Antinuclear antibody IgG indirect immunofluorescence detection method and system

Abstract

The invention belongs to the technical field of biomedicine, and discloses an antinuclear antibody IgG indirect immunofluorescence detection method and system, and the detection standardization degree and the result reproducibility are significantly improved through the full-automatic system design in combination with precise mechanical transmission and an automatic sample adding device. Through an optimized FITC labeling system and a refined washing procedure, non-specific binding is remarkably reduced, and meanwhile, the binding efficiency of the antinuclear antibody and the secondary antibody is improved, so that the detection sensitivity is improved. Through the design of the automatic sample adding and sheet sealing device, the sample treatment efficiency is greatly improved, and simultaneous detection of large-scale samples can be realized. Through combination of a high-resolution imaging system and an intelligent image analysis algorithm, morphological characteristics and distribution rules of fluorescence signals are automatically extracted, and the accuracy and objectivity of a detection result are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to an antinuclear antibody IgG indirect immunofluorescence detection method and system. Background Art

[0002] Traditional antinuclear antibody testing relies on manual labor, resulting in poor reproducibility, low efficiency, and highly subjective results. Nonspecific binding and background fluorescence often reduce the signal-to-noise ratio, impacting sensitivity and accuracy. Due to the limitations of manual labor, traditional methods struggle to meet clinical needs in high-throughput testing scenarios. Traditional microscopic observation relies on manual interpretation, which is subject to significant errors and subjectivity.

[0003] Through the above analysis, the problems and defects of the existing technology are as follows:

[0004] (1) Traditional antinuclear antibody testing relies on manual operation, which has problems such as poor repeatability, low detection efficiency, and highly subjective results.

[0005] (2) In traditional detection methods, nonspecific binding and background fluorescence often lead to a decrease in the signal-to-noise ratio, affecting the sensitivity and accuracy of the detection.

[0006] (3) Due to the limitations of manual operation, traditional methods are difficult to meet clinical needs in high-throughput detection scenarios.

[0007] (4) Traditional microscopic observation relies on manual interpretation, which has the problems of large errors and strong subjectivity. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides a method and system for indirect immunofluorescence detection of antinuclear antibodies IgG.

[0009] The present invention is achieved in that an indirect immunofluorescence detection method for antinuclear antibody IgG comprises:

[0010] Step 1, sample preparation and loading:

[0011] The biological slides are pre-fixed in a specific slide box; each biological slide adopts an independent closed system;

[0012] Step 2, sample incubation and transfer;

[0013] Step 3, preliminary washing and labeling;

[0014] Step 4, secondary washing and sealing;

[0015] Step 5: image acquisition and automatic scanning;

[0016] Step 6: Data analysis and result output.

[0017] Furthermore, the sample incubation and transfer:

[0018] Each sample is precisely loaded onto the surface of a bioslide using a fully automated loading system and transferred to a specific incubation chamber for light-proof incubation. The incubation temperature and humidity are maintained stable using a PID control algorithm.

[0019] Further, the preliminary washing and labeling:

[0020] After incubation, a standardized washing procedure is used to remove unbound antibodies to ensure the optimization of the signal-to-noise ratio; then, the system adds FITC-labeled goat anti-human IgG secondary antibody through an automatic sample injection needle and incubates again in the dark.

[0021] Furthermore, the secondary washing and sealing:

[0022] The washing procedure is achieved by multiple PBS buffer washes, while controlling the liquid flow rate and washing time to ensure the complete removal of nonspecific binding; the washed biological slide is added with the sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure.

[0023] Furthermore, the image acquisition and automatic scanning:

[0024] After sealing, the biological slides are imaged using a fully automated fluorescence microscope system; the system uses a highly sensitive CCD or CMOS sensor combined with a multi-channel filter to collect FITC signals; precise positioning of the slides is achieved using an XYZ stage driven by a stepper motor.

[0025] Furthermore, the data analysis and result output:

[0026] Image analysis algorithms are used to quantitatively analyze images and extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report including the positive or negative results of antinuclear antibodies and their concentration levels.

[0027] Another object of the present invention is to provide an antinuclear antibody IgG indirect immunofluorescence detection system comprising:

[0028] The sample preparation module is used to pre-fix biological slides in a specific slide box to ensure the consistency and reproducibility of sample preparation; each biological slide uses an independent closed system;

[0029] The sample incubation module is used to accurately load each sample onto the surface of the bioslide through a fully automated loading system and transfer it to a specific incubation chamber for light-proof incubation. The incubation temperature and humidity are kept stable by a PID control algorithm.

[0030] The washing module is used to remove unbound antibodies using a standardized washing procedure after incubation to ensure the optimization of the signal-to-noise ratio. Subsequently, the system adds FITC-labeled goat anti-human IgG secondary antibody through an automated sample injection needle and incubates again in the dark.

[0031] The sealing module is used for washing procedures, which are achieved through multiple PBS buffer washes while controlling the liquid flow rate and washing time to ensure the complete removal of non-specific binding. After washing, the biological slide is filled with sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure.

[0032] The image acquisition module is used to image sealed biological slides using a fully automated fluorescence microscope system. The system uses a high-sensitivity CCD or CMOS sensor combined with a multi-channel filter (such as a 488nm excitation wavelength) to capture FITC signals. Precise positioning of the slide is achieved using an XYZ stage driven by a stepper motor.

[0033] The data analysis module is used to perform quantitative analysis of images using image analysis algorithms to extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report, including the positive or negative results of antinuclear antibodies and their concentration levels.

[0034] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0035] The present invention provides a fully automated antinuclear antibody (IgG) detection system based on indirect immunofluorescence, aiming to achieve high-throughput, standardized, and automated antinuclear antibody detection. The core steps of the system include sample loading, incubation, labeling, washing, sealing, and image acquisition and analysis.

[0036] The present invention significantly improves the standardization of detection and the reproducibility of results through a fully automated system design combined with precise mechanical transmission and an automated sample adding device.

[0037] The present invention significantly reduces nonspecific binding through an optimized FITC labeling system and a refined washing procedure, while increasing the binding efficiency between antinuclear antibodies and secondary antibodies, thereby improving detection sensitivity.

[0038] The present invention greatly improves the sample processing efficiency through the design of an automated sample adding and sealing device, and can realize large-scale simultaneous detection of samples.

[0039] The present invention combines a high-resolution imaging system with an intelligent image analysis algorithm to automatically extract the morphological characteristics and distribution patterns of fluorescent signals, greatly improving the accuracy and objectivity of the detection results.

[0040] The present invention combines a fully automated system design with an automatic dilution program to achieve automatic redilution and detection of ultra-high titer samples, simplifying manual operations while improving the accuracy of titer reporting.

[0041] The biological slides in the biological slide box provided by the present invention are independent single-person portions, which can be tested at any time, greatly shortening the detection time and reporting time limit.

[0042] 1. Detection efficiency is significantly improved:

[0043] Compared to traditional manual testing methods, this invention achieves a fully automated antinuclear antibody detection process. This improves detection efficiency by approximately five times. Based on the number of incubation wells designed for the automated instrument, a single run can simultaneously process over 100 samples, and it is projected that over 800 tests can be completed per day. This significantly reduces testing time and labor costs.

[0044] 2. Detection sensitivity and specificity are greatly improved:

[0045] The optimized FITC labeling system and refined washing steps effectively reduced the background noise of nonspecific binding. In a comparative experiment of standard samples and clinical samples, the detection sensitivity of the present invention increased by 30% and the specificity increased by 25%.

[0046] 3. Intelligent and accurate data processing:

[0047] Using a convolutional neural network (CNN) and a multi-scale feature extraction algorithm, the system achieved automatic classification and quantitative analysis of antinuclear antibody distribution patterns. Test results showed that the system achieved a detection accuracy of 98.7%, significantly outperforming traditional manual interpretation methods.

[0048] 4. High-throughput detection capabilities:

[0049] The automated detection system of the present invention can process multiple samples simultaneously, has good scalability and high throughput characteristics, and can significantly improve the laboratory's detection capabilities and service efficiency in clinical applications.

[0050] 5. High reproducibility and standardization:

[0051] Fully automated operation eliminates manual errors and ensures high reproducibility of each test through algorithm optimization. Statistical analysis has reduced the standard deviation of test results by 40%.

[0052] 6. Security and convenience:

[0053] The introduction of light-proof and humidity-control devices improves the storage stability of samples. At the same time, the automated system reduces the need for direct operation by experimenters, improving safety and detection convenience.

[0054] 7. Broad application prospects:

[0055] The present invention is applicable to antibody detection of various autoimmune diseases, including systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, etc., and has wide clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a flow chart of the indirect immunofluorescence detection method for antinuclear antibody IgG provided in an embodiment of the present invention.

[0057] Figure 2 4 is a flow chart of the sample incubation and transfer method provided by an embodiment of the present invention.

[0058] Figure 3 This is a structural block diagram of an indirect immunofluorescence detection system for antinuclear antibody IgG provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0060] Compared to traditional ANA indirect immunofluorescence, this protocol first achieved a key breakthrough in the specimen loading platform. Hep-2 cell sheets and simian liver tissue slices were fixed to a polycarbonate microcavity slide under vacuum isotonic conditions and then immediately sealed with nitrogen. Cytoskeleton and nuclear membrane antigens were briefly treated with a controlled cross-linker, increasing epitope integrity to over 96%. This protocol also avoided fluorescence attenuation caused by humidity fluctuations during conventional open-slide storage, laying the foundation for subsequent quantitative readings.

[0061] After dilution, the serum to be tested enters a constant-temperature microfluidic channel. Through the combined effects of stagnant diffusion and surface tension, the serum uniformly wets the entire cell sheet within 90 seconds. The microfluidic environment minimizes antibody concentration gradients, suppressing "edge effects" and reducing false negatives in blank areas, ensuring consistent kinetics of antigen-antibody complex formation across every field of view.

[0062] The initial rinsing stage uses pulsed laminar shearing, and the liquid flow velocity and fluid dynamic radius are adjusted to 0.9mms by instantaneous feedback. -1 , sufficient to disrupt nonspecific electrostatic adsorption without disturbing the cell monolayer. A fluorescein- and rhodamine-labeled anti-human IgG secondary antibody was then injected into the same chamber, maintaining a stable fluorescence ratio (F:P) of 4.2. This dual-labeling system avoids single-channel oversaturation and enhances the linear detection range for high-titer samples.

[0063] A secondary rinse using a low-ionic strength buffer rapidly displaces any residual unbound secondary antibody, followed by the direct application of a refractive index-matched mounting medium into the chamber. The mounting medium solidifies into an optically uniform layer within 45 seconds, eliminating the need for manual coverslipping and preventing fluorescence decay and air bubble artifacts.

[0064] The entire slide cassette is fed into the confocal scanning module. An adaptive laser beam samples the image line by line along a pre-set spiral trajectory. A real-time autofocus system uses polarization interferometry to measure Z-axis defocus and instantaneously compensates for it, ensuring the imaging plane remains focused on the midsection of the cell nucleus. Excitation-emission separation is accomplished using a multi-band filter, and the two-dimensional fluorescence matrix is ​​stitched together into a seamless full-slide digital image using a GPU pipeline, achieving a spatial resolution of 0.23 μm pxel-1.

[0065] A deep convolutional network then performs pattern recognition and quantification on the digital images. The model leverages frequency-domain features and spatial conformal transformations to simultaneously detect twelve classic fluorescence patterns, including homogeneous, fine granular, nucleolar, and centromere patterns. The model then calculates the integrated intensity value and converts it to international units (IUmL-1). The results are automatically reported via the LIMS, maintaining a coefficient of variation below 6%. This eliminates the subjective variability of manual microscopy readings, significantly shortens laboratory turnaround time, and improves interlaboratory comparability.

[0066] like Figure 1 As shown, an indirect immunofluorescence detection method for antinuclear antibody IgG provided by an embodiment of the present invention comprises the following steps:

[0067] S101, sample preparation and loading:

[0068] Biological slides (Hep-2 or monkey liver tissue slices) are pre-fixed in a specific slide box to ensure consistency and reproducibility of sample preparation. Each biological slide uses an independent closed system to prevent the influence of light and humidity on the sample.

[0069] S102, sample incubation and transfer;

[0070] S103, preliminary washing and labeling;

[0071] S104, secondary washing and sealing;

[0072] S105, image acquisition and automatic scanning;

[0073] S106, data analysis and result output.

[0074] Step 1: Sample preparation and loading

[0075] First, bioluminescent slides (IFslides) uniformly plated with HEp-2 cells or other human cell lines and fixed with methanol / ethanol are installed in a multi-channel automated slide cassette using a precision snap-fit ​​system. This system utilizes an airtight microcavity enclosure to ensure antigen structure stability and minimize cross-contamination between samples during operation. Each slide unit is uniquely identified by a 2D code / barcode, enabling traceability and system integration during testing.

[0076] Step 2: Sample incubation and transfer

[0077] Using an automated sample dispenser module (dispensing arm + visual positioning) equipped with a high-precision positioning system, the serum sample to be tested is precisely dispensed into the wells of the slide at a set dilution factor (e.g., 1:80). The slide is then transferred by a robotic arm to a constant temperature and humidity incubation chamber and incubated in the dark at 37°C and >95% RH for 30 minutes. All incubation parameters are controlled in a closed loop using a PID (proportional-integral-derivative) control algorithm to ensure temperature and humidity fluctuations are less than ±0.2°C and ±2% RH.

[0078] Step 3: Initial washing and fluorescent labeling

[0079] After incubation, the slide is automatically transferred to the multi-channel oscillating washing unit and washed with PBS or PBST buffer for 3 to 5 rounds to remove non-specifically bound serum antibodies. Subsequently, the automatic loading system loads the fluorescently labeled anti-human IgG secondary antibody (such as FITC-labeled goat anti-human IgG) and enters the incubation chamber again for a second incubation, which is controlled within 30 minutes to ensure the adequacy and specificity of the fluorescent labeling reaction.

[0080] Step 4: Secondary washing and sealing

[0081] After the second incubation, the slide undergoes a three-step gradient wash process to remove residual fluorescent antibodies. The pH of the buffer is controlled between 7.2 and 7.4 to maintain staining stability. A glycerol-based mounting medium with anti-fade reagent is then automatically added, and the negative pressure sealing module is used to evenly seal the slide, forming a stable optical interface, preventing fluorescence bleaching, and improving subsequent imaging clarity and accuracy.

[0082] Step 5: Image acquisition and automatic scanning

[0083] After sealing, the slides are transferred to a high-throughput image acquisition module. This module utilizes a fluorescence microscope system with multiple filter sets, combined with a digital CMOS camera, to scan the entire slide using autofocus, regional positioning, and image stitching algorithms. The acquisition system captures images at single-cell resolution (<1μm) and supports simultaneous multi-channel fluorescence acquisition, ensuring high-fidelity recording of typical antinuclear antibody patterns, such as speckled, perinuclear, and nucleolar patterns.

[0084] Step 6: Data analysis and result output

[0085] Image data is processed using an AI image recognition algorithm combined with a traditional morphological feature extraction model, employing a CNN + traditional SVM combined classifier for pattern recognition. The system also extracts fluorescence intensity grayscale values ​​for semi-quantitative judgments (e.g., +, ++, +++). Integrating with the LIMS system, sample identification, test information, and report output are synchronized, ultimately generating a structured, automated interpretation report that supports manual review and a closed-loop feedback optimization mechanism.

[0086] like Figure 2 As shown, the sample incubation and transfer provided by the embodiment of the present invention:

[0087] S201, each sample (serum) is accurately loaded onto the surface of the biological slide by a fully automatic sample loading system and transferred to a specific incubation chamber for light-proof incubation;

[0088] S202, the incubation temperature and humidity are kept stable by a PID control algorithm to ensure optimal conditions for the antigen-antibody reaction.

[0089] Preliminary washing and labeling provided by the embodiment of the present invention:

[0090] After incubation, a standardized washing procedure (such as three PBS buffer washes) is used to remove unbound antibodies to ensure the optimization of the signal-to-noise ratio; then, the system adds FITC-labeled goat anti-human IgG secondary antibody (dilution optimized to 1:200 to 1:500) through an automatic sample injection needle and incubates again in the dark.

[0091] Secondary washing and sealing provided by the embodiment of the present invention:

[0092] The washing procedure is achieved by multiple PBS buffer washes, while controlling the liquid flow rate and washing time to ensure the complete removal of nonspecific binding; the washed biological slide is added with the sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure.

[0093] The image acquisition and automatic scanning provided by the embodiment of the present invention:

[0094] After sealing, the biological slides are imaged using a fully automated fluorescence microscope system. The system uses a highly sensitive CCD or CMOS sensor in combination with a multi-channel filter (such as a 488nm excitation wavelength) to capture the FITC signal. Precise positioning of the slide is achieved using an XYZ stage driven by a stepper motor, ensuring the reproducibility and accuracy of each scan.

[0095] Data analysis and result output provided by the embodiment of the present invention:

[0096] Image analysis algorithms (such as the maximum inter-class variance method Otsu segmentation and convolutional neural network (CNN)) are used to quantitatively analyze images and extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report, including the positive or negative results of antinuclear antibodies and their concentration levels.

[0097] like Figure 3 As shown, an indirect immunofluorescence detection system for antinuclear antibody IgG provided by an embodiment of the present invention includes:

[0098] The sample preparation module is used to pre-fix biological slides (Hep-2 or monkey liver tissue slices) in a specific slide box to ensure consistency and reproducibility of sample preparation. Each biological slide uses an independent closed system to prevent the influence of light and humidity on the sample.

[0099] The sample incubation module is used to accurately load each sample (serum) onto the surface of the bioslide through a fully automated loading system and transfer it to a specific incubation chamber for light-proof incubation. The incubation temperature and humidity are kept stable by a PID control algorithm to ensure optimal conditions for the antigen-antibody reaction.

[0100] The washing module is used to remove unbound antibodies after incubation using a standardized washing procedure (e.g., three PBS washes) to optimize the signal-to-noise ratio. Subsequently, the system adds FITC-labeled goat anti-human IgG secondary antibody (optimized dilution of 1:200 to 1:500) via an automated sample injection needle and incubates again in the dark.

[0101] The sealing module is used for washing procedures, which are achieved through multiple PBS buffer washes while controlling the liquid flow rate and washing time to ensure the complete removal of non-specific binding. After washing, the biological slide is filled with sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure.

[0102] The image acquisition module is used to image sealed biological slides using a fully automated fluorescence microscope system. The system uses a high-sensitivity CCD or CMOS sensor combined with a multi-channel filter (such as a 488nm excitation wavelength) to capture FITC signals. Precise positioning of the slide is achieved using an XYZ stage driven by a stepper motor, ensuring reproducibility and accuracy of each scan.

[0103] The data analysis module is used to perform quantitative analysis of images using image analysis algorithms (such as the maximum inter-class variance method Otsu segmentation and convolutional neural network (CNN)) to extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report, including the positive or negative results of antinuclear antibodies and their concentration levels.

[0104] This invention is suitable for the auxiliary diagnosis and screening of autoimmune diseases, especially the detection of antinuclear antibodies (ANA) for diseases such as systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, and rheumatoid arthritis. Related products include, but are not limited to: automated indirect immunofluorescence detection systems for clinical immunology laboratories, integrated in vitro diagnostic (IVD) devices, intelligent fluorescence pattern recognition terminals, and full-process immunofluorescence training platforms for teaching and scientific research.

[0105] The present invention realizes a high-throughput, low-error, and highly repeatable antinuclear antibody detection process by integrating six functional modules: automated sample loading, incubation, washing, sealing, imaging, and analysis. The detection consistency is better than that of manual operation, the signal-to-noise ratio is improved by more than 15%, and the repeatability error is controlled within ±5%. By comparing the test with the traditional manual method (n = 100 serum samples), the positive detection rate of this system is increased by about 12%, especially in low-titer samples. It has a stronger discrimination ability. In addition, the AI-assisted analysis system achieves a consistency of more than 95% with manual judgment in the identification of antinuclear antibody patterns.

[0106] Human cell lines (such as HEp-2) or frozen sections of monkey liver that meet IF standards are used as antigen substrates. After ethanol dehydration and freeze-drying, they are fixed to glass slides. Each slide is inserted into a slide cassette using a silicone snap-fit ​​system. A polymer sealing membrane isolates the slide from ambient humidity and visible light interference, effectively maintaining the stability of the antigen configuration and fluorescence background. The system automatically identifies the slide information and binds it to the sample number, enabling LIS information traceability.

[0107] A diluted serum sample (e.g., 1:80) is evenly dispensed onto the slide surface using a dispenser needle controlled by an XYZ positioning system, ensuring that the droplet covers the antigen area and avoids edge loss. The slide is then transferred by a robotic arm to a PID-controlled incubation chamber, maintained at 37±0.2°C and 95% relative humidity, for 30 minutes to ensure that the IgG antibodies fully bind to the nuclear antigen.

[0108] After sample incubation, the slides enter a rotating wash module, where unbound IgG is washed from the surface using three cycles of high-speed flow of PBS or PBST buffer. Subsequently, a FITC-conjugated goat anti-human IgG secondary antibody at an optimized dilution (1:200-1:500) is applied, and the incubation process is repeated in the dark. This step ensures a balance between signal enhancement and inhibition of nonspecific binding.

[0109] After a second standardized wash, the system automatically dispenses a glycerol-based mounting solution containing an antifade agent such as DABCO or p-phenylenediamine onto the center of the slide. A vacuum pressure device then smoothly applies the cover slip, creating a uniform optical interface that inhibits fluorescence bleaching and enhances image clarity. The film thickness is controlled within the 80–100 μm range to accommodate imaging with high-NA objectives.

[0110] Completed coverslip slides are automatically loaded onto a CCD or CMOS imaging platform equipped with a high-NA (>0.75) fluorescence objective. The system integrates a 488nm excitation light source and FITC filter set, combined with autofocus and a 2D motorized stage for full-field scanning. Image acquisition utilizes a multi-frame averaging algorithm for signal enhancement and supports flat-field correction and edge denoising.

[0111] The acquired fluorescence images are automatically segmented using the Otsu method, and a trained CNN image recognition model extracts morphological features (such as particle density, fluorescence intensity, and pattern distribution) to determine the type of antinuclear antibody (such as speckled, nucleolar, or homogeneous). The data is automatically exported to the LIMS system and a structured report is generated, supporting terminal review by physicians and result feedback, achieving closed-loop management of the entire results process.

[0112] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for indirect immunofluorescence detection of antinuclear antibody IgG, characterized in that: The following steps are involved: (1) Sample preparation and loading: The human cell biological slide fixed with methanol or ethanol is mounted in an automated slide box with an independent closed structure; (2) Sample incubation and transfer: The diluted serum sample is loaded onto the slide surface by a fully automatic sample loading system. The slide is then transferred to a constant temperature and humidity incubation chamber and incubated in the dark at 37°C and relative humidity greater than 95%. The temperature and humidity are controlled in real time by a closed-loop system based on a PID control algorithm. (3) Preliminary washing and fluorescent labeling: The unbound antibody is washed away with a buffer solution through an oscillating washing module, and then a FITC-labeled anti-human IgG secondary antibody is automatically loaded and incubated for a second time; (4) Secondary washing and sealing: After washing with gradient PBS to remove excess fluorescent antibodies, the slides were loaded with anti-fluorescence quenching sealing solution and sealed evenly using a negative pressure sealing module; (5) Image acquisition and automatic scanning: The sealed slide is transferred to the fluorescence microscopy image acquisition module, and the entire slide is scanned using autofocus, multiple filter sets, and a digital imaging system to obtain multi-channel fluorescence images; (6) Data analysis and result output: By integrating convolutional neural networks with traditional morphological analysis methods, the image data is subjected to pattern recognition and quantitative evaluation of fluorescence intensity, and the sample identification code is combined with the LIMS system to output a structured test report.

2. The indirect immunofluorescence detection method for antinuclear antibody IgG according to claim 1, wherein: The sample incubation and transfer: Each sample is precisely loaded onto the surface of a bioslide using a fully automated loading system and transferred to a specific incubation chamber for light-proof incubation. The incubation temperature and humidity are maintained stable using a PID control algorithm.

3. The indirect immunofluorescence detection method for antinuclear antibody IgG according to claim 1, wherein The initial washing and labeling: After incubation, a standardized washing procedure is used to remove unbound antibodies to ensure the optimization of the signal-to-noise ratio; then, the system adds FITC-labeled goat anti-human IgG secondary antibody through an automatic sample injection needle and incubates again in the dark.

4. The indirect immunofluorescence detection method for antinuclear antibody IgG according to claim 1, wherein: The secondary washing and sealing: The washing procedure is achieved by multiple PBS buffer washes, while controlling the liquid flow rate and washing time to ensure the complete removal of nonspecific binding; the washed biological slide is added with the sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure.

5. The indirect immunofluorescence detection method for antinuclear antibody IgG according to claim 1, wherein: Image acquisition and automatic scanning: After sealing, the biological slides are imaged using a fully automated fluorescence microscope system; the system uses a highly sensitive CCD or CMOS sensor combined with a multi-channel filter to collect FITC signals; precise positioning of the slides is achieved using an XYZ stage driven by a stepper motor.

6. The indirect immunofluorescence detection method for antinuclear antibody IgG according to claim 1, wherein: The data analysis and result output: Image analysis algorithms are used to quantitatively analyze images and extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report including the positive or negative results of antinuclear antibodies and their concentration levels.

7. An indirect immunofluorescence detection system for antinuclear antibody IgG for implementing the indirect immunofluorescence detection method for antinuclear antibody IgG according to any one of claims 1 to 6, characterized in that: The antinuclear antibody IgG indirect immunofluorescence detection system comprises: The sample preparation module is used to pre-fix biological slides in specific slide boxes to ensure consistency and reproducibility of sample preparation; each biological slide uses an independent closed system; The sample incubation module is used to accurately load each sample onto the surface of the bioslide through a fully automated loading system and transfer it to a specific incubation chamber for light-proof incubation. The incubation temperature and humidity are kept stable by a PID control algorithm. The washing module is used to remove unbound antibodies using a standardized washing procedure after incubation to ensure the optimization of the signal-to-noise ratio. Subsequently, the system adds FITC-labeled goat anti-human IgG secondary antibody through an automated sample injection needle and incubates again in the dark. The sealing module is used for washing procedures, which are achieved through multiple PBS buffer washes while controlling the liquid flow rate and washing time to ensure the complete removal of non-specific binding. After washing, the biological slide is filled with sealing medium by an automatic sample injection needle and automatically covered with a glass slide to form a stable sealing structure. The image acquisition module is used to image sealed biological slides using a fully automated fluorescence microscope system. The system uses a high-sensitivity CCD or CMOS sensor combined with a multi-channel filter (such as a 488nm excitation wavelength) to capture FITC signals. Precise positioning of the slide is achieved using an XYZ stage driven by a stepper motor. The data analysis module is used to perform quantitative analysis of images using image analysis algorithms to extract information such as fluorescence intensity, morphological characteristics, and position distribution; the system automatically generates a report, including the positive or negative results of antinuclear antibodies and their concentration levels.

Images