阅读说明：本技术 基于量子点聚苯乙烯微球检测抗aqp4抗体的方法 (Method for detecting anti-AQP 4 antibody based on quantum dot polystyrene microspheres ) 是由 刘天才 李鹏 陈振华 于 2019-09-10 设计创作，主要内容包括：一种基于量子点聚苯乙烯微球检测抗AQP4抗体的方法,将新型的量子点纳米探针与流式细胞分析技术相结合的免疫方法用于检测自身抗AQP4抗体,突破了传统的以有机荧光染料为检测工具的瓶颈。同时利用了流式细胞分析技术具有快速检测易于标准化和自动化等优点,弥补了现有AQP4抗体检测技术的不足。能够简单快速、可重复检测AQP4抗体。(A method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres is characterized in that an immune method combining a novel quantum dot nano probe and a flow cytometry analysis technology is used for detecting the anti-AQP 4 antibody, and the bottleneck of taking organic fluorescent dye as a detection tool in the prior art is broken through. Meanwhile, the flow cytometry analysis technology is utilized, and the advantages of being rapid in detection, easy to standardize and automate and the like are utilized, so that the defects of the existing AQP4 antibody detection technology are overcome. The AQP4 antibody can be detected simply, rapidly and repeatedly.)

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.

Technical Field

The invention relates to the technical field of bioanalytical chemistry and nano biology, in particular to a method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres.

Background

The human body autoantibody is used as an important serological marker of the autoimmune disease, and the rapid detection of the human body autoantibody has important application value for the diagnosis, treatment measure and prognosis evaluation of the autoimmune disease. With the improvement of the sensitivity and specificity of autoantibody detection methods, the diagnostic criteria for related autoimmune diseases are also being updated and adjusted accordingly.

Neuromyelitis optica (NMO) is an acute or subacute inflammatory demyelinating disease in which the optic nerve is affected simultaneously or sequentially with the spinal cord. In 2004 Lennon et al found NMO-IgG antibodies, i.e. anti-aquaporin 4(AQP4) antibodies, for the first time in the serum of NMO patients. AQP4 is the major aquaporin of the central nervous system, located on the foot processes of astrocytes, and is the major target for NMO-IgG. The AQP4 antibody enters the central nervous system through the permeable part of the blood brain barrier, encounters astrocytes and causes a cell-dependent cytotoxic reaction, the foot processes of the astrocytes are degraded by NMO-IgG and complement, macrophages, eosinophils and neutrophils are activated to generate a series of cytokines, oxygen radicals and the like to cause vascular and parenchymal damage, and finally, the damage of white and gray matter including axon and oligodendrocyte is caused. Serum anti-AQP 4 antibodies have been included as a supportive condition in NMO diagnostic standards in 2015.

The detection method for clinically detecting AQP4 at present mainly comprises the following two methods: (1) an Indirect immunofluorescence assay (IIF) method taking a rat brain tissue section highly expressing AQP4 protein as a matrix has high sensitivity, but the method is complex in operation, has high requirements for manufacturing the tissue section, is difficult to control, and requires abundant neurophysiological knowledge of inspectors to accurately judge a detection result. (2) The cell-based assay (CBA) method using the cells successfully expressing the AQP4 protein as the matrix has relatively high specificity, but in the actual detection process, although a negative control is set, the interpretation of the detection result can be influenced due to low protein expression level, and due to inconsistent transfection efficiency, the establishment of a uniform quality control standard is difficult, so that the large-scale commercial production is difficult to realize.

Therefore, in order to overcome the defects in the prior art, it is necessary to provide a method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres.

Disclosure of Invention

The invention aims to avoid the defects of the prior art and provides a method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres, which has the characteristics of simple and rapid preparation process and capability of repeatedly detecting the AQP4 antibody.

The object of the invention is achieved by the following technical measures.

The method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere 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.

Preferably, in the step (1), QPs microspheres are prepared by a chemical infiltration method, and the specific process includes:

(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.

Preferably, the step (2) specifically includes:

(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.

Preferably, the step (2.2) specifically includes the steps of:

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 more than 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.

Preferably, the step (2.3) specifically includes the steps of: selecting positive clones, rotating the culture medium at the 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.

Preferably, the step (3) specifically includes the steps of:

(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;

specifically, the pH value range of the PBS reagent is 7.2 to 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;

specifically, the pH value range of the PBS reagent is 7.2 to 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;

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

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

Preferably, the interpretation of the results is based on:

(4.1) the QPs microspheres are uniform in size, high in red fluorescence intensity, free of any doped fluorescence and excellent in performance as detection probes.

(4.2) the invention adopts an indirect method to form an immune complex consisting of functionalized QPs microspheres-anti-AQP 4 antibody-FITC labeled goat anti-human IgG, and a red-green dual fluorescence localization system is used for facilitating the interpretation of the detection result.

The invention relates to a Flow-type immunization method for detecting an anti-AQP 4 antibody based on Quantum dot polystyrene microspheres, which utilizes the advantages that Quantum Dot (QD) nano materials have high Quantum yield, large fluorescence intensity, wide excitation spectrum range, narrow emission spectrum, long fluorescence life and the like, and combines a novel Quantum dot nano probe with Flow Cytometry/on AQP4-QPs (Flow Cytometry/on AQP4-QPs) to detect the anti-AQP 4 antibody, thereby breaking through the bottleneck of taking organic fluorescent dye as a detection tool in the prior art; meanwhile, the flow cytometry analysis technology is utilized to have the advantages of being rapid in detection, easy to standardize and automate and the like, and the defects of the existing AQP4 antibody detection technology are overcome. The reagent has the advantages of being capable of simply, rapidly and repeatedly detecting the AQP4 antibody.

Drawings

The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.

FIG. 1 shows the molar excitation spectrum and fluorescence emission spectrum of QPs microspheres in the method for detecting an anti-AQP 4 antibody based on quantum dot polystyrene microspheres.

FIG. 2 is a western-blotting electrophoresis identification chart of the expression of AQP4 protein induced by IPTG in the method for detecting the anti-AQP 4 antibody based on the quantum dot polystyrene microsphere.

FIG. 3 is a graph showing the results of flow cytometry for the detection of anti-AQP 4 antibodies in serum samples.

Detailed Description

The invention is further illustrated by the following examples.