of using organic fluorescent dyes as the basis. The bottleneck of detection tools; at the same time, it also utilizes the advantages of rapid detection, easy standardization and automation of flow cytometry technology, which makes up for the shortcomings of existing AQP4 antibody detection technology. Enables simple, rapid and reproducible detection of AQP4 antibodies.

Example 4.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, comprising the following steps:

(1) Preparation of quantum dot polystyrene QPs microspheres;

(2) obtaining the functional fragment target gene of AQP4 protein for expression;

(3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

(4) Perform flow cytometric detection through the red and green dual fluorescence localization system of oil-soluble CdSe quantum dots with a fluorescence emission peak of 650nm and FITC-labeled goat anti-human IgG, and interpret the detection results of the functionalized QPs microspheres .

Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

(1.1) Disperse and suspend 10 mg of PS microspheres in 1 mL of 99.99% saturated n-butanol solution to prepare PS microsphere suspension;

(1.2) Dissolve 1 mg of quantum dot QD in 100 μL of 99.99% saturated dichloromethane solvent and shake and mix to make QD-dichloromethane solution;

(1.3) Add the QD-dichloromethane solution dropwise to the PS microsphere suspension at a drop rate of 2.5 μL/sec to 3.5 μL/sec, and shake at room temperature for 4 to 5 hours;

(1.4) Place the shaken mixture in a water bath at a temperature of 55°C for 25 hours;

(1.5) The mixture after the water bath was centrifuged and washed with an ethanol solution with a volume fraction of 80%, and the precipitate was collected to obtain QPs microspheres.

Step (2) specifically includes:

(2.1) artificially synthesize the target gene of the functional fragment of AQP4 protein, and insert the synthesized target gene into the pET32a plasmid vector to obtain the recombinant plasmid of AQP4-pET32a;

(2.2) Transform the recombinant plasmid of AQP4-pET32a into BL21 strain, and add IPTG inducer to induce expression;

(2.3) centrifuging the protein product obtained after the induced expression, and taking the centrifuged centrifuged supernatant and precipitate respectively for identification by polyacrylamide gel electrophoresis;

(2.4) The protein product obtained after the induced expression was purified through a nickel column, and identified again by western-blotting.

Wherein, step (2.2) specifically comprises the following steps:

Mix 0.5 μg of AQP4-pET32a recombinant plasmid with 100 μL of 50% volume fraction of BL21 strain and mix on ice for more than 30 minutes, then place the mixed bacteria solution in water at 45°C for 95 seconds, and then place it at 0°C Put it in an ice bath for 2 to 3 minutes, rotate the mixed bacterial solution at 35°C to 40°C at a speed of 220 rpm for more than 45 minutes, inoculate the bacterial solution on an agar plate containing ampicillin, and place the agar plate on Incubate at 35°C to 40°C for more than 24 hours.

Wherein, the step (2.3) specifically includes the following steps: picking positive clones, specifically rotating the culture medium at a speed of 220 rpm in an environment of 40°C, incubating until the culture medium is slightly turbid, and dividing the monoclonal bacterial liquid into six tubes And put 1mL bacterial solution in each tube, and group them according to the induction time and IPTG dilution ratio. The induction time is divided into 4 hours and 6 hours, and the IPTG dilution ratio is divided into 0, 1:1000, and 1:100. details as following:

1) The induction time is 4 hours, and the IPTG dilution ratio is 0 as a group;

2) The induction time is 4 hours, and the IPTG dilution ratio is 1:1000 as a group;

3) The induction time is 4 hours, and the IPTG dilution ratio is 1:100 as a group;

4) The induction time is 6 hours, and the IPTG dilution ratio is 0 as a group;

5) The induction time is 6 hours, and the IPTG dilution ratio is 1:1000 as a group;

6) The induction time is 6 hours, and the IPTG dilution ratio is 1:100 as a group.

Wherein, step (3) specifically comprises the following steps:

(3.1) Pretreatment of QPs microspheres: Take 1 mg of QPs microspheres and add them to 1 mL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L for mixing, and place the obtained mixture at a speed of 18,000 rpm Rotate in the environment for more than 5 minutes, and perform centrifugation;

(3.2) Activation of QPs microspheres: Reset the centrifuged product to 500 μL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L, and add 20 μg/μL of EDC reagent and 20 μg/μL of NHS The reagents are mixed, and the mixed product is shaken at room temperature for more than 30 minutes;

(3.3) Termination of activation: place the shaken mixed product in an environment of 18,000 rpm for more than 5 minutes, perform centrifugation, and add 1 mL of PBS reagent to the centrifuged product for washing 2 to 3 times ;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

(3.4) QPs microspheres coupled with AQP4 protein: mix AQP4 protein and QPs microspheres in 1 mL of PBS reagent, and place the mixed product at 45°C overnight or shake at room temperature for 2 hours By 4 hours, the operation of coupling AQP4 protein to QPs microspheres was completed;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

(3.5) Sealing: add BSA reagent with a mass concentration of 5% to the mixed product after shaking, and shake the mixture for more than 30 minutes at room temperature;

(3.6) Termination of the reaction: place the product obtained after shaking with 5% BSA reagent at a mass concentration of 18,000 rpm for more than 5 minutes for centrifugation, and add 1 mL of PBS reagent to the centrifuged product. Wash for 2 to 3 times, and finally add 500 μL of PBST reagent to resuspend the functionalized QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

Specifically, the pH range of the PBST reagent is 7.4, and the concentration is 0.01 mol/L.

The results are based on:

(4.1) The size of the QPs microspheres is uniform and its red fluorescence intensity is high, and there is no doped fluorescence, so it has excellent performance as a detection probe.

(4.2) The present invention uses an indirect method to form an immune complex composed of functionalized QPs microspheres-anti-AQP4 antibody-FITC-labeled goat anti-human IgG, and helps to interpret the detection results by means of a red-green dual fluorescent positioning system .

The invention adopts the technique of combining the novel quantum dot probe with the flow cytometry analysis technique, and breaks through the traditional bottleneck of using organic fluorescent dyes as detection tools. On the one hand, the use of QD nanomaterials as a detection tool overcomes the shortcomings of organic fluorescent dyes such as weak fluorescence intensity, short fluorescence lifetime and easy quenching. At the same time, the use of QPs microspheres as a detection carrier can effectively avoid the inconsistency of detection results caused by batch differences On the other hand, analyzing the molecular structure of AQP4 protein, the use of prokaryotic expression system can realize large-scale production, which is conducive to commercialization; finally, combined with fast and sensitive flow cytometry analysis technology, it can quickly and accurately realize the detection of single QPs-AQP4 microsphere probe The detection greatly improves the sensitivity and accuracy of the detection, and is beneficial to the automation and standardization of the detection of self-anti-AQP4 antibodies.

The present invention cites the common method of fluorescent microspheres to prepare chemical osmosis to improve the oil-soluble QD, and to increase the water solubility of the QD by wrapping PS microspheres on the surface of the QD. Therefore, the present invention uses n-butanol as the dispersion system and dichloromethane as the dispersion system. The swelling agent is easy to operate, and the prepared fluorescent microspheres have stable properties and high fluorescence intensity.

The invention constructs a stable AQP4 protein expression system, which overcomes the limitation of the method relying on tissues or cells with high protein expression as matrix. At the same time, the present invention uses EDC-NHS to couple quantum dot polystyrene microspheres QPs and AQP4 protein to generate stable functionalized QPs microspheres; the large specific surface area of QPs and the covalent combination of AQP4 protein can greatly improve the detection method sensitivity and detection range. In addition, the combination of QPs microsphere probes and flow cytometry makes the detection process easy to control and automate, effectively avoids various errors, and is helpful for clinical auxiliary diagnosis of the occurrence and development of NMO and prognosis assessment.

The flow immunological method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention uses quantum dot (Quantum dot, QD) nanomaterials to have high quantum yield, high fluorescence intensity, wide excitation spectrum range and narrow emission spectrum, Due to the advantages of long fluorescence lifetime, the method of combining new quantum dot nanoprobes with flow cytometry (FlowCytometry/on AQP4-QPs) is used to detect self-anti-AQP4 antibodies, which breaks through the traditional method of using organic fluorescent dyes as the basis. The bottleneck of detection tools; at the same time, it also utilizes the advantages of rapid detection, easy standardization and automation of flow cytometry technology, which makes up for the shortcomings of existing AQP4 antibody detection technology. Enables simple, rapid and reproducible detection of AQP4 antibodies.

Example 5.

A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, comprising the following steps:

(1) Preparation of quantum dot polystyrene QPs microspheres;

(2) obtaining the functional fragment target gene of AQP4 protein for expression;

(3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

(4) Perform flow cytometric detection by the red-green dual fluorescence positioning system of oil-soluble CdSe quantum dots with a fluorescence emission peak of 645nm and FITC-labeled goat anti-human IgG, and interpret the detection results of the functionalized QPs microspheres .

Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

(1.1) Disperse and suspend 10 mg of PS microspheres in 1 mL of 99.99% saturated n-butanol solution to prepare PS microsphere suspension;

(1.2) Dissolve 1 mg of quantum dot QD in 50 μL of 99.99% saturated dichloromethane solvent and shake and mix to make QD-dichloromethane solution;

(1.3) Add the QD-dichloromethane solution dropwise to the PS microsphere suspension at a drop rate of 3.0 μL/sec, and shake at room temperature for 4 hours;

(1.4) Place the shaken mixture in a water bath at a temperature of 50°C for 20 hours;

(1.5) The mixture after the water bath was centrifuged and washed with an ethanol solution with a volume fraction of 50%, and the precipitate was collected to obtain QPs microspheres.

Step (2) specifically includes:

(2.1) artificially synthesize the target gene of the functional fragment of AQP4 protein, and insert the synthesized target gene into the pET32a plasmid vector to obtain the recombinant plasmid of AQP4-pET32a;

(2.2) Transform the recombinant plasmid of AQP4-pET32a into BL21 strain, and add IPTG inducer to induce expression;

(2.3) centrifuging the protein product obtained after the induced expression, and taking the centrifuged centrifuged supernatant and precipitate respectively for identification by polyacrylamide gel electrophoresis;

(2.4) The protein product obtained after the induced expression was purified through a nickel column, and identified again by western-blotting.

Wherein, step (2.2) specifically comprises the following steps:

Mix 0.2 μg of AQP4-pET32a recombinant plasmid with 50 μL of BL21 strain with a volume fraction of 50% and mix on ice for 30 minutes, then place the mixed bacteria solution in water at 42°C for 90 seconds, and then place at 0°C Ice-bath for 2 to 3 minutes in the environment, rotate the mixed bacterial solution at 220 rpm for 45 minutes at a temperature of 37°C, inoculate the bacterial solution on an agar plate containing ampicillin, and place the agar plate on Incubate at 37°C for 24 hours.

Wherein, step (2.3) specifically includes the following steps: picking positive clones, specifically rotating the culture medium at a speed of 220 rpm at a temperature of 37°C, incubating until the culture medium is slightly turbid, and dividing the monoclonal bacterial liquid into six 1 mL of bacterial solution was placed in each tube, and grouped according to the induction time and IPTG dilution ratio. The induction time was divided into 4 hours and 6 hours, and the IPTG dilution ratio was divided into 0, 1:1000, and 1:100. The grouping is as follows:

1) The induction time is 4 hours, and the IPTG dilution ratio is 0 as a group;

2) The induction time is 4 hours, and the IPTG dilution ratio is 1:1000 as a group;

3) The induction time is 4 hours, and the IPTG dilution ratio is 1:100 as a group;

4) The induction time is 6 hours, and the IPTG dilution ratio is 0 as a group;

5) The induction time is 6 hours, and the IPTG dilution ratio is 1:1000 as a group;

6) The induction time is 6 hours, and the IPTG dilution ratio is 1:100 as a group.

Wherein, step (3) specifically comprises the following steps:

(3.1) Pretreatment of QPs microspheres: Take 1 mg of QPs microspheres and add them to 0.5 mL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L for mixing, and place the obtained mixture at 18,000 rpm Rotate in the rotating speed environment for 5 minutes, carry out centrifugal operation;

(3.2) Activation of QPs microspheres: Reset the centrifuged product to 400 μL of MES buffer with a pH value of 6.1 and a concentration of 50 mmol/L, and add 10 μg/μL of EDC reagent and 10 μg/μL of NHS The reagents are mixed, and the mixed product is shaken for 30 minutes at room temperature;

(3.3) Termination of activation: place the shaken mixed product in an environment of 18,000 rpm for 5 minutes, perform centrifugation, and add 0.5 mL of PBS reagent to the centrifuged product for washing 2 to 3 times ;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

(3.4) QPs microspheres coupled with AQP4 protein: mix AQP4 protein and QPs microspheres in 0.5 mL of PBS reagent, and place the mixed product at 4°C overnight or shake at room temperature for 2 Hours to 4 hours, complete the operation of coupling AQP4 protein with QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

(3.5) Sealing: add BSA reagent with a mass concentration of 4% to the shaken mixed product, and shake the mixture for 30 minutes at room temperature;

(3.6) Termination of the reaction: place the product obtained after shaking with 4% BSA reagent at a mass concentration of 18,000 rpm for 5 minutes for centrifugation, and add 1 mL of PBS reagent to the centrifuged product Wash for 2 to 3 times, and finally add 400 μL of PBST reagent to resuspend the functionalized QPs microspheres;

Specifically, the pH range of the PBS reagent is 7.4, and the concentration is 0.01mol/L;

Specifically, the pH range of the PBST reagent is 7.4, and the concentration is 0.01 mol/L.

The results are based on:

(4.1) The size of the QPs microspheres is uniform and its red fluorescence intensity is high, and there is no doped fluorescence, so it has excellent performance as a detection probe.

(4.2) The present invention uses an indirect method to form an immune complex composed of functionalized QPs microspheres-anti-AQP4 antibody-FITC-labeled goat anti-human IgG, and helps to interpret the detection results by means of a red-green dual fluorescent positioning system .

The invention adopts the technique of combining the novel quantum dot probe with the flow cytometry analysis technique, and breaks through the traditional bottleneck of using organic fluorescent dyes as detection tools. On the one hand, the use of QD nanomaterials as a detection tool overcomes the shortcomings of organic fluorescent dyes such as weak fluorescence intensity, short fluorescence lifetime and easy quenching. At the same time, the use of QPs microspheres as a detection carrier can effectively avoid the inconsistency of detection results caused by batch differences On the other hand, analyzing the molecular structure of AQP4 protein, the use of prokaryotic expression system can realize large-scale production, which is conducive to commercialization; finally, combined with fast and sensitive flow cytometry analysis technology, it can quickly and accurately realize the detection of single QPs-AQP4 microsphere probe The detection greatly improves the sensitivity and accuracy of the detection, and is beneficial to the automation and standardization of the detection of self-anti-AQP4 antibodies.

The present invention cites the common method of fluorescent microspheres to prepare chemical osmosis to improve the oil-soluble QD, and to increase the water solubility of the QD by wrapping PS microspheres on the surface of the QD. Therefore, the present invention uses n-butanol as the dispersion system and dichloromethane as the dispersion system. The swelling agent is easy to operate, and the prepared fluorescent microspheres have stable properties and high fluorescence intensity.

The invention constructs a stable AQP4 protein expression system, which overcomes the limitation of the method relying on tissues or cells with high protein expression as matrix. At the same time, the present invention uses EDC-NHS to couple quantum dot polystyrene microspheres QPs and AQP4 protein to generate stable functionalized QPs microspheres; the large specific surface area of QPs and the covalent combination of AQP4 protein can greatly improve the detection method sensitivity and detection range. In addition, the combination of QPs microsphere probes and flow cytometry makes the detection process easy to control and automate, effectively avoids various errors, and is helpful for clinical auxiliary diagnosis of the occurrence and development of NMO and prognosis assessment.

The flow immunological method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres of the present invention uses quantum dot (Quantum dot, QD) nanomaterials to have high quantum yield, high fluorescence intensity, wide excitation spectrum range and narrow emission spectrum, Due to the advantages of long fluorescence lifetime, the method of combining new quantum dot nanoprobes with flow cytometry (FlowCytometry/on AQP4-QPs) is used to detect self-anti-AQP4 antibodies, which breaks through the traditional method of using organic fluorescent dyes as the basis. The bottleneck of detection tools; at the same time, it also utilizes the advantages of rapid detection, easy standardization and automation of flow cytometry technology, which makes up for the shortcomings of existing AQP4 antibody detection technology. Enables simple, rapid and reproducible detection of AQP4 antibodies.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that Modifications or equivalent replacements are made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (6)

1. A method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres comprises the following steps:

(1) preparing quantum dot polystyrene QPs microspheres;

(2) obtaining a functional fragment target gene of AQP4 protein for expression;

(3) activating carboxyl on the surface of the QPs microsphere by using EDC-NHS, and coupling the carboxyl on the surface of the activated QPs microsphere with amino of AQP4 protein to prepare a functionalized QPs microsphere;

(4) performing flow detection by an oil-soluble CdSe quantum dot with a fluorescence emission peak of 600-650 nm and a red-green dual fluorescence positioning system of FITC-labeled goat anti-human IgG, and interpreting the detection result of the functionalized QPs microspheres.

2. The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere as claimed in claim 1, wherein the step (1) adopts a chemical infiltration method to prepare the QPs microsphere, and the specific process comprises:

(1.1) dispersing and suspending 1mg to 10mg of PS microspheres in 0.5mL to 1mL of 99.99% saturated n-butyl alcohol solution to prepare PS microsphere suspension;

(1.2) dissolving 0.1-1 mg of quantum dots QD in 50-100 μ L of 99.99% saturated dichloromethane solvent, and performing shaking mixing to prepare QD-dichloromethane solution;

(1.3) dripping the QD-dichloromethane solution into the PS microsphere suspension at the dripping speed of 2.5 to 3.5 mu L/s, and shaking at room temperature for 4 to 5 hours;

(1.4) heating the mixture after shaking in a water bath at the temperature of between 45 and 55 ℃ for 20 to 25 hours;

(1.5) centrifuging and washing the mixture after the water bath by using an ethanol solution with the volume fraction of 20% to 80%, and collecting precipitates to obtain QPs microspheres.

3. The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere as claimed in claim 2, wherein the step (2) specifically comprises:

(2.1) artificially synthesizing an AQP4 protein functional fragment target gene, and inserting the synthesized target gene into a pET32a plasmid vector to obtain a recombinant plasmid of AQP4-pET32 a;

(2.2) transforming the recombinant plasmid of AQP4-pET32a into a BL21 strain, and adding an IPTG inducer for induced expression;

(2.3) carrying out centrifugal operation on the protein product obtained after induction expression, and respectively taking the centrifuged supernatant and the centrifuged precipitate for polyacrylamide gel electrophoresis identification;

and (2.4) purifying the protein product obtained after the induction expression by a nickel column, and re-identifying by adopting western-blotting.

4. The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere as claimed in claim 3, wherein the step (2.2) specifically comprises the following steps:

0.1 to 0.5 mu g of AQP4-pET32a recombinant plasmid and 50 to 100 mu L of BL21 strain with 50 percent volume fraction are mixed evenly and iced for at least 30 minutes, then the mixed bacterial liquid is put into water with the temperature of 40 to 45 ℃ for water bath for 85 to 95 seconds, then the mixed bacterial liquid is put into the environment with the temperature of 0 ℃ for ice bath for 2 to 3 minutes, the mixed bacterial liquid is cultured at the temperature of 35 to 40 ℃ for more than 45 minutes by rotating at the rotating speed of 220 rpm, the bacterial liquid is inoculated to an agar plate coated with ampicillin, and the agar plate is put into the environment with the temperature of 35 to 40 ℃ for more than 24 hours.

5. The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere as claimed in claim 4, wherein the step (2.3) specifically comprises the following steps: selecting positive clones, specifically, rotating a culture medium at a rotating speed of 220 rpm under the environment of 35-40 ℃, incubating until the culture medium is slightly turbid, dividing the monoclonal bacterial liquid into six tubes, placing 1mL of bacterial liquid in each tube, and grouping according to different induction time and IPTG dilution ratios, wherein the induction time is divided into 4 hours and 6 hours, the IPTG dilution ratio is divided into three types of 0, 1:1000 and 1:100, and the specific grouping conditions are as follows:

1) the induction time is 4 hours, and the IPTG dilution ratio is 0;

2) the induction time was 4 hours, the IPTG dilution ratio was 1:1000 is a group;

3) the induction time was 4 hours, the IPTG dilution ratio was 1:100 is a group;

4) the induction time is 6 hours, and the IPTG dilution ratio is 0;

5) the induction time was 6 hours, the IPTG dilution ratio was 1:1000 is a group;

6) the induction time was 6 hours, the IPTG dilution ratio was 1:100 are a group.

6. The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere as claimed in claim 5, wherein the step (3) specifically comprises the following steps:

(3.1) pretreatment of QPs microspheres: adding 0.1-1 mg QPs microspheres into 0.5-1 mL MES buffer solution with pH value of 6.1 and concentration of 50mmol/L, mixing, placing the obtained mixture in an environment with rotation speed of 18000 rpm for more than 5 minutes, and performing centrifugation;

(3.2) activating QPs microspheres: the centrifuged product is placed in MES buffer solution with pH value of 6.1 and concentration of 50mmol/L in a range of 100 mu L to 500 mu L again, EDC reagent with a concentration of 5 mu g/mu L to 20 mu g/mu L and NHS reagent with a concentration of 5 mu g/mu L to 20 mu g/mu L are respectively added and mixed, and the mixed product is shaken for more than 30 minutes in a room temperature environment;

(3.3) termination of activation: placing the vibrated mixed product in an environment of 18000 rpm and rotating for more than 5 minutes, carrying out centrifugal operation, and adding 0.5-1 mL of PBS reagent into the centrifuged product to carry out washing operation for 2-3 times;

the pH value range of the PBS reagent is 7.2-7.4, and the concentration is 0.01 mol/L;

(3.4) QPs microsphere coupling of AQP4 protein: putting the AQP4 protein and the QPs microspheres into 0.5-1 mL PBS reagent for mixing, and putting the mixed product into a temperature of 3-45 ℃ overnight or shaking the mixed product at room temperature for 2-4 hours to finish the operation of coupling the QPs microspheres with the AQP4 protein;

the pH value range of the PBS reagent is 7.2-7.4, and the concentration is 0.01 mol/L;

(3.5) sealing: adding a BSA (bovine serum albumin) reagent with the mass concentration of 3-5% into the vibrated mixed product, and vibrating the mixture for more than 30 minutes at room temperature;

(3.6) termination of the reaction: placing a product obtained after adding BSA (bovine serum albumin) reagent with the mass concentration of 3-5% and shaking in a rotating speed of 18000 rpm and rotating for more than 5 minutes for centrifugal operation, adding 0.5-1 mL of PBS (phosphate buffer solution) reagent into the centrifuged product for washing for 2-3 times, and finally adding 100-500 mu L of PBST reagent for re-suspending the functionalized QPs microspheres;

the pH value range of the PBS reagent is 7.2-7.4, and the concentration is 0.01 mol/L;

the pH value of the PBST reagent ranges from 7.2 to 7.4, and the concentration is 0.01 mol/L.