A method for detecting neuroimmunological antibodies based on whole brain tissue array sections

Abstract

The application provides a kind of neuroimmunological antibody detection method based on whole brain tissue, comprising obtaining whole brain tissue of perfusion clearance blood components, after quick freezing embedding, the preparation of standardized section covering at least two brain regions is continuously sectioned, after being fixed with organic solvent, incubated with the sample to be measured, and the non-specific coloring is eliminated by using two-way blocking strategy. The slice array quality control system containing reference antibody binding site, fluorescent microspheres and internal reference brain area is constructed, the staining effectiveness is judged and the fluorescence intensity normalization correction is realized. The same tissue section that completes TBA detection is subjected to mild elution treatment, while maintaining RNA integrity, and then combined with spatial transcriptomics analysis, the spatial registration of fluorescence signal pattern and gene expression profile and the screening of differentially expressed genes are realized. The standardization degree and quantitative reliability of neuroimmunological antibody detection are significantly improved, and a new means for high-throughput screening of autoantibody target antigens is provided.

Description

Technical Field

[0001] This invention relates to the field of immunofluorescence detection technology, specifically to a method for detecting neuroimmunoantibodies based on whole-brain tissue array sections. Background Technology

[0002] The diagnosis and mechanistic study of neuroimmunological diseases, such as autoimmune encephalitis, multiple sclerosis, and neuromyelitis optica spectrum disorders, heavily rely on the detection of autoantibodies in brain tissue. Tissue-based indirect immunofluorescence assay (TBA) is currently the recognized gold standard for screening neuroimmune autoantibodies. It involves incubating serum or cerebrospinal fluid samples with animal brain tissue sections, using fluorescently labeled secondary antibodies to detect anti-neural tissue antibodies in the sample, and using fluorescence signal patterns to aid in disease diagnosis.

[0003] Tissue-based indirect immunofluorescence (TBA) is widely used for detecting antibodies associated with diseases such as autoimmune encephalitis or paraneoplastic syndromes, including antibodies against NMDAR, GABABR, GABAAR, LGI1, and CASPR2. Different staining patterns can be used to localize antibodies to tissues and cells, aiding in the determination of the presence of unknown antibodies. For example, prior art Chinese patent application CN115184326A (published on October 14, 2022) discloses an immunofluorescence detection method based on brain tissue sections. Its basic technical route includes: rapidly freezing animal brain tissue in liquid nitrogen; preparing frozen sections of brain tissue; fixing with paraformaldehyde; incubating with the test sample; fluorescent labeling; and microscopic interpretation. Chinese patent application CN118464566A (published on August 9, 2024) discloses an improved method for detecting TBA anti-brain antibodies. The technical solution includes: selecting male Lewis rats, performing cardiac perfusion with physiological saline, decapitating and removing the brain, washing with PBS and fixing, dehydrating, embedding and freezing, and sectioning the tissue on a cryostat to obtain brain tissue sections for TBA anti-brain antibody detection. The method removes residual blood components in the interstitial spaces of the brain tissue through cardiac perfusion, thereby reducing non-specific background color enhancement during the detection process.

[0004] However, existing TBA methods still have the following technical shortcomings:

[0005] (1) Non-standard brain tissue perfusion and processing: Traditional methods for perfusion of experimental animals lack clear and objective criteria for determining the perfusion endpoint, which can easily lead to residual blood components in the brain and cause background fluorescence interference; inconsistent tissue quick-freezing conditions can easily cause ice crystal damage and affect the quality of the slides.

[0006] (2) Lack of standardization in slice layers: Different laboratories or operators select different brain regions for slices, resulting in poor comparability of test results. In particular, the localization of key brain regions such as the hippocampus, basal ganglia, and thalamus relies on the operator's experience and lacks objective means of confirming anatomical localization.

[0007] (3) Non-specific staining interference: Rheumatoid factor, heterophilic antibodies, tissue endogenous Fc receptors, hydrophobic binding sites, etc. in the sample to be tested can cause false positives or background signals. Existing methods have failed to systematically handle the interference from both the tissue end and the sample end at the same time.

[0008] (4) Lack of effective quality control system: The TBA detection process is long and has many influencing factors, but the existing methods lack built-in quality control markers and quantitative normalization methods. The results of different batches and different slices are difficult to compare directly, which limits the standardization of its clinical and scientific research applications.

[0009] (5) Cannot be combined with downstream omics analysis: After traditional TBA detection, the antibody-fluorescent complex on the tissue section is difficult to remove without destroying the RNA, which makes it impossible to obtain antibody binding mode and spatial gene expression profile information on the same section at the same time, thus restricting the high-throughput screening and functional study of self-antibody target antigens.

[0010] Therefore, developing a standardized, highly reliable, quality-controllable method for detecting neuroimmune antibodies in whole brain tissue that can be combined with spatial transcriptomics has significant clinical application value and scientific research significance. Summary of the Invention

[0011] To address the above problems, this invention provides a method for detecting neuroimmune antibodies based on whole brain tissue, comprising the following steps:

[0012] (1) Obtaining whole brain tissue, which is obtained by perfusing the heart with phosphate buffer or saline to remove blood components from the brain;

[0013] (2) The obtained whole brain tissue was placed in an embedding mold, tissue cryopreservation medium was added, and the tissue was pre-cooled with isopentane and then rapidly frozen uniformly.

[0014] (3) The cryo-embedded whole brain tissue was serially sliced ​​in a cryostat to prepare standardized tissue sections covering at least two different brain regions;

[0015] (4) Use organic solvents to fix the sections by precipitation;

[0016] (5) The fixed sections were incubated with the test sample, washed and then incubated with fluorescently labeled secondary antibody for tissue-based indirect immunofluorescence (TBA) detection;

[0017] Step (5) also includes non-specific staining: the tissue end is blocked with a blocking solution containing Fc receptor blocking components, and the sample end is pretreated to remove rheumatoid factor and / or heterophilic antibodies.

[0018] In step (1), the liver color changes from dark red to light red as the criterion for determining that the perfusion is sufficient; in step (2), the isopentane is pre-cooled by liquid nitrogen, and the embedding mold is immersed in the pre-cooled isopentane for quick freezing.

[0019] The standardized slices in step (3) are sagittal slices with a thickness of 4 to 8 μm. The slices are selected from at least two different layers within a range of 0.5 mm to 2.5 mm from the midsagittal plane. The brain regions are selected from at least two of the hippocampus, basal ganglia, thalamus, brainstem, or cerebellum.

[0020] Further, in step (4), the organic solvent is acetone, methanol or a combination thereof; in step (5), when the sample to be tested is serum, it is diluted at a ratio of 1:5 to 1:20, and when it is cerebrospinal fluid, it is not diluted or diluted at a ratio of 1:1 to 1:5, and the incubation time of the fluorescently labeled secondary antibody is 30 to 90 minutes.

[0021] Meanwhile, in step (3), every 5 to 10 slices, one slice is taken for hematoxylin staining to confirm that the hippocampus has been reached. Hematoxylin staining is used to confirm the arrival of the hippocampus as the anatomical location confirmation point of the slice layer.

[0022] Preferably, the whole brain tissue is selected from rodents or primates, and the tissue cryopreservation medium is OCT embedding agent.

[0023] Furthermore, in step (2), the quick-freezing time is 5 to 15 seconds, and then it is transferred to -80°C for storage.

[0024] Further, in step (3), the standardized slice includes a first standard slice 0.5-1.5 mm from the midsagittal plane and a second standard slice 1.5-2.5 mm from the midsagittal plane.

[0025] Furthermore, in step (5), when the sample to be tested is serum, the dilution ratio of the secondary antibody is 1:300 to 1:800, and when it is cerebrospinal fluid, the dilution ratio of the secondary antibody is 1:100 to 1:300.

[0026] Furthermore, in step (5), the blocking solution also contains a hydrophobic binding blocking component and an endogenous IgG binding site inhibitory component.

[0027] Furthermore, it also includes a mounting observation procedure: after adding the mounting medium, observe under a fluorescence microscope, or observe after storing at 2–8°C in the dark.

[0028] Furthermore, it also includes quality control steps: simultaneous testing with known positive and negative control samples to verify the effectiveness of the testing system.

[0029] In addition, to improve the accuracy of detection, step (3) also includes making a slice array from the prepared standardized tissue slices, the slice array containing at least one detection reaction area and at least one quality control reaction area, the quality control reaction area containing at least one of the following quality control markers: a reference antibody binding site with a pre-set known concentration gradient, fluorescent microspheres embedded in the embedding medium, naturally occurring endogenous internal reference brain region, or independent positive quality control slices and negative quality control slices.

[0030] Meanwhile, step (5) also includes a quality control step: the sample to be tested is simultaneously stained with immunofluorescence along with the detection reaction area and the quality control reaction area, the fluorescence signal intensity of the quality control reaction area is detected to determine the effectiveness of the staining process, and the fluorescence signal of the quality control reaction area is used to normalize and correct the fluorescence intensity of the detection reaction area.

[0031] Furthermore, the reference antibody binding site is obtained by spotting an antigen solution of known concentration onto the non-tissue area at the edge of the slide using a spotting instrument. The spotting diameter is 50-200 μm and includes at least two concentration levels among negative, weakly positive, moderately positive, and strongly positive.

[0032] Furthermore, the fluorescent microspheres are selected from polystyrene fluorescent microspheres, silica fluorescent microspheres, or quantum dot microspheres, and are mixed into the tissue cryopreservation medium before embedding in step (2), with a final concentration of 10. 4 ~10 7 per mL.

[0033] Furthermore, the endogenous reference brain region is selected from the white matter region, the corpus callosum, or the periventricular region.

[0034] Furthermore, each reaction zone of the slice array is equipped with an independent QR code, barcode, or RFID tag for recording animal batch, slice date, operator, slice layer number, and quality control data.

[0035] Furthermore, the criteria for judging the effectiveness of the staining process are as follows: the fluorescence signal intensity of the positive control reaction area is greater than the preset positive threshold, and the fluorescence signal intensity of the negative control reaction area is less than the preset negative threshold, and the fluorescence signal intensity of the reference antibody binding site is monotonically correlated with its preset concentration gradient.

[0036] Furthermore, the normalization correction is performed using the formula: Corrected target brain region fluorescence intensity = (Measured fluorescence intensity of target brain region / Measured fluorescence intensity of internal reference region) × Standard internal reference fluorescence intensity value; where the internal reference region is selected from the white matter region, corpus callosum, or fluorescent microsphere region. The preset standard internal reference fluorescence intensity value is derived from the average internal reference intensity of healthy control slides.

[0037] It also includes a method for combining antibody detection and gene expression profiling analysis of whole brain tissue sections, comprising: using the whole brain tissue-based neuroimmunoantibody detection method to detect fluorescence signal patterns of whole brain tissue sections by tissue-based indirect immunofluorescence (TBA), then performing a mild elution process on the same tissue sections to remove antibodies and fluorescent markers while maintaining RNA integrity, then performing spatial transcriptomics analysis on the eluted same tissue sections to obtain gene expression profiles, and then performing spatial registration and joint analysis of fluorescence signal patterns and gene expression profiles to identify differentially expressed genes.

[0038] A kit for a method of combining antibody detection and gene expression profiling analysis of whole brain tissue sections, comprising reagents for performing the whole brain tissue-based neuroimmunoantibody detection method, as well as elution buffer for mild elution and reagents for spatial transcriptome analysis.

[0039] Furthermore, the mild elution process uses 0.05–0.2 M glycine-hydrochloric acid buffer (pH 2.5–3.0) and incubation at 4°C for 5–15 minutes; preferably, the elution buffer also contains 0.5–2 M urea, 0.01–0.1% Triton X-100, and 1–5 mM DTT. These treatment conditions effectively remove antibodies and fluorescent markers while maintaining the integrity of tissue RNA.

[0040] It should be noted that antibodies can also be removed by SDS / β-mercaptoethanol treatment or proteinase K treatment, but the following conditions must be strictly controlled to ensure the stability of the detection results. The elution solution is: 1-2% SDS, 50mM Tris-HCl pH6.8, 100mM β-mercaptoethanol, incubated at 55℃ for 10-30 minutes; or proteinase K 1-10μg / mL, incubated at 37℃ for 2-10 minutes.

[0041] Following mild elution, an RNA integrity assessment step is included: Parallel control sections are used, consisting of tissue sections adjacent to the test section, from which RNA was extracted without TBA staining and elution. The RIN value is measured using a bioanalyzer; an RIN ≥ 6.0 is considered acceptable. Parallel control sections reflect the RNA quality of the tissue itself; if they are acceptable, the test section, after elution, will generally also meet the requirements for downstream analysis. Alternatively, tissue can be collected from the same eluted section, and the Ct value of the internal reference gene GAPDH is measured by qPCR. An increase of no more than 3 in the Ct value compared to the untreated control is considered acceptable. The untreated control refers to adjacent sections that have not undergone TBA staining and elution.

[0042] Furthermore, the spatial transcriptomics analysis was selected from the 10x Genomics Visium, Stereo-seq, GeoMxDSP, Slide-seq, or XYZeq platforms.

[0043] Furthermore, the spatial registration employs a feature point matching algorithm, where the feature points are selected from brain tissue boundaries, brain region contours, blood vessel distribution, or endogenous fluorescent markers.

[0044] Furthermore, the joint analysis includes: defining the fluorescent signal positive region detected by TBA as the antibody binding region, performing differential analysis on the gene expression profile of this region and the non-binding region, and screening specifically highly expressed genes as candidate target antigen genes.

[0045] Furthermore, the secondary antibody marker used in the TBA detection is a photolyzable fluorescent dye or a biotin-avidin system.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention uses the change in liver color from dark red to light red as an objective criterion for determining adequate perfusion, avoiding subjective differences based on experience. Liquid nitrogen is used for pre-cooling isopentane for uniform quick-freezing, with a limited quick-freezing time of 5–15 seconds and subsequent storage at -80°C, effectively reducing ice crystal formation and ensuring the integrity of tissue morphology and antigenicity.

[0048] This invention limits the thickness of sagittal sections to 4–8 μm and specifies that sections are taken from at least two different standard planes within a range of 0.5 mm to 2.5 mm from the midsagittal plane. The first standard plane is 0.5–1.5 mm thick, and the second standard plane is 1.5–2.5 mm thick, ensuring comparable brain region combinations across different batches of experiments. Simultaneously, hematoxylin staining at intervals of 5–10 sections confirms the presence of the hippocampus, providing objective anatomical localization points for the section planes.

[0049] This invention simultaneously addresses non-specific interference from both the tissue and sample ends: the tissue end employs a blocking solution containing Fc receptor blocking components, hydrophobic binding blocking components, and endogenous IgG binding site inhibitory components; the sample end undergoes pretreatment to remove rheumatoid factor and / or heterophile antibodies. This bidirectional blocking strategy significantly reduces background fluorescence and improves detection specificity.

[0050] This invention prepares standardized tissue sections into a section array, comprising a detection reaction zone and a quality control reaction zone. Quality control biomarkers include reference antibody binding sites with pre-set concentration gradients, fluorescent microspheres embedded in an embedding medium, an endogenous internal reference brain region, and independent positive / negative quality control sections. Through simultaneous staining and fluorescence signal detection, the effectiveness of the staining process can be determined, and normalization correction is performed using the formula (corrected intensity = measured intensity / internal reference intensity × standard internal reference intensity) to eliminate batch-to-batch differences and achieve quantitative comparability.

[0051] This invention employs a gentle elution process, incubating at 4°C for 5–15 minutes with 0.05–0.2M glycine-hydrochloric acid buffer (pH 2.5–3.0), preferably with the addition of urea, Triton X-100, and DTT, to effectively remove antibodies and fluorescent markers while maintaining tissue RNA integrity. RNA integrity is assessed using parallel control sections or qPCR, with RIN ≥ 6.0 or GAPDH Ct value increase ≤ 3, ensuring the quality of downstream analysis. The same eluted section can be directly used for spatial transcriptomics analysis using 10x Genomics Visium and Stereo-seq, spatially registering fluorescence signal patterns with gene expression profiles to identify differentially expressed genes, providing a high-throughput method for screening autoantibody target antigens.

[0052] This invention also provides a kit for TBA detection and TBA-spatial transcriptome coupling, which includes a standardized slice array, fixative, staining agent, mild elution buffer and spatial transcriptome analysis reagents. It is easy to use and facilitates the standardization and promotion of this technology in different laboratories.

[0053] In summary, this invention significantly improves the standardization, reliability, and quantitative capability of whole-brain tissue neuroimmunological antibody detection, and innovatively realizes the simultaneous use of antibody detection and spatial transcriptomics, which has broad application prospects in the fields of neuroimmunological disease diagnosis, autoantibody target antigen discovery, and mechanism research. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 The experimental results were observed under a fluorescence microscope;

[0056] Figure 2 A comparison chart showing the effects of different fixatives;

[0057] Figure 3 This is a comparison chart of the detection and processing results of the neuroimmune antibody detection method of the present invention;

[0058] Figure 4 This is an example of the detection results of neuroimmunological antibodies in whole brain tissue; in the cortex: neuronal cell nuclei are positive; in the hippocampus: pyramidal cells are positive; in the cerebellum: granular layer is positive; and in the white matter: negative. The above results suggest the possible presence of anti-nuclear antibodies, and interference from antinuclear antibodies needs to be ruled out.

[0059] Figure 5 This is an example of the results of another whole-brain tissue neuroimmunological antibody detection method; Cortex: positive for nerve cell membranes; Hippocampus: positive for molecular layers; Cerebellum: positive for molecular layers; White matter: negative. The above results suggest the possible presence of anti-nerve cell membrane antibodies, and screening for autoimmune encephalitis-related antibodies is recommended. Detailed Implementation

[0060] Example 1:

[0061] A method for detecting neuroimmune antibodies based on whole brain tissue includes the following steps:

[0062] (1) Obtaining whole brain tissue, which is obtained by perfusing the heart with phosphate buffer or saline to remove blood components from the brain;

[0063] (2) The obtained whole brain tissue was placed in an embedding mold, tissue cryopreservation medium was added, and the tissue was pre-cooled with isopentane and then rapidly frozen uniformly.

[0064] (3) The cryo-embedded whole brain tissue was serially sliced ​​in a cryostat to prepare standardized tissue sections covering at least two different brain regions;

[0065] (4) Use organic solvents to fix the sections by precipitation;

[0066] (5) The fixed sections were incubated with the test sample, washed and then incubated with fluorescently labeled secondary antibody for tissue-based indirect immunofluorescence (TBA) detection;

[0067] Step (5) also includes non-specific staining: the tissue end is blocked with a blocking solution containing Fc receptor blocking components, and the sample end is pretreated to remove rheumatoid factor and / or heterophilic antibodies.

[0068] In step (1), the liver color changes from dark red to light red as the criterion for determining that the perfusion is sufficient; in step (2), the isopentane is pre-cooled by liquid nitrogen, and the embedding mold is immersed in the pre-cooled isopentane for quick freezing.

[0069] The standardized slices in step (3) are sagittal slices with a thickness of 4 to 8 μm. The slices are selected from at least two different layers within a range of 0.5 mm to 2.5 mm from the midsagittal plane. The brain regions are selected from at least two of the hippocampus, basal ganglia, thalamus, brainstem, or cerebellum.

[0070] Further, in step (4), the organic solvent is acetone, methanol or a combination thereof; in step (5), when the sample to be tested is serum, it is diluted at a ratio of 1:5 to 1:20, and when it is cerebrospinal fluid, it is not diluted or diluted at a ratio of 1:1 to 1:5, and the incubation time of the fluorescently labeled secondary antibody is 30 to 90 minutes.

[0071] Meanwhile, in step (3), every 5 to 10 slices, one slice is taken for hematoxylin staining to confirm that the hippocampus has been reached. Hematoxylin staining is used to confirm the arrival of the hippocampus as the anatomical location confirmation point of the slice layer.

[0072] Preferably, the whole brain tissue is selected from rodents or primates, and the tissue cryopreservation medium is OCT embedding agent.

[0073] Furthermore, in step (2), the quick-freezing time is 5 to 15 seconds, and then it is transferred to -80°C for storage.

[0074] Further, in step (3), the standardized slice includes a first standard slice 0.5-1.5 mm from the midsagittal plane and a second standard slice 1.5-2.5 mm from the midsagittal plane.

[0075] Furthermore, in step (5), when the sample to be tested is serum, the dilution ratio of the secondary antibody is 1:300 to 1:800, and when it is cerebrospinal fluid, the dilution ratio of the secondary antibody is 1:100 to 1:300.

[0076] Furthermore, in step (5), the blocking solution also contains a hydrophobic binding blocking component and an endogenous IgG binding site inhibitory component.

[0077] Furthermore, it also includes a mounting observation procedure: after adding the mounting medium, observe under a fluorescence microscope, or observe after storing at 2–8°C in the dark.

[0078] Furthermore, it also includes quality control steps: simultaneous testing with known positive and negative control samples to verify the effectiveness of the testing system.

[0079] In addition, to improve the accuracy of detection, step (3) also includes making a slice array from the prepared standardized tissue slices, the slice array containing at least one detection reaction area and at least one quality control reaction area, the quality control reaction area containing at least one of the following quality control markers: a reference antibody binding site with a pre-set known concentration gradient, fluorescent microspheres embedded in the embedding medium, naturally occurring endogenous internal reference brain region, or independent positive quality control slices and negative quality control slices.

[0080] Meanwhile, step (5) also includes a quality control step: the sample to be tested is simultaneously stained with immunofluorescence along with the detection reaction area and the quality control reaction area, the fluorescence signal intensity of the quality control reaction area is detected to determine the effectiveness of the staining process, and the fluorescence signal of the quality control reaction area is used to normalize and correct the fluorescence intensity of the detection reaction area.

[0081] Furthermore, the reference antibody binding site is obtained by spotting an antigen solution of known concentration onto the non-tissue area at the edge of the slide using a spotting instrument. The spotting diameter is 50-200 μm and includes at least two concentration levels among negative, weakly positive, moderately positive, and strongly positive.

[0082] Furthermore, the fluorescent microspheres are selected from polystyrene fluorescent microspheres, silica fluorescent microspheres, or quantum dot microspheres, and are mixed into the tissue cryopreservation medium before embedding in step (2), with a final concentration of 10. 4 ~10 7 per mL.

[0083] Furthermore, the endogenous reference brain region is selected from the white matter region, the corpus callosum, or the periventricular region.

[0084] Furthermore, each reaction zone of the slice array is equipped with an independent QR code, barcode, or RFID tag for recording animal batch, slice date, operator, slice layer number, and quality control data.

[0085] Furthermore, the criteria for judging the effectiveness of the staining process are as follows: the fluorescence signal intensity of the positive control reaction area is greater than the preset positive threshold, and the fluorescence signal intensity of the negative control reaction area is less than the preset negative threshold, and the fluorescence signal intensity of the reference antibody binding site is monotonically correlated with its preset concentration gradient.

[0086] Furthermore, the normalization correction is performed using the formula: Corrected target brain region fluorescence intensity = (Measured fluorescence intensity of target brain region / Measured fluorescence intensity of internal reference region) × Standard internal reference fluorescence intensity value; where the internal reference region is selected from the white matter region, corpus callosum, or fluorescent microsphere region. The preset standard internal reference fluorescence intensity value is derived from the average internal reference intensity of healthy control slides.

[0087] It also includes a method for combining antibody detection and gene expression profiling analysis of whole brain tissue sections, comprising: using the whole brain tissue-based neuroimmunoantibody detection method to detect fluorescence signal patterns of whole brain tissue sections by tissue-based indirect immunofluorescence (TBA), then performing a mild elution process on the same tissue sections to remove antibodies and fluorescent markers while maintaining RNA integrity, then performing spatial transcriptomics analysis on the eluted same tissue sections to obtain gene expression profiles, and then performing spatial registration and joint analysis of fluorescence signal patterns and gene expression profiles to identify differentially expressed genes.

[0088] A kit for a method of combining antibody detection and gene expression profiling analysis of whole brain tissue sections, comprising reagents for performing the whole brain tissue-based neuroimmunoantibody detection method, as well as elution buffer for mild elution and reagents for spatial transcriptome analysis.

[0089] Furthermore, the mild elution process uses 0.05–0.2 M glycine-hydrochloric acid buffer (pH 2.5–3.0) and incubation at 4°C for 5–15 minutes; preferably, the elution buffer also contains 0.5–2 M urea, 0.01–0.1% Triton X-100, and 1–5 mM DTT. These treatment conditions effectively remove antibodies and fluorescent markers while maintaining the integrity of tissue RNA.

[0090] It should be noted that antibodies can also be removed by SDS / β-mercaptoethanol treatment or proteinase K treatment, but the following conditions must be strictly controlled to ensure the stability of the detection results. The elution solution is: 1-2% SDS, 50mM Tris-HCl pH6.8, 100mM β-mercaptoethanol, incubated at 55℃ for 10-30 minutes; or proteinase K 1-10μg / mL, incubated at 37℃ for 2-10 minutes.

[0091] Following mild elution, an RNA integrity assessment step is included: Parallel control sections are used, consisting of tissue sections adjacent to the test section, from which RNA was extracted without TBA staining and elution. The RIN value is measured using a bioanalyzer; an RIN ≥ 6.0 is considered acceptable. Parallel control sections reflect the RNA quality of the tissue itself; if they are acceptable, the test section, after elution, will generally also meet the requirements for downstream analysis. Alternatively, tissue can be collected from the same eluted section, and the Ct value of the internal reference gene GAPDH is measured by qPCR. An increase of no more than 3 in the Ct value compared to the untreated control is considered acceptable. The untreated control refers to adjacent sections that have not undergone TBA staining and elution.

[0092] Furthermore, the spatial transcriptomics analysis was selected from the 10x Genomics Visium, Stereo-seq, GeoMxDSP, Slide-seq, or XYZeq platforms.

[0093] Furthermore, the spatial registration employs a feature point matching algorithm, where the feature points are selected from brain tissue boundaries, brain region contours, blood vessel distribution, or endogenous fluorescent markers.

[0094] Furthermore, the joint analysis includes: defining the fluorescent signal positive region detected by TBA as the antibody binding region, performing differential analysis on the gene expression profile of this region and the non-binding region, and screening specifically highly expressed genes as candidate target antigen genes.

[0095] Furthermore, the secondary antibody marker used in the TBA detection is a photolyzable fluorescent dye or a biotin-avidin system.

[0096] Example 2:

[0097] Rats whole brain tissue perfusion, embedding and quick-freezing

[0098] Experimental animals: Eight-week-old male SD rats (weighing 250-300g) were selected and housed in an SPF-grade animal room with free access to food and water.

[0099] Perfusion: Anesthetize rats by intraperitoneal injection of 3% sodium pentobarbital (40 mg / kg). After the rats' corneal reflex disappears, open the thoracic cavity to expose the heart. Insert the perfusion needle into the ascending aorta through the left ventricle and cut open the right atrial appendage. First, rapidly perfuse with 0.9% physiological saline (4℃) at a pressure of approximately 100 cmH2O and a flow rate of approximately 20 mL / min. Observe the liver color during perfusion. When the liver changes from dark red to light yellow (approximately 150–200 mL of physiological saline is required), it is considered that the blood in the brain has been sufficiently cleared. Then, replace with 4% paraformaldehyde (PFA) fixative (if necessary). If only used for TBA detection, PFA fixation is not required.

[0100] Brain retrieval: After perfusion, the head is severed and the brain is retrieved. The entire brain is quickly placed on ice, and the olfactory bulb, cerebellum (if necessary) and brainstem ends are removed. The surface bloodstains are washed with pre-cooled PBS.

[0101] Embedding and Quick-Freezing: Place the whole brain tissue in an embedding mold (25mm × 30mm × 15mm) and add OCT embedding agent to completely cover the tissue. Take a separate metal container, fill it with isopentane, and pre-cool the container in liquid nitrogen until the isopentane temperature drops to approximately -60°C (pre-cooling in liquid nitrogen for about 5 minutes). Hold the embedding mold with tweezers and immerse it in the pre-cooled isopentane, ensuring the bottom of the mold is completely in contact with the isopentane, and quick-freeze for 10 seconds. During quick-freezing, the OCT changes from transparent to a white solidified state. Remove the mold and immediately transfer it to a -80°C freezer for later storage.

[0102] Standardized slice preparation and hippocampal region localization confirmation

[0103] Microtome preparation: Remove the cryo-embedded tissue block from -80°C and equilibrate it for 30 minutes in a -20°C cryostat (Leica CM1950). Adjust the section thickness to 6 μm and set the anti-roll plate.

[0104] Serial sections: Starting from the midsagittal plane of the brain tissue, serial sections were prepared. After each section, a pre-cooled brush was used to smoothly mount the section onto a SuperFrost Plus anti-detachment slide. Sections were collected at 0.8 mm, 1.2 mm, 1.8 mm, and 2.2 mm from the midsagittal plane, with three serial sections collected from each plane. The 0.8 mm and 1.2 mm midsagittal plane sections mainly contain the hippocampus and basal ganglia; the 1.8 mm and 2.2 mm midsagittal plane sections mainly contain the thalamus, brainstem, and cerebellum.

[0105] Hippocampal region confirmation: Every 8 slides, take one slide for hematoxylin staining: Immerse the slide in Harris hematoxylin solution for 3 minutes, rinse with running water for 5 minutes, differentiate with 1% hydrochloric acid ethanol for 2 seconds, blue-tide with running water for 5 minutes, dehydrate and mount. Observe under an optical microscope; if the pyramidal cell layers of the CA1 and CA3 regions of the hippocampus are clearly visible, the target layer has been confirmed. Record the number of the first slide confirmed to be in the hippocampus; use this as a basis to advance 50 slides as a stable detection area.

[0106] Section fixation and TBA detection

[0107] Fixation: Immerse the prepared slices (air-dried at room temperature for 30 minutes) in pre-cooled (-20°C) acetone for 10 minutes, then remove and allow to evaporate at room temperature for 5 minutes. Alternatively, fix with a 1:1 methanol:acetone mixture for 5 minutes.

[0108] Blocking (tissue end): Prepare blocking solution containing 5% goat serum, 1% BSA, 0.1% Triton X-100, 0.5% Fc receptor blocker (Innovex #NB-101), 0.5% casein (hydrophobic binding blocking component), and 2% normalmouse serum (endogenous IgG binding site inhibitor), dissolved in PBS. Wash the sections three times with PBS for 5 minutes each time, then add the blocking solution and incubate at room temperature for 30 minutes.

[0109] Sample pretreatment (sample end): Serum samples from clinically suspected autoimmune encephalitis patients were diluted 1:10 with PBS. To remove rheumatoid factor and heterophile antibodies, the diluted serum was mixed with an equal volume of a heterophile antibody blocking tube (Scantibodies HBR-1), vortexed at room temperature for 15 minutes, and then centrifuged at 12000g for 5 minutes. The supernatant was collected for later use. Cerebrospinal fluid samples did not require dilution and were directly mixed 1:1 with HBR-1 reagent and centrifuged.

[0110] Primary antibody incubation: Pretreated serum (1:10 dilution) was dropped onto the tissue sections, approximately 150 μL per section, and placed in a humidified chamber for overnight incubation at 4°C (approximately 16 hours). Negative controls were prepared by simultaneous incubation with serum from healthy individuals (1:10 dilution); positive controls were prepared with serum known to be positive for NMDAR antibodies.

[0111] Washing: Wash 3 times for 5 minutes with PBS-T (containing 0.05% Tween-20), then wash once with PBS for 5 minutes.

[0112] Secondary antibody incubation: Add Alexa Fluor 488-labeled goat anti-human IgG (H+L) secondary antibody (Jackson ImmunoResearch) at a dilution of 1:500 (serum sample) or 1:200 (cerebrospinal fluid sample). Incubate at room temperature in the dark for 60 minutes. Use PBS as a background control instead of the secondary antibody.

[0113] Mounting and observation: After washing, add a DAPI-containing anti-fluorescence quenching mounting medium (Vector H-1200) and cover with a coverslip. Observe immediately under a fluorescence microscope (Olympus BX63), or observe after storage at 4°C in the dark (not exceeding 48 hours).

[0114] Preparation and use of quality control arrays

[0115] Slice array preparation: In step (3), slices of different thicknesses (0.8 mm, 1.2 mm, 1.8 mm, and 2.2 mm) were attached to four pre-defined independent reaction zones on the same slide. Each reaction zone had an area of ​​1.5 cm × 1.5 cm and was separated using a hydrophobic pen. At the same time, positive and negative control zones were set at both ends of the slide: positive control zones were attached to known anti-NMDAR antibody-positive rat brain tissue slices, which had been pre-verified by anti-NMDAR antibody immunohistochemistry; negative control zones were attached to blank slices without antibody incubation.

[0116] Preparation of reference antibody binding sites: Using an Arrayjet Mercury spectrometer, known concentrations of human IgG (0, 0.1, 1, 10 μg / mL) were spotted onto the non-tissue areas at the edge of the slide array, at least 2 mm from the tissue edge. Each spot was 100 μm in diameter, with a spot spacing of 500 μm. After spotting, the slides were allowed to dry at room temperature for 30 minutes.

[0117] Fluorescent microsphere embedding: Prior to embedding, carboxyl-modified red fluorescent polystyrene microspheres (Thermo Fisher F8780), 0.5 μm in diameter, were embedded at a final concentration of 10... 6 Fluorescent microspheres were mixed at a density of 1 / mL into the OCT embedding medium, and then embedded in brain tissue. The microspheres were evenly distributed in the OCT medium, and each section contained fluorescent microspheres as an internal control.

[0118] Endogenous reference brain region: The corpus callosum was selected as the reference brain region because it lacks neuronal cell bodies and has uniform background fluorescence. During data analysis, the average fluorescence intensity of the corpus callosum region was measured as a normalization reference.

[0119] Quality control procedure: The sample to be tested is simultaneously added to both the detection reaction area and the quality control reaction area, and blocking, primary antibody incubation, and secondary antibody incubation are performed concurrently. After staining, images of each reaction area are acquired under a fluorescence microscope using the same exposure time of 200 ms. The positive control area should show strong green fluorescence with an average fluorescence intensity >1500, and the negative control area should show weak fluorescence with an average fluorescence intensity <200. The fluorescence intensity of the reference antibody binding site should exhibit a concentration-dependent relationship: 0 μg / mL <200, 0.1 μg / mL 300-500, 1 μg / mL 800-1200, and 10 μg / mL 1500-2000. If any condition is not met, the batch of experiments is considered invalid and must be retested.

[0120] Normalization correction: The mean fluorescence intensity F_target of the target brain region was measured using ImageJ software, and the mean fluorescence intensity F_corpus of the corpus callosum region in the same image was also measured. The preset standard internal reference fluorescence intensity value F_standard was derived from the mean intensity of the corpus callosum from at least 10 healthy control slides (set to 800). Corrected intensity = (F_target / F_corpus) × 800. For example: if the measured intensity in the hippocampus is 1200, and the intensity in the corpus callosum of the same slide is 600, then the corrected intensity = (1200 / 600) × 800 = 1600, indicating that the antibody binding level in this region is higher than the standard.

[0121] TBA in combination with spatial transcriptomics

[0122] TBA detection: First, TBA detection was performed on rat brain tissue sections according to the prescribed method to obtain fluorescence signal patterns. Alexa Fluor 488 was used as the secondary antibody. The distribution and intensity of positive areas were recorded under a fluorescence microscope, and photographs were taken and saved.

[0123] Mild elution treatment: Prepare mild elution buffer: 0.1M glycine-hydrochloric acid (pH 2.8), containing 1M urea, 0.05% Triton X-100, and 2mM DTT, using RNase-free water, and filter through a 0.22μm filter membrane. Immerse the sections that have undergone TBA detection in the pre-cooled (4℃) elution buffer and incubate at 4℃ for 10 minutes. Then wash three times with RNase-free PBS for 5 minutes each time. After elution, observe under a fluorescence microscope to confirm that the fluorescence signal has completely disappeared and the fluorescence intensity of the originally positive areas has decreased to the background level.

[0124] RNA integrity assessment: Parallel control sections (untreated with TBA staining and elution) adjacent to the test section were used to extract total RNA using the RNeasy Micro Kit (Qiagen). RNA integrity was assessed using an Agilent 2100 Bioanalyzer, with a RIN value of 7.2 (≥6.0 is acceptable). If the RIN value of the parallel control section was <6.0, the tissue was re-prepared. Simultaneously, tissue was scraped from a non-critical region (approximately 5 mm²) at the edge of the eluted section, and the GAPDH gene was detected by qPCR: compared to the untreated control section, the Ct value increased to 1.8 (≤3, acceptable).

[0125] Spatial transcriptomics analysis: A 10x Genomics Visium platform was used. Eluted sections were H&E stained, and bright-field scanning was performed to determine tissue morphology. Then, permeabilization, reverse transcription, cDNA amplification, and library construction were performed according to the Visium kit instructions. Sequencing was performed using an Illumina NovaSeq 6000 with a read length of PE150.

[0126] Data registration: TBA fluorescence images were registered with tissue images of the Visium spatial transcriptome. Brain tissue boundaries, hippocampal contours, and vascular branches were selected as feature points, and affine transformation registration was performed using Elastix software. After registration, each Visium spot, with a diameter of 55 μm, corresponds to a TBA fluorescence intensity value.

[0127] Combined analysis: Spots with TBA fluorescence intensity higher than background (mean intensity + 3 standard deviation) were defined as "antibody-binding positive areas," with the rest defined as "negative areas." Differential gene expression analysis between positive and negative areas was performed using Seurat software, with Wilcoxon test and FDR < 0.05. Genes highly expressed in positive areas were identified. For example, in the sample, the LGI1 gene expression level in the positive area was 5.2 times that in the control area (p = 0.001), suggesting the possible presence of anti-LGI1 antibodies in the patient. Further protein validation confirmed this.

[0128] Optimization and Comparative Experiments of Mild Eluent

[0129] To verify the effectiveness of the eluent of this invention, the following comparative experiment was conducted:

[0130] Group A (elution buffer of this invention): 0.1M glycine-hydrochloric acid pH 2.8, 1M urea, 0.05% Triton X-100, 2mM DTT, incubated at 4°C for 10 minutes.

[0131] Group B (SDS elution): 2% SDS, 50mM Tris-HCl pH6.8, 100mM β-mercaptoethanol, incubated at 55°C for 20 minutes.

[0132] Group C (proteinase K treatment): Proteinase K 5 μg / mL, incubated at 37℃ for 10 minutes.

[0133] Group D (blank control): Incubated with PBS at 4°C for 10 minutes.

[0134] Using the same batch of TBA-stained positive sections, anti-NMDAR antibody, and an average fluorescence intensity of 2000, after elution, the residual fluorescence intensity was 150 in group A (a 92.5% decrease), 80 in group B (a 96% decrease), 120 in group C (a 94% decrease), and 1800 in group D (a 10% decrease). This indicates that the elution buffer of this invention effectively removes antibodies while maintaining RNA integrity. Therefore, for experiments requiring subsequent spatial transcriptome analysis, the acidic glycine elution buffer described in this invention must be used.

[0135] Kit composition and usage

[0136] This invention also provides a kit for TBA detection and TBA-spatial transcriptome coupling, comprising the following components:

[0137] Whole brain tissue slice array: a pre-prepared standardized rat sagittal slice array, including the hippocampus, basal ganglia, thalamus, brainstem, and cerebellum. Each slice contains 4 detection reaction areas and 2 quality control reaction areas, and is pre-embedded with fluorescent microspheres and stored at -80℃.

[0138] Fixative: Acetone (10mL / bottle).

[0139] Blocking solution (tissue end): 10 mL, containing Fc receptor blocker, hydrophobic blocking component and endogenous IgG blocking component.

[0140] Sample pretreatment reagents: Heterophilic antibody blocking tubes (HBR-1, 1 mL / tube).

[0141] Fluorescently labeled secondary antibody: Alexa Fluor 488-goat anti-human IgG (50 μL, 1:500 recommended dilution).

[0142] Mounting tablets (containing DAPI): 5 mL.

[0143] Mild elution buffer: 50 mL, 0.1 M glycine-hydrochloric acid pH 2.8, 1 M urea, 0.05% Triton X-100, 2 mM DTT.

[0144] Spatial transcriptome analysis reagents: 10x Genomics Visium kit (individually packaged, optional).

[0145] Instructions for use: Follow the steps for perfusion, sectioning (if fresh preparation is required), TBA detection, gentle elution, and spatial transcriptome analysis. The kit supports 10 assays.

[0146] Example 3:

[0147] See Figures 1 to 3 A method for detecting neuroimmune antibodies based on whole brain tissue, comprising at least the following steps:

[0148] Step 1: Animal anesthesia and standardized perfusion blood collection: Select experimental animals and perform phosphate-buffered saline (PBS) perfusion via the heart under deep anesthesia to fully remove blood from the cerebral capillaries;

[0149] The liver color was observed to change from dark red to light red as a criterion for determining adequate perfusion, and then the whole brain tissue was removed.

[0150] Technical effects: Significantly reduces non-specific fluorescence background caused by hemoglobin, autologous IgG, etc., and improves detection consistency.

[0151] Step 2: Uniform freezing and embedding with isopentane pre-cooling: The extracted whole brain tissue was placed in an embedding mold and tissue freezing embedding medium was added; rapid and uniform freezing was performed using isopentane pre-cooled with liquid nitrogen, and then the tissue was stored at -80℃.

[0152] Technical benefits: Compared to liquid nitrogen contact freezing, it can effectively reduce ice crystal size and maintain tissue structure integrity and antigen conformation stability.

[0153] Step 3: Preparation of standardized frozen sections of the whole brain at multiple planes: Serial sagittal sections are prepared using a cryostat, with the section thickness controlled at approximately 5 μm; hematoxylin staining is used to confirm the hippocampus as a key anatomical landmark. The standardized sagittal sections are selected from a range of approximately 0.5 mm to 2.5 mm from the median sagittal plane, with the first standard plane approximately 0.5–1.5 mm from the median sagittal plane and the second standard plane approximately 1.5–2.5 mm from the median sagittal plane. A combination of sections covering at least two of the following brain regions is prepared: hippocampus, basal ganglia, thalamus, brainstem, or cerebellum.

[0154] Technical effect: By covering the whole brain / multiple brain regions, it improves the detection capability of specific fluorescence patterns of different neuroimmunoantibodies.

[0155] Step 4: Organic solvent precipitation fixation: The sections are fixed at low temperature using the organic solvent acetone precipitation fixation method, instead of paraformaldehyde cross-linking fixation.

[0156] Technical effects: Improves membrane protein epitope exposure, reduces lipid residue and tissue autofluorescence, and enhances the signal-to-noise ratio of antibody binding. For example... Figure 2 As shown, fluorescence signals in the cerebellar molecular layer are compared; the left image shows fixation with organic solvent, and the right image shows fixation with paraformaldehyde.

[0157] Step 5: System processing for non-specific staining. Tissue-side processing: Blocking is performed using a modified blocking solution containing Fc receptor blocking components, hydrophobic binding blocking components, and endogenous IgG binding site inhibitors. Sample-side processing: The serum or cerebrospinal fluid to be tested is pretreated to remove non-specific interfering factors such as rheumatoid factor and heterophilic antibodies.

[0158] Technical benefits: Further reduces the false positive rate and improves detection specificity. For example... Figure 3 As shown, the left image shows the result after processing, and the right image shows the result before processing.

[0159] Example 4:

[0160] See Figures 4 to 5 Instructions for use of TBA (tissue-based assay) in whole brain tissue sections (cell immunofluorescence method)

[0161] Project Name

[0162] Generic Name: TBA (tissue-based assay) detection of nervous system autoantibodies (whole mouse brain tissue)

[0163] Packaging specifications

[0164] 16 servings / box

[0165] Intended Use

[0166] In vitro brain tissue (rodents or primates) contains all natural target antigens. Antibodies in the test sample (cerebrospinal fluid or serum) react with brain tissue sections via antigen-antibody reactions, often used for screening neurological antibodies. The fluorescence patterns of different neurological antibodies are diverse and complex, with some antibodies showing subtle differences; therefore, the accuracy of fluorescence pattern interpretation is crucial and determines the quality of neurological antibody assessment. Because TBA provides a complete antigen spectrum, it offers a powerful tool for discovering unknown neurospecific antibodies through specific fluorescence patterns. The specific significance is as follows:

[0167] ① For the standardized diagnosis and treatment of diseases, autoimmune diseases cannot rely solely on CBA; antibody screening via TBA is also necessary.

[0168] ② A rapid process requires: early detection, early diagnosis, and early treatment, so that patients can receive effective treatment in a short period of time;

[0169] ③ Overlap antibody screening, suspected case analysis, and research needs;

[0170] ④ Detect the affinity between antibodies and tissues;

[0171] ⑤ Isoform antibodies were discovered.

[0172] Detection principle

[0173] The TBA method uses slides containing tens of millions of antigen proteins. The antibodies in the slides are homologous to human antibodies. Human antibodies can recognize and bind to the antibodies in the slides. After washing away the non-specific antibodies, fluorescently labeled secondary antibodies are added to observe the specific binding sites. Based on experience, the two most common types of cell antibodies observed are those for nerve cells and glial cells.

[0174] Specimen quantity and requirements for specimen preservation and transportation

[0175] 2 ml of blood and cerebrospinal fluid samples should be stored and transported at 2-8℃.

[0176] Reagent Kit Storage Conditions

[0177] Tissue sections should be stored at -20°C for 12 months.

[0178] Tissue sections should be transported at 2℃-8℃ for no more than 4 days.

[0179] The accompanying reagents can be stored at 2℃-8℃ until their expiration date.

[0180] Production batch number and expiration date: see the outer label and bottle label of the reagent kit.

[0181] Detection methods

[0182] 1. Reagent preparation:

[0183] 1) Preparation of detergent

[0184] Dissolve one packet of PBS powder completely in 2L of deionized water. The diluted PBS solution can be stored at 2-8℃ for one month. Take precautions to prevent contamination during storage.

[0185] 2) Collect serum or cerebrospinal fluid samples according to standard procedures.

[0186] Before testing, dilute the serum sample with the prepared PBS buffer at a ratio of 1:10. Cerebrospinal fluid does not need to be diluted.

[0187] 2. Staining Procedure

[0188] 1) Rewarming: Place the slide in a humidified chamber and allow the reagents and samples to equilibrate to room temperature.

[0189] 2) Washing: Add PBS to the reaction wells, then use a pipette to remove the PBS and wipe the non-reaction area dry with a paper towel. Throughout the entire procedure, avoid contact with the fixed tissue.

[0190] 3) Sample serum incubation: Add 1 drop of diluted serum or cerebrospinal fluid stock solution to each reaction well, ensuring complete coverage of the well, and incubate at 37°C for 1 hour.

[0191] 4) Cleaning: Use a pipette to remove the sample to be tested, avoiding cross-contamination between the sample and other reaction wells. Immerse the slide completely in a cleaning bath filled with PBS for 5 minutes to thoroughly clean the slide. Repeat 3 times after changing the PBS solution. Carefully dry the slide without touching the reaction wells. Keep the coated tissue in the reaction wells moist throughout the reaction process.

[0192] 5) Secondary antibody incubation: Add 200 μL of diluted secondary antibody to each reaction well (serum 1:500 dilution, cerebrospinal fluid 1:200 dilution (PBS buffer dilution)) and place in a humidified chamber. Incubate at 37°C for 1 h.

[0193] 6) Cleaning: Same as 4

[0194] 7) Add 1 drop of PBS to the reaction area and quickly observe the experimental results under a fluorescence microscope. If the results cannot be observed immediately, the slide should be mounted and stored for later observation.

[0195] Result Interpretation

[0196] Application and Explanation Section

[0197] Figure 4 The results indicate the following: Cortex: positive for neuronal cell nuclei; Hippocampus: positive for pyramidal cells; Cerebellum: positive for granular layer; White matter: negative. These results suggest the possible presence of antinuclear antibodies, and interference from antinuclear antibodies needs to be ruled out.

[0198] Figure 5 The results indicate the following: Cortex: positive for nerve cell membranes; Hippocampus: positive for molecular layers; Cerebellum: positive for molecular layers; White matter: negative. These results suggest the possible presence of anti-neuronal cell membrane antibodies, and screening for autoimmune encephalitis-related antibodies is recommended.

[0199] Limitations of the detection method

[0200] This kit can be used for qualitative detection and titer determination of antibodies. This kit is suitable for detecting clinical specimens. The results are affected by specimen acquisition and storage, proper use of the testing instrument, and accurate reading of positive signals. Clinical results from this kit are for clinical reference only and should not be used as the sole basis for determining the presence of immune-related antibodies in the nervous system. Negative results cannot rule out different matrix factors; results need to be correlated with clinical findings.

[0201]

Product Performance

[0202] 1. Repeatability: The results should be consistent when the same sample is measured 10 times.

[0203] 2. Inter-batch precision: The same sample should be tested using three different batches of the kit, with each batch repeated 10 times, and the results should be consistent.

[0204] 3. Negative Conformity Rate: When testing the company's negative reference samples, the reaction results should be consistent and all be negative.

[0205] 4. Positive Concordance Rate: When testing the company's positive reference samples, the reaction results should be consistent, all being positive.

[0206] 5. Specificity: The cells transfected with the target antigen should be coated with three other antibodies (IgG) and the results should be negative.

[0207] Thus far, the description of the above embodiments has been provided for illustrative and descriptive purposes. This is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of particular embodiments are generally not limited to those particular embodiments, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. In many respects, the same elements or features may also be varied. Such variations are not considered a departure from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0208] Example embodiments are provided so that this disclosure will become thorough and will fully convey the scope to those skilled in the art. Numerous details, such as examples of specific parts, apparatus, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that the specific details are not required, and the example embodiments may be implemented in many different forms, neither of which should be construed as limiting the scope of this disclosure. In some example embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0209] Technical terms are used herein for the purpose of describing specific exemplary embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein may also refer to the plural forms. The terms “comprising” and “having” are inclusive and therefore specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or additional having of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof. Unless expressly indicated in order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the specific order discussed and shown. It should also be understood that additional or optional steps may be employed.

Claims

1. A method for detecting neuroimmunoantibodies based on whole brain tissue, characterized in that, Includes the following steps: (1) Obtaining whole brain tissue, which is obtained by perfusing the heart with phosphate buffer or saline to remove blood components from the brain; (2) The obtained whole brain tissue was placed in an embedding mold, tissue cryopreservation medium was added, and the tissue was pre-cooled with isopentane and then rapidly frozen uniformly. (3) The cryo-embedded whole brain tissue was serially sliced ​​in a cryostat to prepare standardized tissue sections covering at least two different brain regions; (4) Use organic solvents to fix the sections by precipitation; (5) The fixed sections were incubated with the test sample, washed and then incubated with fluorescently labeled secondary antibody for tissue-based indirect immunofluorescence (TBA) detection; In step (5), non-specific staining is also included: the tissue end is blocked with a blocking solution containing Fc receptor blocking components, and the sample end is pretreated to remove rheumatoid factor and / or heterophilic antibodies.

2. The method according to claim 1, characterized in that, In step (1), the liver color changes from dark red to light red as the criterion for determining that the perfusion is sufficient; in step (2), the isopentane is pre-cooled by liquid nitrogen, and the embedding mold is immersed in the pre-cooled isopentane for quick freezing.

3. The method according to claim 1, characterized in that, The standardized slices in step (3) are sagittal slices with a thickness of 4 to 8 μm. The slices are selected from at least two different layers within a range of 0.5 mm to 2.5 mm from the midsagittal plane. The brain regions are selected from at least two of the hippocampus, basal ganglia, thalamus, brainstem, or cerebellum.

4. The method according to claim 1, characterized in that, In step (4), the organic solvent is acetone, methanol or a combination thereof; in step (5), when the sample to be tested is serum, it is diluted at a ratio of 1:5 to 1:20, and when it is cerebrospinal fluid, it is not diluted or diluted at a ratio of 1:1 to 1:

5. The incubation time of the fluorescently labeled secondary antibody is 30 to 90 minutes.

5. The method according to claim 3, characterized in that, In step (3), one slice is taken every 5 to 10 slices for hematoxylin staining to confirm that the hippocampus has been reached. Hematoxylin staining is used to confirm the arrival of the hippocampus as the anatomical location confirmation point of the slice layer.

6. The method according to claim 1, characterized in that, The whole brain tissue was selected from rodents or primates, and the tissue cryopreservation medium was OCT embedding agent.

7. The method according to any one of claims 1-6, characterized in that, Step (3) further includes preparing a slice array from the standardized tissue slices, the slice array comprising at least one detection reaction zone and at least one quality control reaction zone, the quality control reaction zone comprising at least one of the following quality control markers: a reference antibody binding site with a pre-set known concentration gradient, fluorescent microspheres embedded in the embedding medium, naturally occurring endogenous internal reference brain region, or independent positive and negative quality control slices.

8. The method according to claim 7, characterized in that, The step (5) also includes a quality control step: the sample to be tested is simultaneously stained with immunofluorescence along with the detection reaction area and the quality control reaction area, the fluorescence signal intensity of the quality control reaction area is detected to determine the effectiveness of the staining process, and the fluorescence signal of the quality control reaction area is used to normalize and correct the fluorescence intensity of the detection reaction area.

9. A method for combining antibody detection and gene expression profiling analysis of whole brain tissue sections, characterized in that, include: After obtaining the fluorescence signal pattern by tissue-based indirect immunofluorescence (TBA) detection of whole brain tissue sections using the method described in any one of claims 1-6, the same tissue sections are subjected to mild elution to remove antibodies and fluorescent markers while maintaining RNA integrity. Then, the same eluted tissue sections are subjected to spatial transcriptomics analysis to obtain gene expression profiles. The fluorescence signal patterns and gene expression profiles are spatially registered and jointly analyzed to identify differentially expressed genes.

10. A kit for use in the method of claim 9, characterized in that, It includes reagents for carrying out the method according to any one of claims 1-6, as well as elution buffers for mild elution and reagents for spatial transcriptome analysis.

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