DAB immune color development kit with enhanced color development performance and product stability and application of DAB immune color development kit

Abstract

The invention discloses a DAB immune color development kit capable of enhancing color development performance and product stability and application of the DAB immune color development kit, and relates to the technical field of immunohistochemical staining. The DAB immune chromogenic kit provided by the invention comprises a DAB A solution, a DAB B solution and a DAB enhancer, the DAB A liquid comprises hydrochloric acid, 3, 3 '-diaminobenzidine and a specific polymer component; the DAB solution B comprises hydrogen peroxide, disodium ethylene diamine tetraacetate, tris (hydroxymethyl) aminomethane, hydrochloric acid and imidazole. A specific polymer component is introduced to form a compound with DAB, so that the rigidity of a precipitation product is remarkably enhanced, the problems of deformation and collapse caused by water loss in a dry sealing process are effectively solved, and the morphological characteristics of cell nucleuses are completely maintained. By optimizing the pH ratio system of the solution A and the solution B, the sensitivity and the dyeing strength of the chromogenic reaction are optimized, and the chromogenic strength and the sensitivity are further improved in cooperation with the copper sulfate enhancer. The kit is particularly suitable for nuclear antigen labeling items such as Ki-67 and the like, and is suitable for manual and automatic platforms.

Description

Technical Field

[0001] This invention relates to the field of immunohistochemical staining technology, and in particular to a DAB immunochromatographic kit with enhanced staining performance and product stability, and its application. Background Technology

[0002] Immunohistochemical (IHC) staining is an indispensable technique in pathological diagnosis. The 3,3'-diaminobenzidine (DAB) chromogenic system, due to its ability to produce an insoluble brown precipitate, is widely used to label target antigens in tissue sections. This brown precipitate, after a series of processes including dehydration, clearing, and mounting, can be observed and analyzed under a microscope. However, the chromogenic products formed by the traditional DAB system have significant defects in their physical structure, mainly manifested in insufficient rigidity and resistance to deformation.

[0003] In practice, the precipitate formed by traditional DAB reactions is highly susceptible to deformation during the "dry sealing" process. "Dry sealing" refers to removing moisture from the slides by air drying or blow-drying before mounting. During this process, as moisture evaporates, the DAB precipitate, lacking structural rigidity and the support of its liquid environment, is prone to collapse, shrinkage, and even the formation of filamentous or blocky artifacts. This deformation severely affects the clear presentation of cell nuclei, especially in nuclear positive markers such as Ki-67, leading to inaccurate positive cell counts or deviations in staining intensity assessment, thus interfering with pathologists' diagnostic judgment.

[0004] Mounting procedures are generally divided into two methods: "wet mounting" and "dry mounting." "Wet mounting" refers to mounting tissue sections directly with resin-containing mounting media after clearing, without a drying step; while "dry mounting" requires thorough drying of the sections after clearing before mounting. In recent years, with the improvement of automation in pathological testing, more and more hospitals have begun to use fully automated staining and mounting machines. These devices mostly use a "wet mounting" process, which helps improve testing consistency, reduce human error, and minimize reagent exposure risks for operators. However, in many small and medium-sized hospitals and primary care pathology departments, manual "dry mounting" remains the mainstream method due to equipment limitations. This method is highly dependent on operator experience, but even so, deformation of traditional DAB products during dry mounting is difficult to avoid. Furthermore, if there is residual moisture on the sections during mounting (e.g., incomplete dehydration and clearing) or improper use of mounting media, air bubbles, crystals, or other artifacts may be introduced, further affecting the quality of microscopic examination.

[0005] In summary, the deformation issues exposed by existing DAB chromogenic systems under dry-mount conditions have become a key limiting factor affecting staining stability and interpretation accuracy. Therefore, there is an urgent need to develop a novel DAB immunochromogenic kit that not only possesses highly sensitive chromogenic performance but also forms a precipitate with high structural rigidity and strong resistance to deformation to adapt to different mounting methods (especially dry-mounting), while also being compatible with automated detection platforms, thereby providing more reliable technical support for pathological diagnosis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing DAB immunochromatographic technology has problems such as insufficient rigidity of the chromogenic products, easy deformation after dry sealing, and difficulty in balancing staining sensitivity and stability. The present invention provides a novel DAB immunochromatographic kit with enhanced chromogenic performance and product stability and its application.

[0007] To address the above problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides a DAB immunochromatographic kit, the DAB staining kit comprising DAB A solution, DAB B solution and DAB enhancer.

[0008] In the DAB immunochromogenic kit of the present invention, the DAB A solution comprises hydrochloric acid, 3,3'-diaminobenzidine (DAB), and a polymer selected from one or more of polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP).

[0009] Furthermore, the pH value of the DAB A solution is 2-3, and the acidic environment is conducive to the stable storage of DAB and polymers.

[0010] Furthermore, the volume concentration of hydrochloric acid in the DAB A solution is 0.3-3%, and the molar concentration of 3,3'-diaminobenzidine is 5-100 mM.

[0011] Furthermore, the molar concentration of the polymer in the DAB A solution is 0.3-1.2 mM, and the molecular weight is 300-10000. The introduction of the polymer can form a complex network structure with the DAB oxidation products during the color development process, significantly enhancing the rigidity of the precipitate, effectively resisting deformation during the dry sealing and dehydration process, and maintaining the integrity of the cell nucleus morphology.

[0012] In the DAB immunochromatographic kit of the present invention, the DAB B solution comprises hydrogen peroxide (H2O2), disodium ethylenediaminetetraacetate (EDTA), tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid, and imidazole.

[0013] Furthermore, the pH value of the DAB B solution is 5.5-6.5.

[0014] Further, the DAB B solution contains hydrogen peroxide at a volume concentration of 0.06-0.24%, disodium ethylenediaminetetraacetate at a molar concentration of 0.3-2 mM, tris(hydroxymethyl)aminomethane at a molar concentration of 30-80 mM, hydrochloric acid at a volume concentration of 6-12%, and imidazole at a molar concentration of 30-80 mM.

[0015] In the DAB B solution, H2O2 acts as an oxidant participating in the enzymatic reaction, EDTA acts as a metal ion chelating agent to stabilize the reaction system, Tris and hydrochloric acid together form a buffer system to maintain the reaction pH, and imidazole acts as a catalyst to reduce the activation energy required for enzyme catalysis of H2O2. Through hydrogen bonding and conjugated systems, it forms proton channels, accelerating proton transfer and influencing the deposition kinetics of DBA. The pH of the DABB solution is between 5.5 and 6.5, which can maintain a relatively stable buffer solution environment.

[0016] In the DAB immunochromatographic kit of the present invention, the DAB enhancer includes copper sulfate.

[0017] Furthermore, the molar concentration of copper sulfate in the DAB enhancer is 50-150 mM, for example, 60 mM, 80 mM, 100 mM, 120 mM, etc.

[0018] The enhancer is not added during the preparation of the working solution, but rather applied separately to the tissue section after solution A and solution B are mixed and the conventional colorimetric reaction steps are completed. This invention does not have strict requirements on the amount of DAB enhancer added; the amount sufficient to completely cover and wet the entire surface of the tissue section is acceptable. Those skilled in the art can adjust the amount according to the size and thickness of the section. For example, for a standard tissue section with an area of ​​approximately 1 cm × 1 cm, 120 μL of copper sulfate solution is typically applied. This invention utilizes the Cu in copper sulfate... 2+ As a complexation center, it further complexes with the oxidized and deposited DAB complex. This process can significantly deepen the color of the DAB precipitate (from brownish-yellow to dark brown), effectively enhancing the contrast of the color development results and making them easier to observe and interpret. It is particularly suitable for the detection of antigens with low expression levels.

[0019] The present invention also provides an immunohistochemical staining method, using the aforementioned DAB immunochromogenic kit, and comprising the following steps: (i) Mix DAB A solution and DAB B solution at a volume ratio of 1:1-3 to obtain DAB working solution; (ii) Apply DAB working solution to tissue sections that have been incubated with primary and secondary antibodies to perform a colorimetric reaction; (iii) After the colorimetric reaction is complete, add DAB enhancer to the system; (iv) Perform hematoxylin counterstaining; (v) Use blue reversion solution to perform the blue reversion operation; (vi) To seal a film.

[0020] A further technical solution is that, in step (i), the volume ratio of the DAB A liquid to the DAB B liquid is 1:1, 1:1.5, 1:2, or 1:3.

[0021] Furthermore, the pH of the DAB working solution is 3-5. The DAB working solution is prepared by mixing DAB A solution and DAB B solution at a specific volume ratio, and the pH value of the resulting DAB working solution needs to be precisely controlled within an optimized range of 3-5. This pH range helps to enhance the catalytic activity of horseradish peroxidase (HRP) while ensuring the efficiency of polymer-DAB complex formation, thereby synergistically improving the sensitivity and staining intensity of the colorimetric reaction.

[0022] The present invention also provides the application of the DAB immunochromatographic kit in the field of immunohistochemical (IHC) staining.

[0023] Compared with the prior art, the technical effects achieved by the present invention include: The DAB immunochromatographic kit provided by this invention introduces a specific polymer component, which forms a complex with DAB. This significantly improves the problems of DAB precipitation collapse and shrinkage during the dry sealing process, and greatly enhances the mechanical strength and deformation resistance of the DAB chromogenic product. It is particularly suitable for the morphology preservation and quantitative analysis of nuclear antigens such as Ki-67. Furthermore, by precisely controlling the pH environment of DAB A solution, DAB B solution, and the mixed working solution, and by using copper sulfate for complexation enhancement, the sensitivity, specificity, staining intensity, and contrast of the chromogenic reaction are all significantly improved.

[0024] This invention provides a DAB colorimetric solution that simultaneously meets the requirements of high rigidity, high sensitivity, and high contrast through multi-component synergistic optimization, and has significant clinical application value and market prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 2 and Comparative Example 6 of this invention. In the figures, A is a wet-mounted slide of tonsil tissue (Ki-67 antibody) from Comparative Example 6; B is a wet-mounted slide of tonsil tissue (Ki-67 antibody) from Example 2; C is a dry-mounted slide of tonsil tissue (Ki-67 antibody) from Comparative Example 6; D is a dry-mounted slide of tonsil tissue (Ki-67 antibody) from Example 2; E is a dry-mounted slide of tonsil tissue (CKpan antibody) from Comparative Example 6; F is a dry-mounted slide of tonsil tissue (CKpan antibody) from Example 2; G is a dry-mounted slide of tonsil tissue (D2-40 antibody) from Comparative Example 6; and H is a dry-mounted slide of tonsil tissue (D2-40 antibody) from Example 2.

[0027] Figure 2 The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 1 and Comparative Example 1 of this invention. In the images, A and C are dry-mounted slides of tonsil tissue (Ki-67 antibody) from Comparative Example 1; B and D are dry-mounted slides of tonsil tissue (Ki-67 antibody) from Example 1.

[0028] Figure 3 The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 2 and Comparative Example 2 of this invention. In the images, A is a dry-mounted slide of cervical tissue (P16 antibody) from Comparative Example 2; B is a dry-mounted slide of cervical tissue (P16 antibody) from Example 2; C is a dry-mounted slide of tonsil tissue (CD20 antibody) from Comparative Example 2; and D is a dry-mounted slide of tonsil tissue (CD20 antibody) from Example 2.

[0029] Figure 4 The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 3 and Comparative Example 3 of this invention. In the images, A is a dry-mounted slide of colorectal tissue (PD-L1 antibody) from Comparative Example 3; B is a dry-mounted slide of colorectal tissue (PD-L1 antibody) from Example 3; C is a dry-mounted slide of tonsil tissue (Bcl-2 antibody) from Comparative Example 3; and D is a dry-mounted slide of tonsil tissue (Bcl-2 antibody) from Example 3.

[0030] Figure 5 The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 3 and Comparative Example 4 of this invention. In the figures, A is a dry-mounted slide of tonsil tissue (CKpan antibody) from Comparative Example 4; B is a dry-mounted slide of tonsil tissue (CKpan antibody) from Example 3; C is a dry-mounted slide of tonsil tissue (P53 antibody) from Comparative Example 4; and D is a dry-mounted slide of tonsil tissue (P53 antibody) from Example 3.

[0031] Figure 6The images show a comparison of the immunohistochemical staining results of the DAB immunochromatographic kits used in Example 4 and Comparative Example 5 of this invention. In the images, A is a dry-mounted slide of tonsil tissue (CK5 / 6 antibody) from Comparative Example 5; B is a dry-mounted slide of tonsil tissue (CK5 / 6 antibody) from Example 4; C is a dry-mounted slide of tonsil tissue (Vimentin antibody) from Comparative Example 5; and D is a dry-mounted slide of tonsil tissue (Vimentin antibody) from Example 4. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Experimental methods in the following examples, unless otherwise specified, were generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents and instruments used are commercially available standard products.

[0034] Example 1 The DAB immunochromatographic kit provided in this embodiment includes DAB A solution, DAB B solution, and DAB enhancer.

[0035] The preparation method of DAB A solution is as follows: Dissolve DAB (molar concentration: 10 mM) and PAA (molar concentration: 1 mM, Mw=2000) in purified water as required for the preparation, and adjust the pH to 3.0 with hydrochloric acid, wherein the molar concentration of the hydrochloric acid is 1 mol / L.

[0036] The preparation method of DAB B solution is as follows: Dissolve Tris (molar concentration: 50 mM) and EDTA (molar concentration: 0.8 mM) in purified water as required for the preparation, add imidazole (molar concentration: 50 mM) and 4 mL / L H2O2, adjust the pH to 6.5 with hydrochloric acid, and the concentration of the hydrogen peroxide solution is 30%.

[0037] The preparation method of DAB reinforcing agent is as follows: Dissolve copper sulfate (60 mM) in purified water to prepare a copper sulfate solution.

[0038] Immunohistochemical staining was performed using the DAB immunochromatographic kit provided in Example 1, as follows: 1) Mix DAB A solution and DAB B solution at a volume ratio of 1:1 (DAB working solution pH≈5.0), and incubate the tissue sections that have been dewaxed, repaired, blocked by the blocking agent, labeled with the primary antibody, and amplified with the secondary antibody using the DAB working solution for 6 min. 2) After rinsing with deionized water, incubate the tissue sections that have undergone DAB staining with DAB enhancer for 6 minutes to enhance staining; 3) After rinsing with deionized water, counterstain the tissue sections with hematoxylin for 2 min and then with blue solution for 4 min; 4) Choose any of the following methods for sealing: 4.1) Dry mounting: Dehydrate the tissue sections with conventional graded ethanol. After ethanol dehydration, remove the tissue sections, dry them with a hair dryer, and then mount them with a neutral resin mounting medium. 4.2) Wet mounting: Dehydrate the tissue sections with conventional graded ethanol, then soak them in xylene to clear them. While the tissue is still covered with xylene (i.e., not dry), immediately add a neutral resin mounting medium to mount the sections.

[0039] Example 2 The DAB immunochromatographic kit provided in this embodiment includes DAB A solution, DAB B solution, and DAB enhancer.

[0040] The preparation method of DAB A solution is as follows: Dissolve DAB (molar concentration: 10 mM) and PEI (molar concentration: 1 mM, Mw=1800) in purified water as required for the preparation, and adjust the pH to 3.0 with hydrochloric acid, wherein the molar concentration of the hydrochloric acid is 1 mol / L.

[0041] The preparation method of DAB B solution is as follows: Dissolve Tris (molar concentration: 50 mM) and EDTA (molar concentration: 0.8 mM) in purified water as required for the preparation, add imidazole (molar concentration: 50 mM) and 4 mL / L H2O2, adjust the pH to 6.5 with hydrochloric acid, and the concentration of the hydrogen peroxide solution is 30%.

[0042] The preparation method of DAB reinforcing agent is as follows: Dissolve copper sulfate (60 mM) in purified water to prepare a copper sulfate solution.

[0043] Immunohistochemical staining was performed using the DAB immunochromatographic kit provided in Example 2, as follows: 1) Mix DAB A solution and DAB B solution at a volume ratio of 1:1 (DAB working solution pH≈5.0), and incubate the tissue sections that have been dewaxed, repaired, blocked by the blocking agent, labeled with the primary antibody, and amplified with the secondary antibody using the DAB working solution for 6 min. 2) After rinsing with deionized water, incubate the tissue sections that have undergone DAB staining with DAB enhancer for 6 minutes to enhance staining; 3) After rinsing with deionized water, counterstain the tissue sections with hematoxylin for 2 min and then with blue solution for 4 min; 4) Choose any of the following methods for sealing: 4.1) Dry mounting: Dehydrate the tissue sections with conventional graded ethanol. After ethanol dehydration, remove the tissue sections, dry them with a hair dryer, and then mount them with a neutral resin mounting medium. 4.2) Wet mounting: Dehydrate the tissue sections with conventional graded ethanol, then soak them in xylene to clear them. While the tissue is still covered with xylene (i.e., not dry), immediately add a neutral resin mounting medium to mount the sections.

[0044] Example 3 The DAB immunochromatographic kit provided in this embodiment includes DAB A solution, DAB B solution, and DAB enhancer.

[0045] The preparation method of DAB A solution is as follows: Dissolve DAB (molar concentration: 10 mM) and PEI (molar concentration: 1 mM, Mw=1800) in purified water as required for the preparation, and adjust the pH to 2.5 with hydrochloric acid, wherein the molar concentration of the hydrochloric acid is 1 mol / L.

[0046] The preparation method of DAB B solution is as follows: Dissolve Tris (molar concentration: 50 mM) and EDTA (molar concentration: 0.8 mM) in purified water as required for the preparation, add imidazole (molar concentration: 50 mM) and 4 mL / L H2O2, adjust the pH to 6.0 with hydrochloric acid, and the concentration of the hydrogen peroxide solution is 30%.

[0047] The preparation method of DAB reinforcing agent is as follows: Dissolve copper sulfate (60 mM) in purified water to prepare a copper sulfate solution.

[0048] Immunohistochemical staining was performed using the DAB immunochromatographic kit provided in Example 3, as follows: 1) Mix DAB A solution and DAB B solution at a volume ratio of 1:1 (DAB working solution pH≈3.5), and incubate the tissue sections that have been dewaxed, repaired, blocked by the blocking agent, labeled with the primary antibody, and amplified with the secondary antibody using the DAB working solution for 6 min. 2) After rinsing with deionized water, incubate the tissue sections that have undergone DAB staining with DAB enhancer for 6 minutes to enhance staining; 3) After rinsing with deionized water, counterstain the tissue sections with hematoxylin for 2 min and then with blue solution for 4 min; 4) Choose any of the following methods for sealing: 4.1) Dry mounting: Dehydrate the tissue sections with conventional graded ethanol. After ethanol dehydration, remove the tissue sections, dry them with a hair dryer, and then mount them with a neutral resin mounting medium. 4.2) Wet mounting: Dehydrate the tissue sections with conventional graded ethanol, then soak them in xylene to clear them. While the tissue is still covered with xylene (i.e., not dry), immediately add a neutral resin mounting medium to mount the sections.

[0049] Example 4 The DAB immunochromatographic kit provided in this embodiment includes DAB A solution, DAB B solution, and DAB enhancer.

[0050] The preparation method of DAB A solution is as follows: Dissolve DAB (molar concentration: 10 mM) and PEI (molar concentration: 1 mM, Mw=1800) in purified water as required for the preparation, and adjust the pH to 2.5 with hydrochloric acid, wherein the molar concentration of the hydrochloric acid is 1 mol / L.

[0051] The preparation method of DAB B solution is as follows: Dissolve Tris (molar concentration: 50 mM) and EDTA (molar concentration: 0.8 mM) in purified water as required for the preparation, add imidazole (molar concentration: 50 mM) and 4 mL / L H2O2, adjust the pH to 6.0 with hydrochloric acid, and the concentration of the hydrogen peroxide solution is 30%.

[0052] The preparation method of DAB reinforcing agent is as follows: Dissolve copper sulfate (molar concentration: 120 mM) in the required amount of purified water to prepare a copper sulfate solution.

[0053] Immunohistochemical staining was performed using the DAB immunochromatographic kit provided in Example 4, as follows: 1) Mix DAB A solution and DAB B solution at a volume ratio of 1:1 (DAB working solution pH≈3.5), and incubate the tissue sections that have been dewaxed, repaired, blocked by the blocking agent, labeled with the primary antibody, and amplified with the secondary antibody using the DAB working solution for 6 min. 2) After rinsing with deionized water, incubate the tissue sections that have undergone DAB staining with DAB enhancer for 6 minutes to enhance staining; 3) After rinsing with deionized water, counterstain the tissue sections with hematoxylin for 2 min and then with blue solution for 4 min; 4) Choose any of the following methods for sealing: 4.1) Dry mounting: Dehydrate the tissue sections with conventional graded ethanol. After ethanol dehydration, remove the tissue sections, dry them with a hair dryer, and then mount them with a neutral resin mounting medium. 4.2) Wet mounting: Dehydrate the tissue sections with conventional graded ethanol, then soak them in xylene to clear them. While the tissue is still covered with xylene (i.e., not dry), immediately add a neutral resin mounting medium to mount the sections.

[0054] Comparative Example 1 The DAB immunochromatographic kit provided in Comparative Example 1 differs from that in Example 1 in that the polymer is replaced by polyethylene glycol (molar concentration: 1 mM, Mw=2000) instead of PAA. All other reagents and procedures are the same as in Example 1.

[0055] Comparative Example 2 The DAB immunochromatographic kit provided in Comparative Example 2 differs from that in Example 2 in that the polymer is replaced by PEI (molar concentration: 1 mM, Mw=1800) with PEI (molar concentration: 1 mM, Mw=100000). All other reagents and procedures are the same as in Example 2.

[0056] Comparative Example 3 The DAB immunochromatographic kit provided in Comparative Example 3 differs from that in Example 3 in that the pH of DAB A solution, which was adjusted to 2.5 with hydrochloric acid, is adjusted to 4.0 instead, and the pH of DAB B solution, which was adjusted to 6.0 with hydrochloric acid, is adjusted to 8.5 instead (the pH of the DAB working solution is approximately 7.0). All other reagents and procedures are the same as in Example 3.

[0057] Comparative Example 4 The DAB immunochromatographic kit provided in Comparative Example 4 differs from that in Example 3 in that the pH of DAB A solution, which was adjusted to 2.5 with hydrochloric acid, is adjusted to 1.5 instead, and the pH of DAB B solution, which was adjusted to 6.0 with hydrochloric acid, is adjusted to 4.0 instead (DAB working solution pH≈2.5). All other reagents and procedures are the same as in Example 4.

[0058] Comparative Example 5 The DAB immunochromogenic kit provided in Comparative Example 5 is different from that in Example 4 in that it does not contain a DAB enhancer.

[0059] Comparative Example 6 The immunochromogenic kit provided by Shenzhen Dameng Biotechnology Co., Ltd. (product model: DMRD4044, record number: Yue Shen Xie Bei 20210832) was selected as Comparative Example 6.

[0060] Performance detection (1) The immunochromogenic kits provided in Example 2 of the present invention and Comparative Example 6 were used to perform immunohistochemical staining of three antibodies, Ki-67, CKpan, and D2-40, on tonsil tissues respectively, to systematically evaluate the performance of the DAB immunochromogenic kit of the present invention and the commercially available kit under various conditions. The specific results are as Figure 1 shown.

[0061] In the staining of the strongly positive-expressing Ki-67 antibody, under the wet-sealing condition, both the kit of Example 2 of the present invention and the commercially available kit could maintain the relatively intact nuclear morphology without obvious shrinkage. However, under the dry-sealing condition, the nuclei in Comparative Example 6 showed obvious collapse and shrinkage, accompanied by filamentous and块状 artifacts; while Example 2 of the present invention basically maintained the original nuclear structure without obvious deformation or artifacts.

[0062] In the staining of the non-strongly positive-expressing CKpan antibody, the staining intensity presented by the kit of the present invention was significantly better than that of the commercially available kit, showing better chromogenic ability.

[0063] In the staining of the D2-40 antibody with low antigen abundance, the detection sensitivity of the kit of the present invention was significantly improved compared with that of the commercially available kit, which was more conducive to the detection of low-abundance antigens.

[0064] (2) The immunochromogenic kits provided in Example 1 of the present invention and Comparative Example 1 were used to perform immunohistochemical staining on tonsil tissues (Ki-67 antibody) to evaluate the effect of different polymer stabilizers on the anti-deformation ability of DAB deposits. The specific results are as Figure 2 shown.

[0065] In the immunochromogenic kit of Comparative Example 1, PEG used as a common polymer stabilizer for DAB solution A only played a role in transferring protons and did not participate in the deposition polymerization process of DAB, and thus could not effectively improve the anti-deformation ability of DAB deposits.

[0066] In contrast, after replacing the polymer PEG with PAA in Example 1, PAA could actively participate in the polymerization reaction of DAB deposition, form a cross-linked network structure complex with DAB molecules, and thus significantly enhance the structural stability of the deposits. From Figure 2The results show that, after drying, although the cell nuclei still shrink to some extent, they basically maintain a relatively complete morphological structure, without obvious collapse, filamentous or blocky artifacts, which can ensure the accuracy of the test results.

[0067] (3) Immunohistochemical staining analysis of cervical tissue (P16 antibody) and tonsil tissue (CD20 antibody) was performed using the immunochromatographic kits provided in Example 2 and Comparative Example 2 of this invention, respectively, to evaluate the effect of polymer molecular weight on staining intensity. The specific results are as follows: Figure 3 As shown.

[0068] Example 2, using a low molecular weight polymer, exhibited significantly better staining intensity than Comparative Example 2, which used a high molecular weight polymer. This is because DAB color development is a rapid, multi-step oxidation process catalyzed by HRP to generate short-lived, highly reactive DAB radical intermediates. Low molecular weight polymers possess higher molecular mobility and a more densely exposed active site (amine group), enabling them to more rapidly and effectively capture and stabilize these transient radicals. These radicals serve as nucleation sites, promoting the efficient formation of insoluble precipitates and thus enhancing color development efficiency.

[0069] In contrast, the molecular chains of high molecular weight PEI are prone to entanglement, which leads to the encapsulation of some active sites. The kinetics of binding with DAB free radicals are relatively slow, and some free radicals undergo side reactions or quenching before participating in precipitation formation, ultimately reducing the overall color development efficiency.

[0070] (4) Immunohistochemical staining analysis of colorectal tissue (PD-L1 antibody) and tonsil tissue (Bcl-2 antibody) was performed using the immunochromatographic kits provided in Example 3 and Comparative Example 3 of this invention, respectively, to evaluate the effect of pH value of DAB working solution on staining performance. Specific results are as follows: Figure 4 As shown.

[0071] As can be seen, in antibody projects with low antigen abundance, the staining intensity and sensitivity of the DAB working solution at pH 3.5 are significantly better than those at pH 7.0. This is because horseradish peroxidase (HRP) is actually a mixture of multiple isoenzymes, and their optimal pH varies. Although the classical optimal pH for most isoenzymes is between 5.0 and 7.0, in complex reaction environments such as immunohistochemistry, some key isoenzymes may be more effectively activated under slightly acidic conditions (such as pH 3.5–4.5), thus exhibiting higher catalytic efficiency. Therefore, the immunochromatographic kit provided by this invention, when used, mixes solution A (pH 2–3) with solution B (pH 5.5–6.5) to form a weakly acidic reaction environment of approximately pH 3.5, which provides an ideal catalytic window for these isoenzymes, thereby significantly accelerating the oxidation rate of DAB and improving the staining intensity.

[0072] Meanwhile, under weakly acidic conditions, DAB molecules have higher solubility, and their oxidation potential changes, making them more easily oxidized by the HRP-H2O2 system. When the pH is approximately 3.5, DAB is in a more easily oxidized protonated state, thereby increasing the overall reaction kinetic rate and generating more oxidized DAB precipitate per unit time, ultimately resulting in a significant enhancement in staining intensity.

[0073] (5) Immunohistochemical staining analysis of tonsil tissue (CKpan antibody) and tonsil tissue (P53 antibody) was performed using the immunochromatographic kits provided in Example 3 and Comparative Example 4 of this invention, respectively, to evaluate the staining performance of DAB working solution under different pH conditions. Specific results are as follows: Figure 5 As shown.

[0074] As can be seen, compared with Example 3, the pH of the DAB working solution in Comparative Example 4 was reduced to 2.5. Although the staining intensity was improved, a significant non-specific background appeared. This is because in a solution environment with extremely low pH values, most proteins (including antigens, antibodies, and enzymes) undergo irreversible denaturation, resulting in the destruction of their three-dimensional conformation. Denatured proteins expose a large number of hydrophobic groups and internal amino acid residues, which become "sticky" sites for non-specific binding, adsorbing DAB precipitates and leading to enhanced background staining. Simultaneously, in an overly acidic environment, the kinetics of the oxidative polymerization reaction of DAB itself may change. It may more easily undergo spontaneous oxidative precipitation or react with other reducing substances in the tissue under non-enzymatic catalysis, forming non-enzymatic background staining. Furthermore, in more extreme pH solution environments, denaturation of primary and secondary antibodies leads to the breakage of previously specifically bound recognition sites, and the HRP enzyme protein structure is also severely damaged, resulting in a sharp decrease in catalytic activity or even complete inactivation, leading to a significant reduction in staining intensity or even failure to develop color. Therefore, the pH of the DAB working solution is not necessarily better the lower it is; the pH of the DAB working solution of this invention is preferably 3-5.

[0075] (6) Using the immunochromatographic kits provided in Example 4 and Comparative Example 5 of this invention, immunohistochemical staining analysis was performed on tonsil tissue (CK5 / 6 antibody) and tonsil tissue (Vimentin antibody), respectively, to evaluate the effect of copper sulfate enhancement treatment on staining effect. Specific results are as follows: Figure 6 As shown.

[0076] As can be seen, compared with Comparative Example 5 which did not use an enhancer, the present invention, by introducing copper sulfate (CuSO4) solution after conventional DAB staining, significantly improved both staining intensity and tissue contrast. The core of this phenomenon lies in the Cu... 2+The ions catalytically oxidized and enhanced the already formed oxidized DAB precipitate, and then complexed it with metals. This continuous catalytic oxidation process elongated the conjugated double bond system of the DAB polymer, making its molecular structure more complex and stable. The expansion of the conjugated system directly caused a red shift in its maximum absorption wavelength, and the appearance color deepened from brownish-yellow to dark brown, thus enhancing the intensity of the staining signal.

[0077] Therefore, the role of the copper sulfate enhancer in this invention is not to participate in the initial immune-specific recognition, but rather to amplify and solidify the specific signal a second time through chemical catalysis and complexation.

[0078] In summary, the DAB immunochromogenic kit provided by this invention achieves a significant improvement in immunohistochemical staining performance through the combination of three core technologies. First, by introducing a specific polymer component (such as polyethyleneimine) into solution A, a composite network structure is formed with the DAB chromogenic product, greatly enhancing the rigidity of the precipitate and effectively solving the industry-wide problem of cell nucleus shrinkage and collapse during dry sealing, making it particularly suitable for the precise quantitative analysis of nuclear antigens such as Ki-67. Second, by precisely controlling the pH formulation of solutions A and B, the working solution pH is optimized to the optimal window of 3-5, significantly improving the catalytic efficiency and chromogenic sensitivity of HRP enzyme, while effectively suppressing non-specific background staining. Third, the post-treatment step using copper sulfate enhancer further amplifies the signal through catalytic oxidation and metal complexation, significantly enhancing the chromogenic intensity and contrast.

[0079] In summary, the reagent kit of this invention has high colorimetric sensitivity, strong resistance to dry sealing and deformation, and the working solution can be stably stored for a long time at 2-8℃. It is suitable for both manual and fully automated staining platforms, significantly improving detection efficiency and reducing usage costs.

[0080] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0081] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A DAB immunochromatographic reagent kit, characterized in that, The DAB staining kit includes DAB A solution, DAB B solution, and DAB enhancer; The DAB A solution comprises hydrochloric acid, 3,3'-diaminobenzidine, and a polymer selected from one or more of polyethyleneimine, polyacrylic acid, polyvinyl alcohol, and polyvinylpyrrolidone. The DAB B solution comprises hydrogen peroxide, disodium ethylenediaminetetraacetate, tris(hydroxymethyl)aminomethane, hydrochloric acid, and imidazole. The DAB enhancer includes copper sulfate.

2. The DAB immunochromogenic kit according to claim 1, characterized in that, The pH value of the DAB A solution is 2-3.

3. The DAB immunochromogenic kit according to claim 1 or 2, characterized in that, The volume concentration of hydrochloric acid in the DAB A solution is 0.3-3%, and the molar concentration of 3,3'-diaminobenzidine is 5-100 mM.

4. The DAB immunochromogenic kit according to claim 3, characterized in that, The polymer in the DAB A solution has a molar concentration of 0.3-1.2 mM and a molecular weight of 300-10000.

5. The DAB immunochromatographic reagent kit according to claim 1, characterized in that, The pH value of the DAB B solution is 5.5-6.

5.

6. The DAB immunochromogenic kit according to claim 1 or 5, characterized in that, The DAB solution contains hydrogen peroxide at a volume concentration of 0.06-0.24%, disodium ethylenediaminetetraacetate at a molar concentration of 0.3-2 mM, tris(hydroxymethyl)aminomethane at a molar concentration of 30-80 mM, hydrochloric acid at a volume concentration of 6-12%, and imidazole at a molar concentration of 30-80 mM.

7. The DAB immunochromogenic kit according to claim 1, characterized in that, The copper sulfate molar concentration in the DAB enhancer is 50-150 mM.

8. An immunohistochemical staining method, characterized in that, Using the DAB immunochromogenic kit as described in any one of claims 1-7, and comprising the following steps: (i) Mix DAB A solution and DAB B solution at a volume ratio of 1:1-3 to obtain DAB working solution; (ii) Apply DAB working solution to tissue sections that have been incubated with primary and secondary antibodies to perform a colorimetric reaction; (iii) After the colorimetric reaction is complete, add DAB enhancer to the system; (iv) Perform hematoxylin counterstaining; (v) Use blue reversion solution to perform the blue reversion operation; (vi) To seal a film.

9. The immunohistochemical staining method according to claim 8, characterized in that, The pH of the DAB working solution is 3-5.

10. The application of the DAB immunochromogenic kit as described in any one of claims 1-7 in the field of immunohistochemical staining.

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