A multiplex immunohistochemical luminescence detection method based on spatial reconstruction

Abstract

The present invention relates to a multiplex immunohistochemical luminescence detection method based on spatial reconstruction. The method comprises spatially separating a sample and incubating the sample with a plurality of magnetic beads modified with different anti-reactants, replacing the fluorescent signal with the chemiluminescent signal of the magnetic beads, thereby overcoming the limitation of overlapping fluorescence emission spectra and achieving multiplex detection. The method also uses chemiluminescence technology to accurately quantify the reactants, thereby resolving the problem that existing multiplex immunohistochemistry and immunofluorescence techniques cannot accurately perform quantitative detection. The accurate quantification and multiplex detection solve the problem of high observer variability existing in current immunohistochemistry and immunofluorescence techniques. Compared with traditional chemiluminescence technology, the method can not only quantify markers but also reflect the relative positioning of markers on tissue sections.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a multiplex immunohistochemical luminescence detection method based on spatial reconstruction. Background Art

[0002] Immunohistochemistry (IHC) is the most common application of immunostaining. It utilizes the principle of specific binding of antibodies to antigens in biological tissues to selectively identify antigens in tissue sections. IHC is primarily used in disease diagnosis and basic research, demonstrating the expression of antigens in tissues in situ. It primarily consists of two formats: chromogenic immunohistochemistry and immunofluorescence (IF).

[0003] Traditional immunohistochemistry can only label one marker at a time, which is insufficient for disease diagnosis, especially tumor diagnosis. Currently, no single marker can clearly indicate the occurrence of a tumor. In the field of diagnosis, a multi-marker combined test is generally required to diagnose a disease. Traditional immunohistochemistry also brings the problem of high observer variability. The person observing the immunohistochemistry section can only subjectively judge the pathological condition of the section based on a single indicator, resulting in different observation results between different observers, and even the same observer under different observation conditions.

[0004] In order to avoid the above problems, some multiple immunohistochemical detection technologies that can mark multiple markers have been developed. However, these technologies are limited by the spectral overlap of emission spectra and can generally only perform multiplex detection on 4-6 markers at the same time, and their multiplex detection capabilities are limited. In order to improve the ability of multiple immunohistochemical multiplex detection, researchers have developed iterative sequential antibody labeling and imaging technology, and developed the MultiOmyx platform (patented technology is in the hands of NeoGenomics and General Electric) for staining and detection. This technology uses repeated incubation-imaging-quenching, imaging two fluorescent dyes each time, and after multiple rounds of incubation-imaging-quenching, multiple imaging results are superimposed on a picture to achieve super-multiple detection (Proceedings of the National Academy of Sciences, 2013, 110 (29): 11982-11987). However, this method is not only time-consuming and labor-intensive, but also has a high degree of difficulty in accurately positioning and superimposing images.

[0005] In addition to the unreliability of a single indicator, the difficulty of quantitative immunohistochemistry can also cause observer variability. Since fluorescence intensity can be adjusted by adjusting the exposure time, the fluorescence intensity of different fluorescent dyes is also different. In addition, changes in incubation conditions, storage conditions, reaction time and other conditions will also cause differences in fluorescence intensity. This makes it difficult to perform accurate quantitative analysis using the current mainstream fluorescence-based multiplex immunohistochemistry. Summary of the Invention

[0006] To address the shortcomings of existing multiple immunohistochemistry techniques, which have difficulty achieving super-multiplexity and accurate quantification, the present invention proposes a multiple immunohistochemical chemiluminescence detection method based on spatial reconstruction, which replaces the fluorescent signal with the chemiluminescence signal of different magnetic beads, thereby breaking through the limitation of overlapping fluorescence emission spectra and achieving multiple detection; using chemiluminescence technology to accurately quantify the reactants, it solves the problem that existing multiple immunohistochemistry and immunofluorescence techniques cannot perform accurate quantitative detection. Through accurate quantification and multiple detection, the problem of high observer variability existing in current immunohistochemistry and immunofluorescence techniques is solved. Compared with traditional chemiluminescence technology, the method of the present invention can not only quantify markers, but also reflect the relative positioning of markers on tissue sections.

[0007] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0008] A multiplex immunohistochemical luminescence detection method based on spatial reconstruction, characterized by comprising:

[0009] S1. Prepare samples;

[0010] S2. incubating the sample to obtain one or more specific reactants;

[0011] S3, performing a spatial separation operation on the sample to form a number of separation areas;

[0012] S4, performing a specific reactant stripping operation on each separated area to obtain a number of specific reactant combinations obtained by stripping the corresponding separated areas;

[0013] S5, reacting each specific reactant combination with a plurality of magnetic beads modified with different anti-reactants;

[0014] S6. Perform chemiluminescence detection on the various magnetic bead combinations after the reaction to obtain the luminescence value of each specific reactant corresponding to each separation area;

[0015] S7. Performing a spatial reconstruction operation on the luminescence value of each specific reactant obtained by the detection according to the separated area where it is located, to obtain the in-situ quantitative detection result of the chemiluminescence of the sample.

[0016] Furthermore, the step S5 includes the following sub-steps:

[0017] S51, splitting the specific reactant combination corresponding to a single separation area into n molecular combinations, where n is not less than the number of magnetic bead types corresponding to the reaction;

[0018] S52, reacting each molecular combination with a magnetic bead respectively to obtain magnetic beads after the specific reactant combination reacts with the anti-reactant modified on the magnetic beads;

[0019] S53, collecting the magnetic beads obtained after the reaction of each sub-combination to obtain multiple magnetic bead combinations after the reaction of specific reactant combinations;

[0020] S54 , performing sub-steps S51 to S53 on the specific reactant combinations corresponding to the respective separation areas, to obtain a plurality of magnetic bead combinations after the specific reactant combinations corresponding to the respective separation areas have reacted.

[0021] Furthermore, the performing chemiluminescence detection on the multiple magnetic beads after the reaction includes:

[0022] Performing chemiluminescence detection on the multiple post-reaction magnetic beads contained in the multiple magnetic bead combinations after the reaction of the specific reactant combination corresponding to each separation area to obtain the luminescence value of the specific reactant corresponding to the reactant magnetic bead;

[0023] According to the corresponding relationship between the target detection object and the specific reactant, the luminescence value of the specific reactant is used to determine the content of the target detection object, that is, the content of the target detection object in the separated area.

[0024] Furthermore, the step S3 further includes:

[0025] Add identification numbers to the separated areas formed.

[0026] Furthermore, the step S4 further includes:

[0027] The corresponding identification number is added to the specific reaction product combination obtained by stripping.

[0028] Furthermore, the sub-step S51 further includes: adding corresponding identification numbers to the sub-combinations obtained by splitting;

[0029] The sub-step S53 further includes: adding corresponding identification numbers to the various magnetic bead combinations.

[0030] Furthermore, the spatial reconstruction operation includes assigning the luminescence value of each specific reactant obtained by detection to a separate area according to the identification number.

[0031] Furthermore, the target detection object includes one or more combinations of antigens, antibodies, nucleic acids, cells, extracellular vesicles, and exosomes.

[0032] Furthermore, the specific reactant is a substance that can react specifically with one or more target detection substances.

[0033] Furthermore, the anti-reactant is a substance that can specifically react with a specific specific reactant.

[0034] Furthermore, the anti-reactant includes one or more combinations of antigens, antibodies, nucleic acids, and amino acid sequences.

[0035] Furthermore, the target detection object is an antigen.

[0036] Furthermore, the specific reactant is an antibody.

[0037] Furthermore, the anti-reactant is an antigen.

[0038] The beneficial effects of the present invention are:

[0039] The multiple immunohistochemical luminescence detection method based on spatial reconstruction described in the present invention is adopted. By replacing the fluorescent signal with the chemiluminescence signal of different magnetic beads, the limitation of overlapping fluorescence emission spectra is broken through, and multiple (multivariate) detection is achieved; the reactants are accurately quantified using chemiluminescence technology, which solves the problem that existing multiple immunohistochemistry and immunofluorescence technologies cannot perform accurate quantitative detection. The spatial reconstruction method is used to achieve chemiluminescence-like in situ detection of immune tissue samples, and the problem of high observer variability in current immunohistochemistry and immunofluorescence technologies is solved through accurate quantification and multiple detection. Compared with traditional chemiluminescence technology, the present invention can not only quantify markers, but also reflect the relative positioning of markers on tissue sections. Compared with the existing technology, the detection method of the present invention does not require the complex operation steps of repeated incubation-imaging-quenching. After the antibody incubation is completed, the antibody only needs to be stripped out and the detection can be carried out directly. The time required is shorter and the operation is simpler. The subsequent data processing does not require image superposition, reducing the demand for image processing capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the process of the multiple immunohistochemical luminescence detection method based on spatial reconstruction of the present invention.

[0041] Figure 2 This is the imaging result of HSP-90 protein in tissue section sample under fluorescence microscope.

[0042] Figure 3A Schematic diagram of the comparison of HSP-90 fluorescence intensity in different compartments of tissue sections.

[0043] Figure 3BSchematic diagram of the comparison of HSP-90 chemiluminescence values ​​in different separated areas of tissue sections.

[0044] Figure 4 It is the result of direct multiplex detection of various specific reactants in a separated area of ​​a tissue section.

[0045] Figure 5 It is the result of indirect multiplex detection of various specific reactants in a separated area of ​​a tissue section. DETAILED DESCRIPTION

[0046] In order to more clearly understand the content of the present invention, it will be described in detail with reference to the accompanying drawings and embodiments.

[0047] like Figure 1 The figure shows a typical process flow diagram of the multiple immunohistochemical luminescence detection method based on spatial reconstruction of the present invention, including:

[0048] S1. Prepare the sample. The sample preparation process is the same as that of traditional immunohistochemistry. The traditional immunohistochemistry sample preparation process and related slice samples can be directly applied to the method of the present invention.

[0049] S2. Incubate the sample to obtain one or more specific reactants. A specific reactant is a substance that can specifically react with the target detection object, which includes one or more combinations of antigens, antibodies, nucleic acids, cells, extracellular vesicles, and exosomes. When the target detection object is preferably an antigen, the corresponding specific reactant is preferably an antibody.

[0050] S3. Perform a spatial separation operation on the sample to form a plurality of separated areas, and add identification numbers to the formed separated areas. Forming a plurality of separated areas means dividing the sample into individual areas, each of which is independent of each other. When performing the subsequent stripping operation (step S4), the individual areas will not affect each other. The separated areas can be selected in the form of regular rows and columns, or in any shape and arrangement. This can avoid some areas with poor staining or poor properties, and can also perform more comprehensive testing on irregular key areas.

[0051] S4. Specific reactant stripping is performed on each separation region to obtain a plurality of specific reactant combinations obtained by stripping the corresponding separation regions. Corresponding identification numbers are assigned to the stripped specific reactant combinations, ensuring that the stripped specific reactant combinations correspond to the separation regions at which they were stripped. Stripping involves removing various specific reactants from the target object using chemical reagents, heating, microwave treatment, or other methods.

[0052] S5, each specific reactant combination is reacted with a variety of magnetic beads modified with different anti-reactants, specifically including the following steps: S51, the specific reactant combination corresponding to the single separation area is split into n sub-combinations and the corresponding identification number is added to the sub-combination obtained by the split, where n is not less than the number of magnetic bead types corresponding to the reaction; S52, each sub-combination is reacted with a magnetic bead to obtain the magnetic beads after the specific reactant combination reacts with the anti-reactant modified by the magnetic bead; S53, the magnetic beads obtained after the reaction of each sub-combination are collected to obtain a variety of magnetic bead combinations after the specific reactant combination reacts, and the corresponding identification number is added to the variety of magnetic bead combinations; S54, the specific reactant combination corresponding to each separation area is executed by sub-steps S51 to S53 to obtain a variety of magnetic bead combinations after the reaction of each specific reactant combination. That is, it is necessary to react each separation area with a variety of magnetic beads to achieve a multivariate detection of each separation area. In actual applications, the number of magnetic bead types used can be adjusted as needed. When the more magnetic bead types are used, the more corresponding specific reactant types are detected, and more diverse detection can be achieved.

[0053] S6, respectively perform chemiluminescence detection on the various magnetic bead combinations after the reaction to obtain the luminescence value of each specific reactant in each corresponding separation area. Specifically, the various magnetic beads contained in the various reaction magnetic beads after the reaction of the specific reactant combination corresponding to each separation area are respectively subjected to chemiluminescence detection to obtain the luminescence value of the specific reactant corresponding to the reactant magnetic bead. According to the corresponding relationship between the target detection object and the specific reactant, the luminescence value of the specific reactant is used to determine the content of the target detection object, that is, the content of the target detection object in the separation area. The luminescence value corresponding to various specific reactants in each separation area refers to determining the type of specific reactant to be detected according to the type of magnetic beads; determining the content of the corresponding target detection object by the height of the chemiluminescence value of a certain magnetic bead, thereby realizing quantitative detection; judging which specific reactants the region contains by the height of the chemiluminescence value of each magnetic bead, thereby realizing multivariate quantitative detection; judging the distribution of various specific reactants by the height of the chemiluminescence value of each magnetic bead in each separation area, realizing in situ multivariate quantitative detection.

[0054] S7. Spatially reconstructing the luminescence values ​​of each specific reactant detected according to the compartments in which they are located, including mapping the luminescence values ​​of each specific reactant detected to the compartments according to their identification numbers, thereby obtaining an in situ quantitative chemiluminescence detection result of the sample. The chemiluminescence value reflects the content of each specific reactant. Spatial reconstruction can be used to map the quantitative chemiluminescence result to the location on the sample, achieving quasi-in situ detection, thereby obtaining the quantitative result of each specific reactant at the corresponding location.

[0055] Chemiluminescence detection can be performed automatically by the instrument and automatically gives data without the need for manual reading, thus avoiding observer variability. At the same time, the chemiluminescence detection results are numerical values ​​of the content of various specific reactants in each area, rather than traditional fluorescent photos.

[0056] The chemiluminescence detection method of the present invention is further illustrated below through specific operation examples.

[0057] The experimental materials and instruments used in the examples include:

[0058] Antigens and antibodies were purchased from Biolegend;

[0059] The instrument model used for fluorescence imaging was SLIDEVIEW VS200 from Olympus;

[0060] The instrument model used for chemiluminescence imaging was Cosmay's SMART 6500;

[0061] Acridinium esters were purchased from McCarthy's reagent;

[0062] Magnetic beads were purchased from Thermo Fisher Dynabeads TM MyOne TM Carboxylic acid magnetic beads;

[0063] Antigen retrieval reagents were purchased from Akoya Biosciences.

[0064] Sample Group 1

[0065] The direct method uses the following steps:

[0066] Antibodies (specific reactants) corresponding to antigens (target detectable substances) highly expressed in the tissue sample to be tested are selected: anti-GAPDH antibody, anti-α-actin antibody, anti-β-actin antibody, anti-α-tubulin antibody, anti-β-tubulin antibody, anti-transferrin antibody, anti-cytokeratin antibody, anti-lamin B1 antibody, anti-HSP-90 antibody, anti-SDHA antibody, and anti-histone antibody.

[0067] The above antibodies were labeled with AE (acridinium ester) according to the manufacturer's instructions;

[0068] Paraffin tissue sections prepared using FFPE were dewaxed with xylene;

[0069] Afterwards, the cells were rehydrated with graded ethanol of 100%, 95%, 85%, and 75%;

[0070] Antigen retrieval was performed by microwave heat retrieval of antigens using preheated epitope retrieval reagent for 30 min;

[0071] Endogenous catalase was inactivated by incubation with 3% H2O2 at room temperature for 20 min;

[0072] The cells were incubated with 10% goat serum at room temperature for 20 min to block nonspecific epitopes.

[0073] The sections were incubated with acridinium ester-labeled primary antibodies in a 4°C refrigerator overnight;

[0074] The samples were washed with TBST for 5 min × 3 times;

[0075] Divide the above sample into several parts (it can be any part, preferably 2 rows and 2 columns in this embodiment, a total of 4 parts);

[0076] Number each partition (e.g. A1, A2, B1, B2);

[0077] Stripping the antibodies from each separated area (any antibody stripping method can be used. In this embodiment, the antibody stripping method is microwave treatment. To strip the antibodies, add antigen retrieval reagent and microwave the sample to 95° C. for 20 minutes).

[0078] The antibodies stripped from each separated area were numbered (corresponding to A1, A2, B1, B2);

[0079] The stripped antibodies are in antigen retrieval reagent.

[0080] The above-mentioned proteins (anti-reactants) highly expressed in tissues are modified on carboxyl magnetic beads for chemiluminescence according to the instructions: GAPDH protein, α-actin, β-actin, α-tubulin, β-tubulin, transferrin, cytokeratin, lamin B1, HSP-90 protein, SDHA protein, and histone. Magnetic beads modified with GAPDH protein, α-actin, β-actin, α-tubulin, β-tubulin, transferrin, cytokeratin, lamin B1, HSP-90 protein, SDHA protein, and histone are obtained respectively; preferably, each magnetic bead is also correspondingly added with its own label (for example, magnetic beads a, magnetic beads b, magnetic beads c, magnetic beads d, magnetic beads e, magnetic beads f, magnetic beads g, magnetic beads h, magnetic beads i, magnetic beads j, and magnetic beads k);

[0081] Divide the antibodies stripped from each separation area into several equal portions. The number of portions should not be less than the number of magnetic bead types (for example, 11 portions, marked as I, II, III, IV, V, VI, VII, VIII, IX, X, and XI).

[0082] By combining the above numbering, each antibody in each compartment can be numbered separately (e.g., A1 I, A1 II, A1 III, A1 IV, A1 V, A1 VI, A1 VII, A1 VIII, A1 IX, A1 X, A1 XI; A2 I, A2 II, A2 III, A2IV, A2 V, A2 VI, A2 VII, A2 VIII, A2 IX, A2 X, A2 XI; B1 I, B1 II, B1 III, B1 IV, B1 V, B1VI, B1 VII, B1 VIII, B1 IX, B1 X, B1 XI; B2 I, B2 II, B2 III, B2 IV, B2 V, B2 VI, B2 VII, B2 VIII, B2 IX, B2 X, B2 XI);

[0083] Correspondingly, the No. I antibody in each separation area reacts with the a magnetic bead, the No. II antibody reacts with the b magnetic bead, the No. III antibody reacts with the c magnetic bead, the No. IV antibody reacts with the d magnetic bead, the No. V antibody reacts with the e magnetic bead, the No. VI antibody reacts with the f magnetic bead, the No. VII antibody reacts with the g magnetic bead, the No. VIII antibody reacts with the h magnetic bead, the No. IX antibody reacts with the i magnetic bead, the No. X antibody reacts with the j magnetic bead, and the No. XI antibody reacts with the k magnetic bead;

[0084] By comparing the luminescence value of each magnetic bead after the reaction, if it is more than 2.3 times higher than the background value, it is considered to contain the antibody corresponding to the magnetic bead, and the antibody is positive; the luminescence value of each antibody can be used to determine the content of the antigen to be tested in the sample, thereby achieving quantitative detection.

[0085] The incubated magnetic beads were detected by chemiluminescence instrument, and the test results were as follows: Figure 4 As shown (taking region A1 as an example), the technical solution of the present invention can simultaneously detect multiple specific reactants in any region (in this embodiment, 11 antibodies are detected simultaneously), breaking the limitation of overlapping fluorescence emission spectra and realizing super-multivariate detection.

[0086] Sample Group 2

[0087] The indirect method includes the following steps:

[0088] Select antibodies (specific reactants) corresponding to antigens (target detection substances) highly expressed in the tissue sample to be tested: anti-GAPDH antibody, anti-α-actin antibody, anti-β-actin antibody, anti-α-tubulin antibody, anti-β-tubulin antibody, anti-transferrin antibody, anti-cytokeratin antibody, anti-lamin B1 antibody, anti-HSP-90 antibody, anti-SDHA antibody, and anti-histone antibody. All of the above antibodies are mouse anti-human antibodies. (The above antibodies are not labeled with AE)

[0089] Paraffin tissue sections prepared using FFPE were dewaxed with xylene;

[0090] Afterwards, the cells were rehydrated with graded ethanol of 100%, 95%, 85%, and 75%;

[0091] Antigen retrieval was performed by microwave heat retrieval of antigens using preheated epitope retrieval reagent for 30 min;

[0092] Endogenous catalase was inactivated by incubation with 3% H2O2 at room temperature for 20 min;

[0093] The cells were incubated with 10% goat serum at room temperature for 20 min to block nonspecific epitopes.

[0094] Incubate the sections with primary antibodies overnight in a 4°C refrigerator;

[0095] The samples were washed with TBST for 5 min × 3 times;

[0096] Divide the above sample into several parts (it can be any part, preferably 2 rows and 2 columns in this embodiment, a total of 4 parts);

[0097] Stripping the antibodies from each partitioned area (any antibody stripping method may be used. In this embodiment, the antibody stripping method is microwave treatment. To strip the antibodies, add antigen retrieval reagent and microwave the sample to 95° C. for 20 minutes).

[0098] Number each partition (e.g. A1, A2, B1, B2);

[0099] The stripped antibodies are in antigen retrieval reagent.

[0100] The above-mentioned proteins (anti-reactants) highly expressed in tissues are modified on carboxyl magnetic beads for chemiluminescence according to the instructions: GAPDH protein, α-actin, β-actin, α-tubulin, β-tubulin, transferrin, cytokeratin, lamin B1, HSP-90 protein, SDHA protein, and histone. Magnetic beads modified with GAPDH protein, α-actin, β-actin, α-tubulin, β-tubulin, transferrin, cytokeratin, lamin B1, HSP-90 protein, SDHA protein, and histone are obtained respectively; preferably, each magnetic bead is also correspondingly added with its own label (for example, magnetic beads a, magnetic beads b, magnetic beads c, magnetic beads d, magnetic beads e, magnetic beads f, magnetic beads g, magnetic beads h, magnetic beads i, magnetic beads j, and magnetic beads k);

[0101] Divide the antibodies stripped from each separation area into several equal portions. The number of portions should not be less than the number of magnetic bead types (for example, 11 portions, marked as I, II, III, IV, V, VI, VII, VIII, IX, X, and XI).

[0102] By combining the above numbering, each antibody in each compartment can be numbered separately (e.g., A1 I, A1 II, A1 III, A1 IV, A1 V, A1 VI, A1 VII, A1 VIII, A1 IX, A1 X, A1 XI; A2 I, A2 II, A2 III, A2IV, A2 V, A2 VI, A2 VII, A2 VIII, A2 IX, A2 X, A2 XI; B1 I, B1 II, B1 III, B1 IV, B1 V, B1VI, B1 VII, B1 VIII, B1 IX, B1 X, B1 XI; B2 I, B2 II, B2 III, B2 IV, B2 V, B2 VI, B2 VII, B2 VIII, B2 IX, B2 X, B2 XI);

[0103] Correspondingly, the No. I antibody in each separation area reacts with the a magnetic bead, the No. II antibody reacts with the b magnetic bead, the No. III antibody reacts with the c magnetic bead, the No. IV antibody reacts with the d magnetic bead, the No. V antibody reacts with the e magnetic bead, the No. VI antibody reacts with the f magnetic bead, the No. VII antibody reacts with the g magnetic bead, the No. VIII antibody reacts with the h magnetic bead, the No. IX antibody reacts with the i magnetic bead, the No. X antibody reacts with the j magnetic bead, and the No. XI antibody reacts with the k magnetic bead;

[0104] The various magnetic beads that captured the stripped antibodies were incubated with the secondary antibody labeled with AE (the secondary antibody in the present invention is an anti-mouse secondary antibody, and any substance that can specifically react with the stripped antibodies can achieve similar effects according to this method);

[0105] By comparing the luminescence value of each magnetic bead after the reaction, if it is more than 2.3 times higher than the background value, it is considered to contain the antibody corresponding to the magnetic bead, and the antibody is positive; the luminescence value of each antibody can be used to determine the content of the antigen to be tested in the sample, thereby achieving quantitative detection.

[0106] The incubated magnetic beads were detected by chemiluminescence instrument, and the test results were as follows: Figure 5 As shown (taking region A1 as an example), the technical solution of the present invention can simultaneously detect multiple specific reactants in any region (in this embodiment, 11 antibodies are detected simultaneously). By separately detecting different antibodies by chemiluminescence, the limitation of overlapping fluorescence emission spectra is broken, and super-multivariate detection is achieved.

[0107] Sample Group 3

[0108] One of the paraffin tissue sections prepared from FFPE was detected using a traditional immunofluorescence detection method, and the other corresponding section was detected using the chemiluminescence method of the present invention.

[0109] Among them, the method of detection using traditional immunofluorescence detection method is as follows:

[0110] Paraffin tissue sections prepared using FFPE were dewaxed with xylene;

[0111] Afterwards, the cells were rehydrated with graded ethanol of 100%, 95%, 85%, and 75%;

[0112] Antigen retrieval was performed by microwave heat retrieval of antigens using preheated epitope retrieval reagent for 30 min;

[0113] Endogenous catalase was inactivated by incubation with 3% H2O2 at room temperature for 20 min;

[0114] The cells were incubated with 10% goat serum at room temperature for 20 min to block nonspecific epitopes.

[0115] The sections were incubated with fluorescently labeled anti-HSP-90 antibodies in a 4°C refrigerator overnight;

[0116] The samples were washed with TBST for 5 min × 3 times;

[0117] Imaging was performed using a SLIDEVIEW VS200 multielement fluorescence microscope.

[0118] The method of multiple immunohistochemical luminescence detection based on spatial reconstruction of the present invention is as follows:

[0119] Paraffin tissue sections prepared using FFPE were dewaxed with xylene;

[0120] Afterwards, the cells were rehydrated with graded ethanol of 100%, 95%, 85%, and 75%;

[0121] Antigen retrieval was performed by microwave heat retrieval of antigens using preheated epitope retrieval reagent for 30 min;

[0122] Endogenous catalase was inactivated by incubation with 3% H2O2 at room temperature for 20 min;

[0123] The cells were incubated with 10% goat serum at room temperature for 20 min to block nonspecific epitopes.

[0124] The sections were incubated with anti-HSP-90 antibody labeled with acridinium ester in a refrigerator at 4°C overnight;

[0125] The samples were washed with TBST for 5 min × 3 times;

[0126] Divide the sample into several portions (any portion is acceptable, in this embodiment, 6 rows and 8 columns, for a total of 48 portions); number each separated area (as shown in FIG3 , numbered A1 to H6);

[0127] Stripping the antibodies from each compartment (any method may be used; in this embodiment, the method for stripping the antibodies is microwave treatment, which involves adding an antigen retrieval reagent and microwave-heating the sample at 95° C. for 20 minutes). Numbering the antibodies stripped from each compartment (numbered A1 to H6, respectively);

[0128] Stripped HSP-90 antibody in antigen retrieval reagent.

[0129] HSP-90 protein was detected according to the method described in sample group 1, and the numbers of the stripped antibodies were matched one by one with the numbers of each separation area, and the detection results of each antibody were restored to the position of each separation area of ​​the sample to achieve in situ detection.

[0130] The results of imaging tissue sections using fluorescence microscopy are as follows Figure 2 As shown, the expression of HSP-90 can be detected in situ on tissue sections. Figure 2 The white area in the middle is the expressed HSP-90. Figure 2 The inability to quantify HSP-90 expression at every location is a problem with existing immunohistochemistry. This inability to quantify and manual interpretation can easily lead to inter-observer variability.

[0131] The results of conventional immunofluorescence imaging and the multiple immunohistochemical luminescence detection based on spatial reconstruction of the present invention are as follows: Figure 3A and Figure 3B As shown, Figure 3A It will Figure 2 The corresponding fluorescence image in is divided into 48 regions, and the average fluorescence intensity of each region; Figure 3B It will be with Figure 3A After the corresponding other slice was divided into 48 areas, the results of chemiluminescence detection were performed in each area. The results showed that the average fluorescence intensity and the chemiluminescence results corresponded to each other, but the fluorescence intensity results could not be quantified, and the distinction between different fluorescence intensities between the separated areas was not high, which was prone to observer variability due to subjective judgment. However, the results of chemiluminescence quantification can be read directly by the machine, and each area has an intuitive and accurate quantitative result, which avoids the observer variability caused by subjective judgment and achieves accurate quantification of the results. In addition, the chemiluminescence results of different areas are not only more direct, but also have higher distinction and contrast, and also achieve in situ detection.

[0132] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention are intended to be covered by the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A multiplex immunohistochemical luminescence detection method based on spatial reconstruction, characterized in that: include: S1. Prepare samples; S2. incubating the sample to obtain one or more specific reactants; S3, performing a spatial separation operation on the sample to form a number of separation areas; S4, performing a specific reactant stripping operation on each separated area to obtain a number of specific reactant combinations obtained by stripping the corresponding separated areas; S5, reacting each specific reactant combination with a plurality of magnetic beads modified with different anti-reactants; S6. Perform chemiluminescence detection on the various magnetic bead combinations after the reaction to obtain the luminescence value of each specific reactant corresponding to each separation area; S7, performing a spatial reconstruction operation on the luminescence value of each specific reactant obtained by the detection according to the separated area where it is located, to obtain the in situ quantitative chemiluminescence detection result of the sample; The step S5 comprises the following sub-steps: S51, splitting the specific reactant combination corresponding to a single separation area into n molecular combinations, where n is not less than the number of magnetic bead types corresponding to the reaction; S52, reacting each molecular combination with a magnetic bead respectively to obtain magnetic beads after the specific reactant combination reacts with the anti-reactant modified on the magnetic beads; S53, collecting the magnetic beads obtained after the reaction of each sub-combination to obtain multiple magnetic bead combinations after the reaction of specific reactant combinations; S54, performing sub-steps S51 to S53 on the specific reactant combinations corresponding to the respective separation regions to obtain a plurality of magnetic bead combinations after the specific reactant combinations corresponding to the respective separation regions have reacted; The chemiluminescence detection of the multiple magnetic beads after the reaction comprises: Performing chemiluminescence detection on the multiple post-reaction magnetic beads contained in the multiple magnetic bead combinations after the reaction of the specific reactant combination corresponding to each separation area to obtain the luminescence value of the specific reactant corresponding to the reactant magnetic bead; Based on the corresponding relationship between the target analyte and the specific reactant, the luminescence value of the specific reactant is used to determine the content of the target analyte, that is, the content of the target analyte in the separated area; The target detection object is an antigen, the specific reactant is an antibody, and the anti-reactant is an antigen.

2. The method according to claim 1, wherein The step S3 further comprises: Add identification numbers to the separated areas formed.

3. The method according to claim 2, wherein The step S4 further includes: The corresponding identification number is added to the specific reaction product combination obtained by stripping.

4. The method according to claim 3, wherein The sub-step S51 further includes: adding corresponding identification numbers to the sub-combinations obtained by splitting; The sub-step S53 further includes: adding corresponding identification numbers to the various magnetic bead combinations.

5. The method according to claim 4, wherein The spatial reconstruction operation includes assigning the luminescence value of each specific reactant obtained by detection to a separate region according to the identification number.

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