New method for detection of anti-nmda receptor antibodies based on microfluidics and stable cell model technology

Abstract

The application belongs to the technical field of precise detection of nerve immune antibodies, and provides a new method for detecting anti-NMDA receptor antibodies based on microfluidic and stable cell model technology, which is characterized by the following steps: a detection device is prepared by integrating a microfluidic chip and a miniaturized microscopic imaging system; a HEK293T stable cell strain is used as an antigen carrier to specifically combine with anti-NMDAR antibodies in a fingertip blood sample; the blood plasma is separated by the microfluidic chip, the cells are fixed, and then quantitative detection is performed by the microscopic imaging system to complete the detection of the anti-NMDA receptor antibodies in the fingertip blood sample. The detection effect of the application on the anti-NMDA receptor antibodies is evaluated by using the above detection device and detection method, and the results show that the application has high detection efficiency and provides a reliable evidence for the rapid clinical diagnosis of NMDAR encephalitis.

Description

Technical Field

[0001] This invention belongs to the field of precision detection technology for neuroimmune antibodies, and relates to the detection of anti-NMDA receptor antibodies, specifically a new method for detecting anti-NMDA receptor antibodies based on microfluidics and stable cell model technology. Background Technology

[0002] Currently, the mainstream technique for clinical detection of anti-NMDAR antibodies is transient transfection cell model assay (CBA), supplemented by radioimmunoprecipitation assay (RIPA) and tissue substrate-based assay (TBA). However, the interpretation of antibody detection results largely relies on visual fluorescence observation or semi-quantitative flow cytometry. These techniques have the following significant drawbacks: At the cell model level: the transient CBA method requires re-transfection of plasmids before each test, and the transfection reagent is cytotoxic, affecting cell viability; the transfection efficiency is affected by many factors such as cell state and operation technique, and the test results of different batches have large errors. The stability and uniformity of the model are extremely low, and the time, technical and economic costs are high. It requires professional operators and is difficult to promote clinically.

[0003] In terms of testing procedures: existing tests often require sending samples to third-party institutions, resulting in high costs for the entire set of tests, which are paid out of pocket by patients; the testing cycle is long (reports take 5 working days), which can easily delay clinical diagnosis and treatment; the sample requirements are large (peripheral blood ≥10 mL, cerebrospinal fluid ≥6 mL), and patients with difficulty in collecting cerebrospinal fluid cannot complete the test, and collecting large amounts of cerebrospinal fluid can easily cause discomfort to patients.

[0004] In terms of detection technology: RIPA cannot capture low-affinity antibodies, the operation is complicated and requires radiation protection measures, making it difficult to conduct batch detection; fluorescence observation depends on the accuracy of the equipment and the subjective judgment of the operator, resulting in poor objectivity of the results; semi-quantitative flow cytometry technology can only achieve semi-quantitative analysis, and the detection process is cumbersome, making it impossible to achieve rapid on-site detection.

[0005] At the equipment integration level: existing detection technologies lack integrated detection systems. Plasma separation, cell incubation and washing, antibody detection, and result imaging analysis must be completed step by step on different devices. This involves many operational steps, which can easily introduce errors. Furthermore, the devices are bulky and cannot be used in a portable manner.

[0006] To address the above problems, this invention provides a novel method for detecting anti-NMDA receptor antibodies based on microfluidics and stable cell model technology. Summary of the Invention

[0007] The purpose of this invention is to provide a new method for detecting anti-NMDA receptor antibodies based on microfluidics and stable cell model technology, to prepare a detection device for detecting anti-NMDA receptor antibodies based on microfluidics and stable cell model technology, and to apply it to the detection of anti-NMDAR antibodies, thereby providing a new rapid detection method for the rapid clinical diagnosis of NMDAR encephalitis.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides an anti-NMDAR antibody detection device based on microfluidics and a stable cell model. The detection device consists of two modules: a detachably connected integrated microfluidic chip and a miniaturized microscopic imaging system. The microfluidic chip has a three-layer composite structure, which, from top to bottom, consists of an upper plasma separation chamber, a middle dual-pore PMM filter membrane, and a lower micro-nano structure reaction chamber. The microfluidic chip has five functional sample inlets on its side and a waste liquid outlet at its bottom. The lower micro-nano structure reaction chamber has a built-in microbead blocking unit at its outlet and is a toothed microfluidic channel.

[0009] Preferably, the five functional injection ports are the microbead inlet, cell inlet, sample inlet, fluorescent secondary antibody inlet, and washing solution inlet.

[0010] Preferably, the microscopic imaging system is positioned directly above the integrated microfluidic chip, precisely corresponding to the detection area of ​​the lower micro / nano structure reaction chamber of the chip; the microscopic imaging system also includes an electrically connected electronic control module, an LED lighting module, a displacement lifting module, and an image acquisition and analysis module, and all of the above modules are integrated and detachably matched with the microfluidic chip.

[0011] The present invention also provides a method for using the anti-NMDAR antibody detection device as described above, wherein the method specifically comprises: (1) Chip pretreatment: 10 μm microbeads are injected into the lower reaction chamber of the chip from the microbead inlet to form a microbead blocking unit; the inner surface of the chip chamber is sealed by Pluronics and BSA. (2) Cell loading: NMDAR stable cell line was injected into the chip through the cell inlet and the flow rate was adjusted to 40 μL / s to make the cells uniformly distributed in a monolayer in the toothed microfluidic channel, thus completing efficient cell capture. (3) Sample processing: Finger blood is injected into the upper plasma separation chamber of the chip through the sample inlet. The plasma and blood cells are quickly separated through the middle small-pore PMM filter membrane. The separated plasma directly enters the lower reaction chamber. (4) Antigen-antibody reaction: Plasma and stable cells are incubated in the lower reaction chamber. Anti-NMDAR antibody specifically binds to the GluN1 subunit on the cell surface. Fluorescently labeled anti-IgG secondary antibody is injected through the fluorescent secondary antibody inlet and incubated in the dark for 20 min to form a GluN1 subunit-anti-NMDAR antibody-fluorescent secondary antibody complex. (5) Washing and purification: Inject washing buffer into the washing solution inlet to rinse the reaction chamber, remove unbound free antibodies and impurities, and discharge the waste liquid from the sample outlet; (6) Imaging analysis: Start the microscopic imaging system, automatically count the number of cells, fluorescence positivity rate and intensity, and output quantitative detection results.

[0012] Preferably, the NMDAR stable cell line is injected at a concentration of 1×10⁻⁶. 6 The flow rate is 20-40 μL / s, the volume of the fingertip blood sample is 50-100 μL, and the flow rate of the washing solution is 30 μL / s.

[0013] The present invention also provides the application of the anti-NMDAR antibody detection device and / or the above-described method of use in the detection of anti-NMDA receptor antibodies.

[0014] The beneficial effects of this invention are: The present invention aims to solve the technical problems existing in the detection technology of anti-NMDAR antibody, such as low stability and uniformity of detection models, high detection cost and cumbersome operation, low detection efficiency and reliance on human subjective judgment, large sample requirements and long detection cycle, and insufficient detection sensitivity and specificity. By optimizing the lentiviral vector design, stable cluster expression of the GluN1 subunit on the surface of HEK293T cells was achieved, maintaining high expression levels even after 10 passages. This replaces the traditional transient cell model, improving the stability and uniformity of detection. The design of serrated micro / nanostructure channels enables high-throughput (up to 40 μL / s) cell capture of HEK293T cells, while the micropillar array prevents cell blockage, ensuring continuous detection. The integrated design of a three-layer composite microfluidic chip integrates plasma separation, cell loading, incubation and washing, and antigen-antibody reaction into a single chip, requiring only finger-prick blood for detection, significantly reducing sample volume and operation time. The customized miniaturized microscopic imaging system integrates fluorescence illumination, autofocus, and imaging analysis, eliminating errors from subjective human judgment. The system is also miniaturized and portable, supporting rapid on-site detection. This invention combines a stable cell model with microfluidic technology, allowing antigen-antibody reactions to occur within micro- and nano-channels. This results in short diffusion distances, rapid reaction speeds, and the entire detection process can be completed within 1.5 hours, far shorter than the 5 working days required by traditional detection methods. Attached Figure Description

[0015] Figure 1 This is a diagram of the construction of stable transgenic strains in this invention (A is a schematic diagram of stable transgenic strain construction; B is a lentiviral vector). Figure 2 This is a structural diagram of the microfluidic chip in this invention (A is a schematic diagram of the microfluidic chip structure; B is a schematic diagram of the microfluidic chip appearance). Figure 3 This is a flowchart of the integrated microfluidic chip detection process in this invention (A is the plasma separation flowchart; B is the operation flowchart). Detailed Implementation

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0017] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] Example 1: Anti-NMDAR Antibody Detection Device Based on Microfluidics and Stable Cell Model

[0020] This detection device is an integrated portable system, mainly composed of three parts: an NMDAR-stabilized cell line, an integrated microfluidic chip, and a miniaturized microscopic imaging system. The structure, connection relationship, and functional coordination of each part are as follows: 1. Composition of core components: NMDAR stable cell line Clusters of stable HEK293T cells expressing the NMDA receptor GluN1 subunit (core assay substrate) Integrated microfluidic chip Upper plasma separation chamber, middle dual-pore Parylene microporous membrane (PMM), lower micro / nano structure reaction chamber, inlet, outlet, and microbead blocking unit. Miniaturized microscopic imaging system LED lighting module, 100-400x microscope lens, displacement and lifting module, electronic control module, image acquisition and analysis module, and portable casing. 2. The location and connection relationship of each component NMDAR stable cell line: It is a liquid suspension cell that is loaded into the cell loading area of ​​the lower micro-nano structure reaction chamber of the integrated microfluidic chip before use, and acts as an antigen carrier to specifically bind to the anti-NMDAR antibody in the sample; Integrated microfluidic chip ( Figure 2 The AB layer in the chip has a three-layer composite structure, consisting of an upper plasma separation chamber, a middle dual-pore PMM filter membrane, and a lower micro-nano structure reaction chamber. Five functional inlets are located on the side of the chip (microbead inlet, cell inlet, sample inlet, fluorescent secondary antibody inlet, and washing solution inlet), and a waste liquid outlet is located at the bottom. A microbead blocking unit (10 μm microbeads) is built into the outlet of the lower reaction chamber to prevent cells from flowing out with the waste liquid. Miniaturized microscopic imaging system: Positioned directly above the integrated microfluidic chip, precisely corresponding to the detection area of ​​the lower micro / nano structure reaction chamber of the chip; the LED illumination module is located below the microscope head, providing adjustable brightness bright-field / fluorescent illumination for the detection area; the displacement and lifting module is connected to the microscope head to achieve lens focal plane adjustment; the electrical control module is electrically connected to the LED illumination module, displacement and lifting module, and image acquisition and analysis module respectively to achieve automated system control; all modules are integrated into a portable housing and are detachably matched with the microfluidic chip.

[0021] 3. Structural parameters of each component (1) NMDAR stable cell line: constructed by optimizing lentiviral vector design ( Figure 1 HEK293T cells contain the GluN1 subunit nucleic acid sequence and can still stably cluster and express the GluN1 subunit after 10 passages, with a cell diameter of about 10-15 μm. (2) Integrated microfluidic chip: The whole is micron-scale and made of polydimethylsiloxane (PDMS); the upper layer is a plasma separation chamber; the middle layer is a PMM filter membrane made of high porosity and high mechanical strength material, with small pore size filter membrane (pore size ≤5μm) used for plasma separation and large pore size filter membrane (pore size 10~15 μm) used for loading stable cells; the lower layer is a micro-nano structure reaction chamber, which is a toothed microfluidic channel to achieve high throughput (40 μL / s) and high efficiency cell capture. The channel is equipped with a microcolumn array for cell dispersion and to avoid channel blockage. (3) Miniaturized microscopic imaging system: The LED illumination module can adjust the visible light / fluorescence wavelength (compatible with DyLight488, Alexa Flour594, Hoechst fluorescent labels), and the brightness is continuously adjustable; the displacement lifting module realizes the lens lifting from 0 to 50 mm, and precisely adjusts the focal plane; the image acquisition and analysis module has built-in quantitative analysis software, which can automatically count the number of cells and fluorescence intensity; the overall shell size is ≤20cm×15cm×10cm, which is easy to carry.

[0022] 4. Product working principle This device uses NMDAR-stabilized cell lines as antigen carriers and leverages the micro / nano structure of an integrated microfluidic chip to achieve efficient cell capture, rapid sample processing, and specific antigen-antibody reactions. A miniaturized microscopic imaging system enables automated imaging and quantitative analysis of the reaction results. The core principle is as follows: The principle of antigen-antibody specific binding: The GluN1 subunits expressed in clusters on the surface of stable transfected cells specifically bind to the anti-NMDAR antibody in the sample. The fluorescent secondary antibody can further bind to the bound antibody, generating a specific fluorescent signal. Microfluidic technology principle: Utilizing the hydrodynamic properties of micro- and nano-scale channels, it enables rapid separation of plasma (finger-tip blood is sufficient), efficient capture and fixation of cells, while shortening the diffusion distance of antigens and antibodies and accelerating the reaction rate; Microscopic imaging and quantitative analysis principle: Fluorescence signals are captured through a customized illumination system and microscope lens, and the fluorescence intensity and positive cell ratio are quantitatively analyzed by software to achieve qualitative and semi-quantitative detection of anti-NMDAR antibodies.

[0023] 5. Product working process All components work together to complete the entire process from sample injection to result output. The overall workflow is as follows: Figure 3 ): (1) Chip pretreatment: 10 μm microbeads are injected into the lower reaction chamber of the chip from the microbead inlet to form a microbead blocking unit to block the cell outflow channel at the outlet; the inner surface of the chip chamber is sealed by Pluronics and BSA to reduce non-specific adsorption. (2) Cell loading: The NMDAR stable cell line was injected into the chip through the cell inlet and the flow rate was adjusted to 40 μL / s so that the cells were uniformly distributed in a monolayer in the lower toothed microfluidic channel. The microcolumn array dispersed the cells to avoid blockage and complete efficient cell capture. (3) Sample processing: Finger blood is injected into the upper plasma separation chamber of the chip through the sample inlet. The plasma and blood cells are quickly separated through the middle small-pore PMM filter membrane. The separated plasma directly enters the lower reaction chamber. (4) Antigen-antibody reaction: Plasma and stable cells are incubated in the lower reaction chamber. Anti-NMDAR antibody specifically binds to the GluN1 subunit on the cell surface. Fluorescently labeled anti-IgG secondary antibody is injected through the fluorescent secondary antibody inlet and incubated in the dark for 20 min to form a "GluN1 subunit-anti-NMDAR antibody-fluorescent secondary antibody" complex. (5) Washing and purification: Inject washing buffer into the washing solution inlet to rinse the reaction chamber, remove unbound free antibodies and impurities, and discharge the waste liquid from the sample outlet; (6) Imaging analysis: Start the miniaturized microscopic imaging system, the LED illumination module provides fluorescence illumination, and adopts bright field + fluorescence dual-mode imaging. The displacement lifting module adjusts the focal plane of the lens, the image acquisition module captures the fluorescence signal in the detection area, and the analysis module automatically counts the number of positive cells and fluorescence intensity, and outputs quantitative detection results.

[0024] Example 2: Method for detecting anti-NMDAR antibodies using the above-described apparatus

[0025] This method, based on the aforementioned integrated detection device and combining immunofluorescence and microfluidic technologies, enables rapid and accurate detection of anti-NMDAR antibodies. It is applicable to the detection of human serum / cerebrospinal fluid samples. The specific steps are as follows: Step 1: Preparation and resuscitation of NMDAR stable cell lines The HEK293T stable cell line expressing the GluN1 subunit was revived from liquid nitrogen and cultured and passaged in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged (1000 r / min, 5 min), and resuspended in PBS to adjust the cell concentration to 1×10⁻⁶ cells / min. 6 Quantity / mL, for later use.

[0026] Step 2: Preprocessing of integrated microfluidic chips Chip cleaning: The PDMS microfluidic chip was ultrasonically cleaned with anhydrous ethanol and deionized water for 10 minutes in sequence, dried with nitrogen, and then sterilized under ultraviolet light for 30 minutes. Surface sealing: Inject PBS blocking solution containing 1% BSA and 0.1% Pluronics into the chip chamber, incubate at 37°C for 1 h, remove the blocking solution, and rinse 3 times with PBS; Microbead blocking: 10 μm polystyrene microbeads are injected from the microbead inlet until a dense microbead layer is formed at the outlet of the reaction chamber in the lower layer of the chip, blocking the cell outflow channel. Excess microbeads are then rinsed with PBS.

[0027] Step 3: Chip loading of stabilized cells The stable cell suspension prepared in step 1 was injected into the chip through the cell inlet, and the flow rate was adjusted to 20~40μL / s by the injection pump, so that the cells flowed slowly in the lower toothed microfluidic channel and were efficiently captured. Observe the cell distribution under a microscope until a single, uniform cell layer forms in the detection area. Then, close the cell inlet and gently rinse the channel with PBS to remove any unattached cells.

[0028] Step 4: Sample injection and plasma separation Collect 50-100 μL of blood from the subject's fingertip and immediately inject it into the upper plasma separation chamber of the chip through the sample inlet; By utilizing the sieving effect of the middle layer small-pore PMM filter membrane, blood cells are retained in the upper layer of the filter membrane, while plasma passes through the filter membrane into the lower reaction chamber, comes into contact with stable cells, and is incubated at 37°C for 30 min, allowing the anti-NMDAR antibody to fully bind to the GluN1 subunit on the cell surface.

[0029] Step 5: Incubation with fluorescent secondary antibody DyLight488-labeled anti-human IgG fluorescent secondary antibody (dilution ratio 1:200) was injected into the fluorescent secondary antibody inlet and incubated at 37°C in the dark for 20 min to allow the fluorescent secondary antibody to specifically bind to the anti-NMDAR antibody bound to the cell surface. At the same time, Hoechst staining solution (1:1000) was added to stain the cell nuclei for cell counting.

[0030] Step 6: Washing and Impurity Removal Inject pre-cooled PBS washing buffer into the washing solution inlet and rinse the lower reaction chamber of the chip three times at a flow rate of 30 μL / s, each time for 5 min, to remove unbound fluorescent secondary antibody, free protein and other impurities. The waste liquid is collected from the sample outlet and disposed of.

[0031] Step 7: Automated Imaging and Quantitative Analysis The pre-processed microfluidic chip is placed on the detection stage of the miniaturized microscopic imaging system, precisely aligned with the detection area, and the system is started. Adjust the LED illumination module to fluorescence mode, select the appropriate excitation wavelength (DyLight488: 488nm, Hoechst: 350nm), and adjust the focal plane of the microscope head through the displacement lifting module until cells and fluorescence signals are clearly observed; The image acquisition module automatically takes pictures of the detection area (≥10 fields of view), and the analysis module automatically counts: ① total number of cells (Hoechst staining positive); ② number of fluorescent positive cells (DyLight488 staining positive); ③ average fluorescence intensity. Set the detection threshold: if the proportion of fluorescent positive cells is ≥5% or the average fluorescence intensity is ≥2 times that of the normal control, it is judged as positive for anti-NMDAR antibody; otherwise, it is negative, and the system will automatically output a test report.

[0032] Step 8: Cleaning and Preservation of the Chip After the test is completed, rinse the chip channel with deionized water 3 times, blow it dry with nitrogen, and store it in a sealed container at 4°C. It can be reused 3 to 5 times.

[0033] Application example: Detecting fingertip blood samples using the apparatus of Example 1 and the method of Example 2.

[0034] Finger-prick blood samples from 30 clinically diagnosed NMDAR encephalitis patients and 15 healthy controls were used to compare the detection performance of the present invention, the clinical transient CBA method, and the radioimmunoassay (RIPA) method. The results (Table 1) show that the present invention has a sensitivity of 96.67%, a specificity of 100.00%, and an overall accuracy of 97.78% for NMDAR antibodies, all of which are significantly superior to the transient CBA method and the RIPA method. Furthermore, the present invention has a lower limit of detection, a smaller intra-assay coefficient of variation, requires only a small amount of finger-prick blood sample, and has a shorter detection time. Overall, its detection performance and practicality are significantly superior to existing routine clinical techniques.

[0035] Table 1. Detection results of different detection methods Number of positive cases / Total number of cases 29 / 30 22 / 30 19 / 30 Number of positive control cases / Total number of cases 0 / 15 1 / 15 2 / 15 Sensitivity 96.67% 73.33% 63.33% Specificity 100.00% 93.33% 86.67% accuracy 97.78% 80.00% 71.11% Positive predictive value (PPV) 100.00% 95.65% 90.48% Negative predictive value (NPV) 93.75% 63.64% 54.17% Limit of detection 0.12 ng / mL 1.5 ng / mL 2.8 ng / mL In-batch CV 2.50% 13.20% 16.70% Sample size 50 μL fingertip blood 5 mL venous blood 5 mL venous blood Detection time 2 h 5 working days 5 working days In summary, the novel method for detecting anti-NMDA receptor antibodies based on microfluidics and stable cell model technology provided by this invention can significantly reduce the detection time of NMDAR antibodies, requires a small amount of blood sample, and has high detection sensitivity, specificity, and accuracy. It can be used for rapid clinical detection of NMDA receptor antibodies, providing reliable evidence for the rapid diagnosis of NMDAR encephalitis patients.

[0036] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A device for detecting anti-NMDAR antibodies based on microfluidics and a stable cell model, characterized in that, The detection device consists of two modules: a detachably connected integrated microfluidic chip and a miniaturized microscopic imaging system. The microfluidic chip has a three-layer composite structure, which consists of an upper plasma separation chamber, a middle dual-pore PMM filter membrane, and a lower micro-nano structure reaction chamber from top to bottom. The microfluidic chip has five functional sample inlets on its side and a waste liquid outlet at its bottom. The lower micro-nano structure reaction chamber has a built-in microbead blocking unit at its outlet and a toothed microfluidic channel.

2. The anti-NMDAR antibody detection device according to claim 2, characterized in that, The five functional injection ports are the microbead inlet, cell inlet, sample inlet, fluorescent secondary antibody inlet, and washing solution inlet.

3. The anti-NMDAR antibody detection device according to claim 1, characterized in that, The microscopic imaging system is positioned directly above the integrated microfluidic chip, precisely corresponding to the detection area of ​​the lower micro-nano structure reaction chamber of the chip. The microscopic imaging system also includes an electrically connected control module, an LED lighting module, a displacement lifting module, and an image acquisition and analysis module. All of these modules are integrated and detachably matched with the microfluidic chip.

4. The method of using the anti-NMDAR antibody detection device as described in claims 1-3, characterized in that, The method is specifically as follows: (1) Chip pretreatment: 10 μm microbeads are injected into the lower reaction chamber of the chip from the microbead inlet to form a microbead blocking unit; the inner surface of the chip chamber is sealed by Pluronics and BSA. (2) Cell loading: NMDAR stable cell line was injected into the chip through the cell inlet and the flow rate was adjusted to 40 μL / s to make the cells uniformly distributed in a monolayer in the toothed microfluidic channel, thus completing efficient cell capture. (3) Sample processing: Finger blood is injected into the upper plasma separation chamber of the chip through the sample inlet. The plasma and blood cells are quickly separated through the middle small-pore PMM filter membrane. The separated plasma directly enters the lower reaction chamber. (4) Antigen-antibody reaction: Plasma and stable cells are incubated in the lower reaction chamber. Anti-NMDAR antibody specifically binds to the GluN1 subunit on the cell surface. Fluorescently labeled anti-IgG secondary antibody is injected through the fluorescent secondary antibody inlet and incubated in the dark for 20 min to form a GluN1 subunit-anti-NMDAR antibody-fluorescent secondary antibody complex. (5) Washing and purification: Inject washing buffer into the washing solution inlet to rinse the reaction chamber, remove unbound free antibodies and impurities, and discharge the waste liquid from the sample outlet; (6) Imaging analysis: Start the microscopic imaging system, automatically count the number of cells, fluorescence positivity rate and intensity, and output quantitative detection results.

5. The method of use according to claim 4, characterized in that, The NMDAR stable cell line was injected at a concentration of 1×10⁻⁶. 6 The flow rate is 20-40 μL / s, the volume of the fingertip blood sample is 50-100 μL, and the flow rate of the washing solution is 30 μL / s.

6. The use of any one of the anti-NMDAR antibody detection devices as described in claims 1-3 and / or any one of the usage methods as described in claims 4-5 in the detection of anti-NMDA receptor antibodies.

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