An immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction

Abstract

The application relates to an immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction, and relates to the field of immunohistochemistry. The application first constructs a solid-phase Fc binding target by anchoring an IgG1 subtype primary antibody on a tissue section, maintains IgG4-Fc homologous interaction activity in a buffer system containing reduced glutathione, specifically recognizes tissue-fixed immunoglobulin by using a biotin-labeled IgG4 probe, and realizes immunoglobulin tracing staining independent of a Fab antigen recognition channel; two sets of schemes, namely an IgG4 verification scheme and a papain segmented tracing verification scheme, are matched synchronously, and it is confirmed that the signal is derived from IgG4 specific binding and the interaction domain is an Fc fragment. The application is compatible with conventional pathological laboratory equipment and reagents, can correct false positive misjudgment of IgG4 antibody immunohistochemistry, can be used as a new technical means for pathological mechanism research on tumor and autoimmune disease tissue immunoglobulin deposition, is standardized in operation, is high in result reliability, and has good clinical and scientific research application prospects.

Description

Technical Field

[0001] This invention belongs to the field of immunohistochemistry, specifically relating to an immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction. Background Technology

[0002] Immunohistochemical staining is a technique that utilizes antigen-antibody reactions. By labeling antibodies with chromogenic substances, a chemical reaction causes the antibody chromogenic agent to develop color, thereby detecting the presence of target antigen proteins in cells or tissues and studying their localization and relative quantification. In routine pathological interpretation, specific positive staining results are usually attributed to the specific recognition of antigenic epitopes by the Fab region of the primary antibody. For example... Figure 1 As shown in A, the traditional IHC colorimetric signal is generated solely by the antibody Fab fragment specifically recognizing tissue antigens, resulting in only one layer of binding reaction and weak colorimetric intensity.

[0003] However, the inventors repeatedly observed an abnormal staining phenomenon during long-term experiments: when antibodies or probes were prepared with human IgG4 subtype, in addition to the expected antigen-positive areas, reproducible additional staining signals often appeared in the tissue. Unlike traditional random background staining, these signals showed tissue selectivity, anatomical localization and reproducibility, suggesting that there is a mechanism behind it that is independent of classical antigen-antibody recognition.

[0004] Existing reports clearly demonstrate that IgG4 possesses several unique biological characteristics, including Fab arm exchangeability and the ability to engage in Fc-Fc interactions with immobilized immunoglobulins under specific solid-phase conditions. Therefore, when experimental probes or primary antibodies are prepared using the IgG4 isoform, they may exhibit Fc-dependent interactions with pre-existing tissue-associated immunoglobulins, resulting in positive staining results independent of the target antigen. Thus, this seemingly non-specific staining may actually reflect the interaction between the antibody and tissue-bound immunoglobulins, rather than direct binding to the target antigen. Accurately distinguishing between these two types of staining is technically crucial; ignoring this mechanism can lead to misinterpretations of immunohistochemical results. This also suggests that such staining may provide important information regarding the quantity, distribution, and pathological significance of tissue-associated immunoglobulins. The existing technology has two major flaws: First, it cannot distinguish between two independent staining signals: Fab-mediated target antigen-specific staining and non-target staining produced by the Fc-Fc interaction between IgG4-Fc fragments and tissue-fixed immunoglobulins. Confusion between the two can easily lead to misinterpretation of pathological results. Second, it lacks standardized experimental methods to utilize the unique Fc interaction characteristics of IgG4, making it impossible to specifically trace immunoglobulins deposited in tissues. At the same time, it lacks a complete verification protocol to prove that the staining signal originates from the Fc-Fc interaction, and cannot eliminate experimental artifacts.

[0005] Currently, only in vitro biochemical studies have reported that IgG4 can undergo Fc homologous binding. No immunohistochemical practice system adapted to paraffin tissue sections has been established, nor are there any supporting methods for specificity verification and functional domain tracing verification, making it difficult to apply to clinical pathology and basic research. Summary of the Invention

[0006] The purpose of this invention is to provide an immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction, which solves the problem that existing technologies cannot effectively distinguish between traditional antigen-antibody recognition staining and IgG4-mediated Fc-Fc interaction staining.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] An immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction includes the following steps:

[0009] S1. Biotin was used to label the full-molecule IgG4 and IgG1 as target antibodies, respectively.

[0010] S2. Pre-treat and block the tissue sections, then incubate the tissue sections with IgG antibodies at low temperature to anchor IgG proteins in the tissue.

[0011] S3. Incubate the tissue slices anchored to IgG protein with a mixture of the target antibody and reduced glutathione.

[0012] S4. After incubation, add HPR enzyme-labeled streptavidin for reaction, and then add chromogenic substrate for color development.

[0013] As a further optimization of the above invention, it also includes S5, which verifies that the positive signal of the staining method originates from IgG4-Fc interaction rather than a detection artifact, specifically through the following steps:

[0014] S5.1 Select tissue sections with low IgG expression for pretreatment and blocking, and then incubate the tissue sections with IgG primary antibody at low temperature.

[0015] S5.2. The tissue section is incubated with unlabeled intact IgG4 antibody and reduced glutathione, and then anti-human IgG4 antibody is added for further incubation.

[0016] S5.3, then add enzyme-labeled secondary antibody and incubate, then add chromogenic substrate for color development;

[0017] The significantly enhanced positive signal in the tissue section proves that the added unlabeled IgG4 does indeed interact with the tissue-fixed IgG via Fc-Fc interaction and remain on the tissue.

[0018] As a further optimization of the above invention, it also includes S6, which is used to verify whether the IgG4-Fc terminus or Fab reacts with tissue-fixed immunoglobulins, including the following steps:

[0019] S6.1. The whole IgG4 molecule is enzymatically digested into two parts, IgG4-Fab and IgG4-Fc, and together with IgG4 and IgG1, they are used as target antibodies. The target antibodies are then biotinylated.

[0020] S6.2 Pre-treat and block the tissue sections, and pre-incubate the pre-treated tissue sections with IgG primary antibody at low temperature;

[0021] S6.3. The tissue sections are incubated with a mixture of the target antibody and reduced glutathione.

[0022] S6.4 After incubation, add HPR enzyme-labeled streptavidin for reaction, and then add chromogenic substrate for color development.

[0023] When IgG1 and IgG4-Fab groups show no specific staining, while IgG4 whole molecules and IgG4-Fc groups produce specific positive signals, it indicates that IgG4 can specifically bind to the Fc segment of tissue-fixed IgG.

[0024] As a further optimization of the above invention, the biotin is biotin;

[0025] The pretreatment consists of routine immunohistochemical dewaxing, dehydration, and repair; the blocking is goat serum blocking.

[0026] The low-temperature incubation is overnight incubation at 4°C; the incubation is 0.5–2 hours incubation at 37°C.

[0027] The concentration of the reduced glutathione is 3–6 mM.

[0028] The chromogenic substrate is AEC or DAB, and the staining is observed under a light microscope.

[0029] As a further optimization of the above invention, step S4 is replaced by: using fluorescently conjugated streptavidin as a secondary antibody, incubating DAPI, and then observing under a fluorescence microscope.

[0030] As a further optimization of the above invention, in step S2, the IgG antibody is a protein positively expressed in tissue sections; in step S3, the concentration of the target antibody is 0.05–0.2 mg / mL.

[0031] As a further optimization of the above invention, the IgG primary antibody in step S5.1 is a protein positively expressed in tissue sections; the concentration of the unlabeled intact IgG4 in step S5.2 is 0.1-0.2 mg / mL; the anti-human IgG4 antibody is a rabbit anti-human IgG4 antibody; and the enzyme-labeled secondary antibody in step S5.3 is a horseradish peroxidase-labeled goat anti-rabbit secondary antibody.

[0032] As a further optimization of the above invention, step S6.1 describes the whole-molecule IgG4 being digested into Fab and Fc ends using a papaya digestion kit, and the fragments obtained after digestion are used only after meeting the purity standards.

[0033] As a further optimization of the above invention, the immunohistochemical staining method can be applied to any of the following scenarios:

[0034] (1) Quality control of immunohistochemical pathological results, used to distinguish between IgG4 antibody-mediated Fc-Fc false positives and target antigen-specific staining;

[0035] (2) Scientific tracing of the distribution and relative quantification of immunoglobulin deposition in tissues;

[0036] (3) Research on the pathological mechanisms of IgG4-related diseases, tumor immunity, and autoimmune diseases;

[0037] (4) Evaluation of the background effect of IgG4 subtype therapeutic antibody tissue staining.

[0038] As a further optimization of the above invention, the tumor includes breast cancer and lung cancer.

[0039] As a further optimization of the above invention, the tissue section is breast cancer epithelial tissue, and the breast cancer epithelial tissue is selected as anti-CK7.

[0040] The beneficial effects of this invention are as follows:

[0041] (1) This invention utilizes the property that IgG4 subtype antibody can specifically interact with tissue-anchored IgG to develop a staining method that can specifically display the distribution of bound immunoglobulins in tissues.

[0042] (2) By introducing the IgG4 verification experiment, it can be effectively confirmed that the positive signal comes from the specific Fc-Fc interaction mediated by IgG4, rather than non-specific background or detection artifact, which significantly improves the reliability of the results.

[0043] (3) This invention uses papain to digest the entire IgG4 molecule into Fab and Fc ends, which more intuitively proves that it is the Fc end of IgG4 that reacts with the immunoglobulins bound in the tissue. For the first time, it has been confirmed in tissue that IgG4 can have a specific Fc-Fc interaction with fixed IgG. The operation procedure of this method is compatible with conventional immunohistochemistry and can be applied to routine pathological testing without special equipment modification. It can provide a new technical means for studying pathological processes related to immunoglobulin deposition in tissues. Attached Figure Description

[0044] Figure 1 This is a schematic diagram illustrating the mechanism of the improved immunohistochemical method and results of the present invention; (A) Conventional antigen-specific staining is a Fab-terminal binding pathway; (B) IgG4 interacts with IgG1 bound to antigens on the tissue via Fc-Fc interaction; (C) There is no fixed IgG on the tissue, so IgG4 cannot bind; (D) IgG1 cannot interact with fixed IgG via Fc-Fc interaction; (E) The Fc-Fc interaction between the full-molecule IgG4 and IgG1 bound to the tissue is mediated by its Fc terminus, and the Fab terminus cannot bind to IgG1 on the tissue.

[0045] Figure 2 The image shows an example of the improved immunohistochemistry method of the present invention, specifically the staining results of breast cancer and placental tissues, wherein biotin-labeled IgG1 is used as a negative control.

[0046] Figure 3 The following is an example diagram of the IgG4 verification experiment of this invention, specifically the staining results of breast tissue; A: Breast cancer epithelial tissue expresses IgG1 / IgG4, and no positive staining phenomenon occurs when biotin-labeled IgG4 is directly added; B-1: Breast cancer epithelial gland results show CK7 (IgG1 protein); B-2: After pre-incubation with CK7 antibody, biotin-labeled IgG4 is added, and a positive staining phenomenon occurs; B-3: The antibody that shows a positive staining phenomenon with CK7 antibody is IgG4; C is a specific mechanism diagram.

[0047] Figure 4 The images shown are examples of the binding phenomenon of IgG4-Fc ends to verify the present invention, specifically the staining results of lung cancer and breast cancer. Group A is lung cancer tissue, and Group B is placental tissue. Both tissues express IgG1. IgG4 can exhibit positive staining with fixed IgG1, and the object of positive staining is -IgG4-Fc rather than IgG4-Fab. Detailed Implementation

[0048] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0049] At least one embodiment of the present invention discloses an immunohistochemical staining method based on IgG4 antibody Fc-dependent interaction, comprising the following steps:

[0050] S1. Biotin was used to label the full-molecule IgG4 and IgG1 as target antibodies, respectively.

[0051] S2. Pre-treat and block the tissue sections, then incubate the tissue sections with IgG antibodies at low temperature to anchor IgG proteins in the tissue; the IgG antibodies are proteins positively expressed in the tissue sections.

[0052] S3. Tissue sections anchored to IgG protein are incubated with a mixture of target antibody and reduced glutathione; the concentration of the target antibody is 0.05–0.2 mg / mL.

[0053] S4. After incubation, add HPR enzyme-labeled streptavidin for reaction, followed by the addition of a chromogenic substrate for color development. Step S4 can be replaced by using fluorescently conjugated streptavidin as a secondary antibody, incubating with DAPI, and then observing under a fluorescence microscope.

[0054] At least one embodiment of the present invention discloses the following steps for verifying that a positive signal in a staining method originates from IgG4-Fc interaction rather than a detection artifact:

[0055] S5.1 Select tissue sections with low IgG expression for pretreatment and blocking, and then pre-incubate the tissue sections with IgG primary antibody at low temperature; the IgG primary antibody is the protein positively expressed in the tissue section;

[0056] S5.2 Incubate tissue sections with unlabeled intact IgG4 antibody and reduced glutathione, followed by incubation with anti-human IgG4 antibody; the concentration of unlabeled intact IgG4 is 0.1–0.2 mg / mL;

[0057] S5.3. Add enzyme-labeled secondary antibody and incubate, then add chromogenic substrate for color development; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-rabbit secondary antibody.

[0058] The significantly enhanced positive signal in the tissue section proves that the added unlabeled IgG4 does indeed interact with the tissue-fixed IgG via Fc-Fc interaction and remain on the tissue.

[0059] At least one embodiment of the present invention discloses the following method for verifying whether the IgG4-Fc terminus or Fab reacts with tissue-fixed immunoglobulins, including the following steps:

[0060] S6.1. Digest the whole IgG4 molecule into two parts, IgG4-Fab and IgG4-Fc, and use them together with IgG4 and IgG1 as target antibodies. Label the target antibodies with biotin. Specifically, digest the whole IgG4 molecule into Fab and Fc ends using a papaya digestion kit. The fragments obtained after digestion must meet the purity requirements before use.

[0061] S6.2 Pre-treat and block the tissue sections, and pre-incubate the pre-treated tissue sections with IgG primary antibody at low temperature;

[0062] S6.3. After incubating the tissue sections with a mixture of the target antibody and reduced glutathione, the tissue sections were incubated.

[0063] S6.4 After incubation, add HPR enzyme-labeled streptavidin for reaction, and then add chromogenic substrate for color development.

[0064] When IgG1 and IgG4-Fab groups show no specific staining, while IgG4 whole molecules and IgG4-Fc groups produce specific positive signals, it indicates that IgG4 can specifically bind to the Fc segment of tissue-fixed IgG.

[0065] Biotin is the name of the product.

[0066] Pretreatment consisted of routine immunohistochemical dewaxing, dehydration, and repair; blocking was performed with goat serum.

[0067] Low-temperature incubation is performed at 4°C overnight; incubation is performed at 37°C for 0.5–2 hours.

[0068] The concentration of reduced glutathione is 3–6 mM;

[0069] The chromogenic substrate is AEC or DAB, and the staining is observed under a light microscope.

[0070] The above immunohistochemical staining methods can be applied to any of the following scenarios:

[0071] (1) Quality control of immunohistochemical pathological results, used to distinguish between IgG4 antibody-mediated Fc-Fc false positives and target antigen-specific staining;

[0072] (2) Scientific tracing of the distribution and relative quantification of immunoglobulin deposition in tissues;

[0073] (3) Research on the pathological mechanisms of IgG4-related diseases, tumor immunity, and autoimmune diseases;

[0074] (4) Evaluation of the background effect of IgG4 subtype therapeutic antibody tissue staining.

[0075] I. Explanation

[0076] 1. Experimental reagents of this invention

[0077] (1) Basic buffer PBS (pH 7.2~7.4): NaCl 8.0g, KCl 0.2g, Na2HPO4 1.44g, KH2PO4 0.24g, dissolved in 1L deionized water, filtered through a 0.22μm filter membrane for sterilization, and stored at 4℃;

[0078] (2) 5mM reduced glutathione (GSH) working buffer: Weigh the reduced GSH powder, add sterile PBS to dissolve it, and make a final concentration of 5mM. Prepare and use immediately, and incubate in the dark.

[0079] (3) 3% hydrogen peroxide blocking solution: 30% H2O2 stock solution diluted with PBS at a volume ratio of 1:9, freshly prepared at room temperature;

[0080] (4) Blocking solution: 5% normal goat serum blocking solution, diluted with PBS, containing 0.1% Triton X-100, used to block the adsorption of non-specific proteins in tissues;

[0081] (5) Tris-EDTA antigen retrieval solution (pH 9.0): Tris 1.21g, EDTA disodium 0.37g, dissolved in 1L deionized water;

[0082] (6) AEC colorimetric reagent kit (AEC-0037), hematoxylin counterstain (CTS-1097), glycerol gelatin mounting medium (C0187);

[0083] (7) Biotin labeling kit (HY-D0802), papain antibody digestion kit (44685);

[0084] (8) Antibody raw materials: human IgG1 (Chengdu Rongsheng Pharmaceutical Co., Ltd., specification: 5% 50ml 2.5g / bottle), human IgG4 complete monoclonal antibody (IVIG source, Chengdu Rongsheng Pharmaceutical Co., Ltd., specification: 5% 50ml 2.5g / bottle); HER2-IgG1 primary antibody (kit-0043), HLA-G-IgG1 primary antibody (Ab26034), anti-CK7(IgG1) primary antibody (ZM-0069), rabbit anti-human IgG4 specific primary antibody (ab109493), HRP-labeled streptavidin (A0303), HRP goat anti-rabbit secondary antibody (KT5020);

[0085] (9) Verification reagents: Western blot protein electrophoresis, transfer membrane, blocking: complete set of secondary antibody color development reagents.

[0086] Marker (AR1210); Blocking solution BSA (Cat#A8020); NC membrane (10600002); Secondary antibody (926-68079);

[0087] 2. General tissue pretreatment method of this invention

[0088] (1) Section preparation: The tissue paraffin block was continuously sectioned with a section thickness of 4μm, attached to a glass slide to prevent detachment, and baked at 60℃ for 2h;

[0089] (2) Dewaxing and rehydration: Xylene I 10 min → Xylene II 10 min → Anhydrous ethanol 5 min → 95% ethanol 5 min → 80% ethanol 5 min → Rinse with deionized water for 3 min;

[0090] (3) Antigen retrieval: The slides were immersed in Tris-EDTA retrieval solution, subjected to high pressure retrieval at 121℃ for 3 min, and then allowed to cool naturally to room temperature;

[0091] (4) Blocking of endogenous peroxidase: Incubate with 3% H2O2 at room temperature in the dark for 10 min, wash with PBS 3 times, 3 min each time;

[0092] (5) Serum blocking: Add 5% goat serum blocking solution, block at room temperature for 30 minutes, pour off the blocking solution, do not wash, and proceed directly to the next incubation step.

[0093] It should also be noted that, unless otherwise specified, the methods used in this invention are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products; the instruments used, such as paraffin microtome, constant temperature water bath, 4°C constant temperature incubator, optical microscope, high-speed centrifuge, gel imaging system, high-pressure antigen retrieval pot, pipette, light-proof incubation humidifier, etc., are all conventional instruments known to those skilled in the art, unless otherwise specified.

[0094] II. Experiment

[0095] 1. Immunohistochemical staining method based on IgG4-Fc interaction

[0096] Paraffin-embedded tissue sections of HER2-positive human breast cancer and full-term placenta; three parallel control sections were set up for each tissue group, as follows:

[0097] Control group 1: Incubation only with anchored IgG primary antibody (anti-HER2 / anti-HLA-G);

[0098] Control group 2: Anchored IgG primary antibody + biotin-labeled IgG1;

[0099] Experimental group: anchored IgG primary antibody + biotin-labeled IgG4.

[0100] Preparation of target antibody probes: Purified intact human IgG1 and intact human IgG4 antibodies were covalently labeled with biotin using a commercial biotin labeling kit according to the instructions. After labeling, free biotin was removed by dialysis, the antibodies were concentrated by ultrafiltration, and the protein concentration was determined by the BCA method. The antibodies were aliquoted and stored at -20℃. Before use, the antibodies were diluted with PBS containing 5mM GSH to a working concentration of 0.1mg / mL, mixed thoroughly, and stored in the dark. The IgG1 probe was used as a negative control throughout the process.

[0101] (1) Pretreatment of tissue sections

[0102] Complete the pretreatment steps described above, including baking, dewaxing and rehydration, high-pressure antigen retrieval, hydrogen peroxide blocking, and goat serum blocking.

[0103] (2) Construction of tissue-anchored IgG molecules

[0104] Breast cancer tissue sections were treated with diluted anti-HER2 IgG1 primary antibody; placental tissue sections were treated with anti-HLA-G IgG1 primary antibody.

[0105] Place the slides in a humidified, light-protected chamber and incubate overnight at 4°C (14-16 h). After incubation, wash the slides three times with PBS for 5 min each time to thoroughly remove unbound free primary antibody. The IgG1 primary antibody specifically binds to the tissue target antigen (HER2 / HLA-G) through the Fab segment, stably anchoring on the tissue slides, exposing the free Fc domain, and forming a solid-phase target site for IgG4Fc binding.

[0106] (3) Biotin-labeled IgG probe was incubated in the dark.

[0107] Two probe working solutions were prepared: ① 0.1 mg / mL biotin-IgG1 (5 mM GSH-PBS); ② 0.1 mg / mL biotin-IgG4 (5 mM GSH-PBS). IgG1 working solution was added to slides in control group 2, IgG4 working solution was added to slides in the experimental group, and blank PBS containing 5 mM GSH was added to slides in control group 1. The slides were incubated in a humidified chamber at 37°C for 60 min, protected from light. After incubation, the slides were washed thoroughly with PBS four times for 5 min each time to remove unspecifically bound free IgG probes. Reduced GSH is a necessary reducing microenvironment for Fc-Fc homologous interactions of IgG4. Without GSH buffer, the degree of Fc binding is low, and specific positive staining is difficult to detect with the naked eye.

[0108] (4) HRP-labeled streptavidin signal amplification

[0109] All slides were incubated with HRP-labeled streptavidin working solution at room temperature in the dark for 60 min; washed 4 times with PBS for 5 min each time; streptavidin binds specifically to the biotin group on the probe with high affinity, and the coupled HRP enzyme provides a catalytic site for subsequent color development.

[0110] (5) AEC color development and hematoxylin counterstaining

[0111] Prepare AEC staining solution, evenly cover the slides, and develop at room temperature in the dark for 3-8 minutes. Observe the staining intensity under a microscope in real time. Once the expected staining intensity is reached, immediately immerse the slides in deionized water to stop the staining process. Stain the nuclei with hematoxylin for 30 seconds, rinse with running water for 1 minute to reverse the blue staining. Mount the slides with glycerol gelatin and observe and photograph them under an optical microscope.

[0112] like Figure 1 A, Figure 1 B Figure 1 D and Figure 2 As shown, in breast cancer and placental tissues, compared with control group 1 which was stained with positive antibodies (anti-HER2 and anti-HLA-G) alone, the experimental group, after additional addition of biotin-IgG4 (anti-HER2 + biotin-IgG4 and anti-HLA-G + biotin-IgG4), exhibited significantly enhanced and specifically distributed positive staining signals. In contrast, control group 2, which received biotin-IgG1 as a control, did not show this phenomenon. This suggests that the staining may be due to an Fc-Fc reaction between the Fc fragment of IgG4 and the Fc terminus of anchored IgG.

[0113] Conclusion: This experiment established an immunohistochemical staining system based on the Fc-Fc-dependent homology interaction between IgG4 and tissue-anchored IgG1, which specifically displays the distribution of fixed immunoglobulins in tissues and distinguishes between traditional Fab antigen-specific staining and Fc-Fc-mediated additional positive signals.

[0114] 2. IgG4 Specificity Verification Experiment

[0115] Paraffin sections of benign human breast epithelium with very low endogenous IgG expression were used, with three control sections set up, as detailed below:

[0116] A. Blank control: Only biotin-IgG4 was added, without pre-anchored IgG primary antibody;

[0117] Experimental group B: Biotin-IgG4 was added after pre-incubation with anti-CK7 (IgG1) primary antibody;

[0118] C validation group: pre-incubated with anti-CK7 (IgG1) primary antibody to release unlabeled IgG4-Fc sites, and then developed with anti-human IgG4 antibody.

[0119] Unlabeled intact IgG4 working solution: Dissolve IgG4 in 5mM GSH-PBS to a final concentration of 0.2mg / mL, prepare fresh before use; Rabbit anti-human IgG4 primary antibody: Dilute with PBS to a working concentration of 1:1000; HRP-labeled goat anti-rabbit secondary antibody: Dilute according to the kit's recommended ratio; Other buffers and pretreatment reagents are the same as in Experiment 1.

[0120] (1) General preprocessing of slices

[0121] Complete the pretreatment steps described above, including baking, dewaxing and rehydration, antigen retrieval, hydrogen peroxide blocking, and goat serum blocking.

[0122] (2) Constructing tissue-anchored IgG target sites

[0123] In group B and group C, anti-CK7 (IgG1) subtype primary antibody was added to the slices; in group A, blank PBS was added; the slices were incubated overnight at 4°C in the dark for 14-16 hours; the slices were washed with PBS 3 times, 5 minutes each time; CK7 was specifically expressed in epithelial cells, and the IgG subtype primary antibody was stably anchored on the surface of the epithelial antigen, exposing the free Fc domain.

[0124] Group C tissue sections were treated with 0.2 mg / mL unlabeled intact IgG4 (5 mM GSH-PBS); Groups A and B were treated with equal amounts of blank GSH-PBS; the tissue sections were incubated at 37°C in the dark for 60 min to allow free IgG4 molecules to pre-bind to the Fc binding sites of tissue-anchored IgG, occupying all interaction binding sites; the tissue sections were washed thoroughly with PBS 4 times, 5 min each time, to remove unbound free IgG4.

[0125] (3) Specific IgG4 recognition incubation

[0126] All three groups of slides were added with 1:1000 rabbit anti-human IgG4 specific primary antibody and incubated at 37°C in the dark for 60 min; washed three times with PBS for 5 min each time; this antibody only specifically recognizes IgG4 molecules bound to tissues and does not recognize endogenous IgG1 in tissues.

[0127] (4) Secondary antibody incubation, AEC staining, and hematoxylin counterstaining

[0128] All slides were added with HRP goat anti-rabbit secondary antibody and incubated at room temperature for 45 min; washed with PBS; AEC staining, hematoxylin counterstaining, mounting, and microscopic examination were performed according to the same procedure as in Experiment 1.

[0129] like Figure 3 As shown, in breast cancer epithelial tissue, the original tissue was negative for anti-IgG1 / IgG4 staining, indicating that the expression of endogenous IgG1 / IgG4 in breast epithelium was low. This proves that the biotin-labeled IgG4 added directly could not interact with IgG through Fc-Fc interactions and therefore showed no staining phenomenon. Figure 3A); In the same tissue, Anti-CK7 staining showed a positive signal in epithelial cells ( Figure 3 B-1). Upon addition of biotin-IgG4, additional positive staining appeared in the interstitial region (B-1). Figure 3 B-2). In the verification experiment ( Figure 3 After incubation with Anti-CK7 (B-3), followed by incubation with unlabeled IgG4, the addition of rabbit anti-human IgG4 antibody (anti-IgG4) resulted in significant staining, demonstrating that IgG4 indeed binds to the anchored IgG antibody Anti-CK7 via Fc-Fc interaction and remains on the tissue. This proves that biotin-IgG4 can bind to CK7 and anchor IgG. The staining signal is a product of the specific Fc-Fc interaction between IgG4 and tissue-fixed IgG, eliminating non-specific background artifacts. Figure 1 C Figure 3 C).

[0130] Conclusion: This experiment proves that the positive signal observed in Experiment 1 is not a false appearance caused by tissue non-specific adsorption, reagent contamination, or autofluorescence. The positive staining does indeed originate from the specific Fc-Fc binding of IgG4 molecules to tissue-anchored IgG.

[0131] 3. IgG4-Fc / Fab fragment traceability verification experiment

[0132] Paraffin-embedded tissue sections of human lung cancer and full-term placenta; five parallel control groups were set up for each tissue group, as detailed below:

[0133] Only anchored IgG1 primary antibody;

[0134] Anchoring IgG1+Biotin-IgG1 (negative control);

[0135] Anchoring the full molecule of IgG1+Biotin-IgG4;

[0136] Anchoring IgG1+ Biotin-IgG4-Fab fragment;

[0137] Anchoring IgG1+Biotin-IgG4-Fc fragment.

[0138] Probe and IgG4 fragment preparation - Papain digestion of intact IgG4 antibody preparation: Take purified intact human IgG4 antibody and prepare digestion buffer according to the instructions of the papain digestion kit; mix antibody and papain at a mass ratio of 20:1 and digest at 37℃ for 4 h; terminate the reaction with digestion stop solution, and separate the digestion products by protein A affinity chromatography column: Fc fragment binds to protein A, and Fab fragment is collected by flow-through; elute to obtain purified IgG4-Fab and IgG4-Fc respectively.

[0139] Fragment purity verification by Western blot: SDS-PAGE electrophoresis was performed on whole IgG4 molecules, purified Fab, and purified Fc protein; after transfer and blocking, the membrane was incubated with anti-human IgG-Fab specific antibody and anti-human IgG-Fc specific antibody, respectively; HRP secondary antibody imaging was performed; only the corresponding bands were visible and there were no impurities, which proved that the fragment purity met the standard, and biotin labeling could then be performed.

[0140] Biotin-labeled complete probe kit: Four proteins were simultaneously labeled using a biotin-labeling kit: intact IgG1, intact IgG4, IgG4-Fab, and IgG4-Fc; free biotin was removed by dialysis, and after BCA quantification, the samples were aliquoted and stored at -20℃; before use, the samples were uniformly diluted to 0.1 mg / mL in 5 mM GSH-PBS.

[0141] (1) General preprocessing of slices

[0142] Lung cancer and placental sections were pretreated according to the above steps: baking, dewaxing and rehydration, high-pressure antigen retrieval, 3% H2O2 blocking, and goat serum blocking.

[0143] (2) Construction of tissue-anchored IgG1 target

[0144] All 5 groups of tissue sections were added with universal IgG1 subtype primary antibody; incubated overnight at 4°C in a humidified chamber protected from light for 14-16 hours; washed 3 times with PBS for 5 minutes each time; lung cancer and placental tissue antigens can stably bind to IgG1, exposing free Fc binding sites on the tissue surface.

[0145] (3) Segmented probes were incubated in the dark.

[0146] Each group was added with the corresponding probe working solution (0.1 mg / mL, 5 mg GSH-PBS system); one group was added with blank GSH-PBS; all sections were incubated at 37°C in the dark for 60 min; after incubation, the sections were washed with PBS 4 times for 5 min each time to remove unbound free probe fragments.

[0147] (4) Signal amplification, color development and restaining process

[0148] HRP-labeled streptavidin incubated at room temperature for 60 min → washed thoroughly with PBS → AEC staining → hematoxylin nuclei counterstaining → dehydrated and mounted → observed and photographed under an optical microscope. The operating parameters were exactly the same as in Experiment 1.

[0149] like Figure 1 E and Figure 4As shown, the addition of anti-IgG1 followed by biotinylated IgG4 to lung cancer tissue and placental tissue resulted in significantly deeper and more specific positive staining than anti-IgG1 alone. Biotinylated IgG1, as a negative control, showed no positive staining. Furthermore, sections with biotinylated IgG4-Fab showed no obvious positive staining, similar to the negative control group. However, sections with biotinylated IgG4-Fc all showed obvious and specific positive staining signals, fully demonstrating that this positive staining phenomenon occurred at the Fc end of IgG4 rather than the Fab end, further confirming the characteristic that IgG4-Fc can interact specifically with fixed IgG.

[0150] Conclusion: This experiment distinguishes the contributions of the Fab antigen-binding segment and the Fc segment in the IgG4 molecule to the Fc-Fc homology interaction, directly proving that the positive staining in Experiment 1 is mediated only by the IgG4-Fc domain, and the Fab fragment does not participate in this binding reaction.

[0151] In summary, this invention is the first to fully validate the Fc-Fc homologous interaction between IgG4 and fixed IgG in a paraffin tissue section system, establishing a novel immunohistochemical tracing technique independent of the classic Fab antigen recognition pathway. It also simultaneously provides two supporting schemes: IgG4 specificity verification and IgG4 segmented source tracing verification, completely eliminating experimental artifacts and ensuring reproducible and highly reliable experimental results. It can distinguish between target antigen-specific signals and Fc-Fc false-positive signals in IgG4 antibody staining, correcting immunohistochemical pathological misjudgments. Furthermore, it can specifically trace the deposition location and relative content of immunoglobulins in tumor, breast, lung, and placental tissues, supporting research on the mechanisms of IgG4-related diseases, tumor immunity, and autoimmune diseases.

[0152] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An immunohistochemical staining method based on Fc-dependent interaction of IgG4 antibody, characterized in that, Includes the following steps: S1. Biotin was used to label the full-molecule IgG4 and IgG1 as target antibodies, respectively. S2. Pre-treat and block the tissue sections, then incubate the tissue sections with IgG antibodies at low temperature to anchor IgG proteins in the tissue. S3. Incubate the tissue slices anchored to IgG protein with a mixture of the target antibody and reduced glutathione. S4. After incubation, add HPR enzyme-labeled streptavidin for reaction, and then add chromogenic substrate for color development.

2. The immunohistochemical staining method according to claim 1, characterized in that, It also includes S5, which verifies that the positive signal of the staining method originates from IgG4-Fc interaction rather than a detection artifact, specifically through the following steps: S5.1 Select tissue sections with low IgG expression for pretreatment and blocking, and then incubate the tissue sections with IgG primary antibody at low temperature. S5.

2. The tissue section is incubated with unlabeled intact IgG4 antibody and reduced glutathione, and then anti-human IgG4 antibody is added for further incubation. S5.3, then add enzyme-labeled secondary antibody and incubate, then add chromogenic substrate for color development; The significantly enhanced positive signal in the tissue section proves that the added unlabeled IgG4 does indeed interact with the tissue-fixed IgG via Fc-Fc interaction and remain on the tissue.

3. The immunohistochemical staining method according to claim 2, characterized in that, It also includes S6, used to verify whether the IgG4-Fc terminus or Fab reacts with tissue-fixed immunoglobulins, including the following steps: S6.

1. The whole IgG4 molecule is enzymatically digested into two parts, IgG4-Fab and IgG4-Fc, and together with IgG4 and IgG1, they are used as target antibodies. The target antibodies are then biotinylated. S6.2 Pre-treat and block the tissue sections, and pre-incubate the pre-treated tissue sections with IgG primary antibody at low temperature; S6.

3. The tissue sections are incubated with a mixture of the target antibody and reduced glutathione. S6.4 After incubation, add HPR enzyme-labeled streptavidin for reaction, and then add chromogenic substrate for color development. When IgG1 and IgG4-Fab groups show no specific staining, while IgG4 whole molecules and IgG4-Fc groups produce specific positive signals, it indicates that IgG4 can specifically bind to the Fc segment of tissue-fixed IgG.

4. The immunohistochemical staining method according to any one of claims 1-3, characterized in that, The biotin is biotin; The pretreatment consists of routine immunohistochemical dewaxing, dehydration, and repair; the blocking is goat serum blocking. The low-temperature incubation is overnight incubation at 4°C; the incubation is 0.5–2 hours incubation at 37°C. The concentration of the reduced glutathione is 3–6 mM. The chromogenic substrate is AEC or DAB, and the staining is observed under a light microscope.

5. The immunohistochemical staining method according to claim 1, characterized in that, Step S4 is replaced with: using fluorescently conjugated streptavidin as a secondary antibody, incubating DAPI, and then observing under a fluorescence microscope.

6. The immunohistochemical staining method according to claim 1, characterized in that, In step S2, the IgG antibody is a protein positively expressed in tissue sections; in step S3, the concentration of the target antibody is 0.05–0.2 mg / mL.

7. The immunohistochemical staining method according to claim 2, characterized in that, The IgG primary antibody mentioned in step S5.1 is a protein positively expressed in tissue sections; the concentration of the unlabeled intact IgG4 mentioned in step S5.2 is 0.1-0.2 mg / mL; the anti-human IgG4 antibody is rabbit anti-human IgG4 antibody; the enzyme-labeled secondary antibody mentioned in step S5.3 is horseradish peroxidase-labeled goat anti-rabbit secondary antibody.

8. The immunohistochemical staining method according to claim 3, characterized in that, In step S6.1, the IgG4 whole-molecule is digested into Fab and Fc ends using a papaya digestion kit, and the fragments obtained after digestion are used only after meeting the purity standards.

9. The immunohistochemical staining method according to any one of claims 1-3, characterized in that, The immunohistochemical staining method is applicable to any of the following scenarios: (1) Quality control of immunohistochemical pathological results, used to distinguish between IgG4 antibody-mediated Fc-Fc false positives and target antigen-specific staining; (2) Scientific tracing of the distribution and relative quantification of immunoglobulin deposition in tissues; (3) Research on the pathological mechanisms of IgG4-related diseases, tumor immunity, and autoimmune diseases; (4) Evaluation of the background effect of IgG4 subtype therapeutic antibody tissue staining.

10. The immunohistochemical staining method according to claim 9, characterized in that, The tumors include breast cancer and lung cancer; the tissue section is breast cancer epithelial tissue.

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