Antibody compositions based on semiconductor colloidal quantum wells and methods for detection of t fh and dc function in lymphoid tissues

Abstract

The application discloses a kind of antibody compositions based on semiconductor colloidal quantum well and its method for Tfh and DC function detection in lymphoid tissue.The composition includes semiconductor colloidal quantum well and specific antibody, semiconductor colloidal quantum well is coupled with at least one specific antibody.The preparation method includes (1) synthesis colloidal quantum well;(2) water phase transfer and surface functionalization are carried out using ligand exchange strategy;(3) colloidal quantum well is coupled with specific antibody, target antibody is stably grafted to colloidal quantum well surface;(4) the semiconductor colloidal quantum well antibody conjugate obtained is purified, and stable water-soluble colloidal quantum well antibody composition is obtained.The composition is used for multiple immunofluorescence staining of lymphoid tissue section.The application realizes the simultaneous detection and spatial positioning of Tfh cell proportion, DC autophagy level and its antigen presentation ability in the same tissue section, and significantly improves the signal resolution and image accuracy of multi-target detection.

Description

Technical Field

[0001] This invention belongs to the field of biomarker and immune function detection technology, specifically relating to a fluorescent antibody formed by coupling a semiconductor colloidal quantum well with a specific antibody, and its application method in detecting the functional status of follicular helper T cells (Tfh) and dendritic cells (DC) in lymphoid tissue. Background Technology

[0002] Lymphatic tissue is an important component of the immune system, and can be divided into central lymphatic tissue and peripheral lymphatic tissue based on function and structure. Central lymphatic tissue includes the bone marrow and thymus, with the thymus being the primary site of T cell differentiation and maturation, ensuring T cell immune tolerance through positive and negative selection mechanisms. Peripheral lymphatic tissue includes lymph nodes, the spleen, and mucosa-associated lymphoid tissue. Through complex cell interactions and microenvironment regulation, lymphatic tissue maintains immune homeostasis and is crucial for antigen recognition, immune response, and the formation of immune memory.

[0003] The thymus, as a core organ of the central lymphatic system, plays a crucial role in T cell development and the establishment of immune tolerance. However, thymic disorders (such as thymoma or thymic hyperplasia) lead to significant alterations in its microenvironment, including abnormalities in immune cell composition, cytokine secretion, and antigen presentation. These changes are often closely associated with the pathogenesis of autoimmune diseases such as myasthenia gravis and systemic lupus erythematosus. In patients with thymoma, abnormalities in the thymic microenvironment can lead to the breakdown of immune tolerance, inducing the production of autoantibodies and thus exacerbating disease progression. Therefore, accurate detection of the functional status of immune cells in the thymic and other lymphatic tissue microenvironments is of great significance for elucidating the pathogenesis of autoimmune diseases and developing diagnostic tools.

[0004] Follicular helper T cells (Tfh) are a subset of CD4+ T cells characterized by high expression of CXCR5. They primarily assist in B cell differentiation, maturation, and antibody production in germinal centers of lymphoid tissues such as lymph nodes and the thymus. In autoimmune diseases such as myasthenia gravis, Tfh cell hyperactivity is considered a key factor driving abnormal antibody production. For example, in MG patients, Tfh cells promote the production of anti-acetylcholine receptor antibodies by overactivating B cells, leading to neuromuscular junction dysfunction. Therefore, assessing the number and activation status of Tfh cells in lymphoid tissues is expected to provide important reference data for evaluating the immune status of lymphoid tissues.

[0005] Dendritic cells (DCs) are the body's primary antigen-presenting cells, marked by CD21 and CD23. In lymphoid tissues, they are responsible for antigen capture, processing, and presentation to T cells, initiating adaptive immune responses. Autophagy is a crucial mechanism for maintaining DC homeostasis, and its levels can be assessed using markers such as Beclin and LC3. In autoimmune diseases such as myasthenia gravis, abnormal DC autophagy levels are considered associated with disease progression. Studies have shown that enhanced DC autophagy can promote cellular phenotypic changes, thereby upregulating the expression of co-stimulatory molecules such as CD80 and HLA-DR, as well as MHC molecules, leading to enhanced antigen presentation capacity. This enhancement can induce autoimmune responses. In MG patients, hyperactive DC autophagy may exacerbate disease symptoms by enhancing antigen presentation and driving the activation of autoreactive T cells.

[0006] The enhanced antigen-presenting capacity induced by dendritic cell (DC) autophagy has a dual impact on the immune system. On the one hand, enhanced antigen presentation can improve the efficiency of the immune system's response to exogenous antigens; on the other hand, in autoimmune diseases, this enhancement may lead to the over-presentation of self-antigens, breaking immune tolerance and inducing or aggravating pathological damage. Therefore, accurately detecting the autophagy level and antigen-presenting capacity of DCs (using CD80 or HLA-DR markers) is of great significance for understanding the mechanisms of immune imbalance in the lymphoid tissue microenvironment.

[0007] Abnormalities in the lymphoid tissue (especially the thymus) microenvironment are closely related to the pathogenesis of autoimmune diseases, with the functional status of Tfh cells and dendritic cells (DCs) playing a crucial role. However, traditional organic fluorescent dyes suffer from drawbacks such as poor photostability, easy photobleaching, severe overlap of excitation and emission spectra, and low staining signal-to-noise ratio, making it difficult to meet the needs for efficient and accurate analysis of Tfh cell proportions, DC autophagy levels, and antigen-presenting capabilities. Therefore, developing a highly sensitive detection method based on a semiconductor colloidal quantum well antibody composition to conduct multidimensional assessments of the functional status of Tfh cells and DCs in lymphoid tissues can provide technical support for a deeper understanding of changes in the immune microenvironment and their mechanisms of action in autoimmune diseases.

[0008] In recent years, colloidal quantum dots (CQDs) have been increasingly applied in the field of biolabeling due to their high brightness, narrow-band emission, excellent photostability, and tunable wavelengths. Based on this, two-dimensional colloidal quantum wells (CQWs), such as CdSe, CdSe / CdS, CdSe / CdZnS, and CdZnSe / ZnS, have been developed. These exhibit higher excitation cross-sections, stronger multiphoton responses, and superior optical purity, demonstrating superior performance in multiplex fluorescent labeling and high spatial resolution imaging. In particular, the two-dimensional structure of CQWs provides a larger surface area, significantly increasing antibody-binding sites and enabling efficient and stable antibody binding. This structural characteristic not only enhances labeling specificity, ensuring high affinity binding between antibodies and target antigens, but also significantly improves detection sensitivity, making the detection of trace antigens possible. Furthermore, the unique optical properties of CQWs, through narrow emission spectra and optimized optical characteristics, effectively reduce signal crosstalk in multicolor immunofluorescence and background interference in complex lymphoid tissue sections. This characteristic is particularly crucial in multi-target detection, enabling high-resolution spatial localization. Compared to traditional fluorescent dyes, which are easily limited by tissue autofluorescence and signal overlap, semiconductor colloidal quantum wells perform better in the complex microenvironment of lymphoid tissue, making them especially suitable for immunological research requiring high-precision, multidimensional analysis. However, existing immunofluorescence techniques still have limitations in sensitivity, resolution, and multi-target detection, making it difficult to meet the needs for efficient and accurate analysis of Tfh cell proportions, DC autophagy levels, and antigen presentation capabilities.

[0009] Despite the excellent optical properties of CQWs, their application in immune cell functional imaging is still in the exploratory stage. The main challenges are: how to achieve efficient water solubility and surface functionalization of CQWs, how to stably conjugate high-affinity antibodies, and how to ensure fluorescence intensity, specificity, and tissue compatibility in the imaging system.

[0010] Therefore, there is an urgent need to develop a CQW antibody probe with high stability, high specificity, and multicolor labeling capability to improve the ability to detect the functional status of immune cells such as Tfh and DC in lymphoid tissues. Summary of the Invention

[0011] This invention addresses the shortcomings of existing techniques for detecting Tfh and DC function in lymphoid tissues, such as poor stability in bioimaging, limited labeling channels, and strong imaging background interference. It provides a highly sensitive, high-throughput, and low-interference antibody composition based on a semiconductor colloidal quantum well, and its method for detecting Tfh and DC function in lymphoid tissues. This enables precise quantification and spatial distribution visualization of the functional status of Tfh cells and DCs, significantly improving signal resolution and image accuracy for multi-target detection, and effectively overcoming problems such as signal overlap, fluorescence quenching, and tissue background interference inherent in traditional fluorescent probes. It is particularly suitable for the co-labeling, functional status determination, and spatial distribution studies of Tfh cells and DCs in lymphoid tissues, providing an efficient, sensitive, and scalable detection platform for the functional analysis of immune cells in complex lymphoid tissues.

[0012] The present invention relates to an antibody composition based on a semiconductor colloidal quantum well, and adopts the following technical solution.

[0013] The colloidal quantum well antibody composition comprises a semiconductor colloidal quantum well (CQW) and a specific antibody. The semiconductor colloidal quantum well is coupled to at least one specific antibody to form a semiconductor colloidal quantum well antibody conjugate. The specific antibody selectively recognizes one or more of the following target antibodies: CD4, CXCR5, CD21, CD23, Beclin, LC3, CD80, and HLA-DR, and is covalently linked to the semiconductor colloidal quantum well via EDC / NHS or SMCC chemical conjugation. The specific antibody is a monoclonal antibody IgG or a polyclonal antibody IgG, preferably a monoclonal antibody IgG, to improve labeling specificity.

[0014] The aforementioned colloidal quantum trap antibody composition was subjected to multi-label staining using paraffin sections of lymphoid tissue. This enabled multi-target detection and spatial localization of Tfh cell proportions, DC autophagy levels, and antigen-presenting capacity within the lymphoid tissue sections. Spatial localization and quantitative analysis of three cell subsets were also performed to evaluate the immune status of related diseases (such as thymopathy complicated by autoimmune diseases). The lymphoid tissue sections were selected from paraffin sections of thymic tissue, thymoma tissue, thymic hyperplasia tissue, lymph node tissue, or spleen tissue.

[0015] The semiconductor colloidal quantum well includes CdSe, CdSe / ZnS, CdSe / CdS, CdZnSe / ZnS, and CdSe / CdZnS and their derived structures.

[0016] The semiconductor colloidal quantum well preferably has a CdSe / ZnS structure with an emission wavelength of 450-630nm. The bandgap is controlled by adjusting the Cd precursor concentration and the Se / S ratio, covering blue (450nm), green (520nm), yellow (580nm), or red (630nm) light. It has a broad excitation spectrum and a narrow emission spectrum (half-width of 10nm or less), effectively avoiding signal crosstalk and tissue autofluorescence interference in multicolor immunofluorescence.

[0017] The semiconductor colloidal quantum well antibody conjugate comprises: a 525nm wavelength semiconductor colloidal quantum well with a CD4 antibody conjugate, a 625nm wavelength semiconductor colloidal quantum well with a CXCR5 antibody conjugate, a 525nm wavelength semiconductor colloidal quantum well with a CD21 antibody conjugate, a 585nm wavelength semiconductor colloidal quantum well with a CD23 antibody conjugate, a 625nm wavelength semiconductor colloidal quantum well with a Beclin or LC3 antibody conjugate, and a 625nm wavelength semiconductor colloidal quantum well with a CD80 or HLA-DR antibody conjugate; wherein:

[0018] (1) The following materials were used for Tfh cell detection: 525nm wavelength semiconductor colloidal quantum well with CD4 antibody conjugate and 625nm wavelength semiconductor colloidal quantum well with CXCR5 antibody conjugate; staining was performed in the order of CD4 (525nm wavelength) first and then CXCR5 (625nm wavelength).

[0019] (2) The following are used for detecting DC autophagy levels: 525nm wavelength semiconductor colloidal quantum well with CD21 antibody conjugate, 585nm wavelength semiconductor colloidal quantum well with CD23 antibody conjugate, and 625nm wavelength semiconductor colloidal quantum well with Beclin or LC3 antibody conjugate; staining is performed in the order of CD21 (525nm wavelength), CD23 (585nm wavelength) and Beclin or LC3 (625nm wavelength).

[0020] (3) The following are used for detecting DC antigen presentation ability: 525nm wavelength semiconductor colloidal quantum well with CD21 antibody conjugate, 585nm wavelength semiconductor colloidal quantum well with CD23 antibody conjugate, and 625nm wavelength semiconductor colloidal quantum well with CD80 or HLA-DR antibody conjugate; staining is performed in the order of CD21 (525nm wavelength), CD23 (585nm wavelength), and CD80 or HLA-DR (625nm wavelength).

[0021] The above pairing scheme reduces signal crosstalk to less than 5%, increases signal strength by 3-5 times, and improves spatial resolution.

[0022] The method for preparing the above-mentioned antibody composition based on semiconductor colloidal quantum well includes the following steps:

[0023] (1) Synthesis of Colloidal Quantum Wells (CQWs): Two-dimensional colloidal quantum wells were prepared in the oil phase using a hot-injection method to obtain oil-phase colloidal quantum wells (CQWs). Compared with traditional spherical quantum dots, the two-dimensional structure of the quantum wells endows them with a larger specific surface area, which can significantly improve the density of antibody-coupled sites, enhance target recognition ability, and improve detection sensitivity and specificity, making them suitable for high signal-to-noise ratio detection of low-abundance targets.

[0024] By precisely controlling the thickness and chemical composition of the core and shell layers, adjusting the emission wavelength to 510-650 nm, and optimizing the quantum yield to over 60%, the composition is ensured to have high brightness and narrow emission spectrum.

[0025] (2) Use ligand exchange strategies (such as using mercapto PEG or polyamine ligands) to perform aqueous phase transfer and surface functionalization to achieve stable dispersion and biocompatibility of CQWs in the aqueous environment; through carboxyl ligand functionalization, the antibody binding capacity is increased by 30%-50% compared with semiconductor colloidal quantum wells, and the biocompatibility and coupling efficiency are significantly improved.

[0026] (3) The semiconductor colloidal quantum well synthesized in step (1) is coupled with a specific antibody (using methods such as EDC / NHS or SMCC, using carboxyl-modified CQWs to chemically couple with the amino groups on the antibody molecule), and the target antibody (CD4, CXCR5, CD21, CD23, Beclin, LC3, CD80 or HLA-DR) is stably grafted onto the surface of the colloidal quantum well (CQWs) to form a semiconductor colloidal quantum well antibody conjugate;

[0027] (4) The obtained semiconductor colloidal quantum trap antibody conjugate is purified (by ultrafiltration, chromatography, centrifugation, etc.) to obtain a stable water-soluble fluorescent label, which is the colloidal quantum trap antibody composition.

[0028] The specific process of step (2) is as follows:

[0029] ① Resuspend the oil-phase colloidal quantum wells (CQWs) in chloroform or hexane, and slowly add an alkaline aqueous solution containing mercaptopropionic acid (MPA) or mercaptoacetic acid (TGA). The pH of the alkaline aqueous solution is 10-11.

[0030] ②Irrigate or sonicate at room temperature for 30-60 minutes until the fluorescence signal is transferred to the aqueous phase;

[0031] ③ Free ligands were removed by purification (ultrafiltration (10kDa MWCO) or chromatography) to obtain water-soluble colloidal quantum wells (CQWs) with carboxyl groups on their surface.

[0032] The process of coupling the semiconductor colloidal quantum well with the specific antibody in step (3) includes the following steps:

[0033] ① Dilute the semiconductor colloidal quantum well to 1.0-2.0 μM and place it in PBS buffer at pH 7.2-7.4;

[0034] ② Add pre-dissolved and deaggregated antibody dropwise to the semiconductor colloidal quantum well solution, controlling the molar ratio of semiconductor colloidal quantum well to antibody to be 1:(10-200);

[0035] ③ A two-stage coupling reaction is adopted, wherein: in the first stage, EDC and NHS are added to the semiconductor colloidal quantum well solution for carboxyl preactivation to generate an active ester intermediate; in the second stage, the antibody solution is slowly added to the activation reaction system and gently rotated and incubated at 20-25℃ for 2-4 hours, and 0.1%-0.5% BSA and 0.01% Tween-20 are added as protective agents and surface stabilizers to reduce non-specific adsorption and aggregation of the semiconductor colloidal quantum well.

[0036] ④ After the reaction is complete, centrifuge to remove large particle aggregates, and perform two rounds of concentration and washing through a 100kDa ultrafiltration tube. The concentration ratio in each round shall not exceed 10 times. The washing solution is PBS buffer containing 0.01% Tween-20.

[0037] ⑤ Finally, the volume was adjusted to 50 μL with PBS to obtain a fluorescently stable and sharp signal conjugate antibody semiconductor colloidal quantum well composition.

[0038] To further improve colloidal stability or reduce non-specific adsorption, block polymer ligands (such as HS-PEG-COOH) may be grafted in step (3) to enhance water solubility and biocompatibility.

[0039] The colloidal quantum well antibody composition prepared by the above process exhibits high photostability, multiple labeled channels, and high target recognition capability. It is suitable for multi-target immunofluorescence staining and functional status analysis of Tfh cells and dendritic cells (DCs) in lymphoid tissues, providing an efficient and sensitive detection tool for immunological research. It outperforms traditional methods in terms of conjugation rate, signal intensity, and background suppression, achieving a conjugation efficiency of 75%-88%, with free antibody residue controlled below 5%. The fluorescence of the semiconductor colloidal quantum well remains stable and sharp, without quenching or shift. The conjugate is resuspended in PBS buffer containing 0.1%-1% BSA, with 0.1% sodium azide added for preservation. It is then aliquoted and stored at 4℃ or -20℃ in the dark. Experiments have verified that this method allows the conjugate to be stably preserved for 2-4 weeks, with fluorescence intensity attenuation of less than 10%, antibody activity retention exceeding 95%, and storage at -20℃ for one year.

[0040] To ensure the optical properties, stability, and coupling efficiency of the above-mentioned colloidal quantum well antibody composition, the following characterization was performed:

[0041] (1) The emission wavelength and fluorescence intensity of the colloidal quantum well were confirmed by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy;

[0042] (2) The stability and particle size distribution of the colloid were evaluated by dynamic light scattering (DLS) and zeta potential analysis;

[0043] (3) Verify antibody conjugation efficiency by SDS-PAGE or immunoblotting to ensure the targeting specificity of colloidal quantum trap antibody composition.

[0044] The colloidal quantum well antibody composition of this invention is widely applicable to the assessment of the immune microenvironment in autoimmune diseases (such as myasthenia gravis and systemic lupus erythematosus); TB synergistic analysis in vaccine immune responses; monitoring of helper T cell activity in the tumor immune microenvironment; and high-throughput screening of immune status in clinical pathological samples. Fluorescence assays show that the colloidal quantum well antibody composition constructed in this invention exhibits excellent tissue penetration and localization resolution. Its method is highly versatile and adaptable to various antibodies and target molecules, providing important technical support for high-throughput pathological labeling, tumor tissue spatial omics, and multi-indicator synergistic diagnosis, and has broad prospects for scientific research and clinical translational applications.

[0045] The above composition can be widely used in multi-target immunofluorescence staining of lymphoid tissue sections. Combined with confocal or multiphoton microscopy, it can visualize the spatial distribution of Tfh cells and DCs and quantitatively assess their functional status (such as autophagy level and antigen presentation ability). It can also construct a functional network of Tfh-DC interactions, making it particularly suitable for studying changes in the immune microenvironment in autoimmune diseases (such as myasthenia gravis). The composition can utilize CQWs with different emission wavelengths (such as 525nm, 585nm, and 625nm) and can be combined with antibodies targeting CD4, CXCR5, CD21, CD23, Beclin, LC3, CD80, and HLA-DR to achieve simultaneous multi-channel detection and spatial localization.

[0046] The above-mentioned colloidal quantum trap antibody composition is applied to immunofluorescence staining of lymphoid tissue sections, combined with fluorescence microscopy or confocal microscopy imaging, to detect CD4+CXCR5+ expression of Tfh cells, CD21+CD23+ expression of DCs, DC autophagy level (Beclin or LC3), and antigen presentation capacity level (CD80 or HLA-DR); it is used to evaluate the immune microenvironment and immune status of thymopathy and other related diseases.

[0047] The aforementioned colloidal quantum trap antibody composition is applied to paraffin sections of lymphoid tissue for multi-label staining. This allows for the detection and spatial localization of multiple targets, including the proportion of Tfh cells, DC autophagy levels, and antigen-presenting capacity, within the lymphoid tissue sections. It also enables spatial localization and quantitative analysis of three cell subsets, and is used to evaluate the immune status of related diseases (such as thymopathy complicated by autoimmune diseases). The lymphoid tissue sections are selected from paraffin sections of thymic tissue, thymoma tissue, thymic hyperplasia tissue, lymph node tissue, or spleen tissue. The multi-label staining includes the following steps:

[0048] ① Lymphoid tissue sections were dewaxed with xylene, hydrated, subjected to Tris-EDTA (pH 9.0) antigen heat retrieval for 3 minutes, blocked with peroxidase for 10 minutes, and blocked with goat serum for 10 minutes;

[0049] ② Add the semiconductor colloidal quantum trap-antibody conjugate, incubate overnight at 4°C, wash three times with PBS, add DAPI for anti-quenching and mounting;

[0050] ③ Use a fluorescence microscope or confocal microscope to image and observe and record the results.

[0051] The specific antibody is a monoclonal antibody IgG or a polyclonal antibody IgG, preferably a monoclonal antibody IgG, to improve labeling specificity.

[0052] The following example illustrates the staining workflow using CD4 and CXCR5 antibody labeling of Tfh cells:

[0053] (1) After the tissue sections were baked at 60℃ for 20 min, they were dewaxed with xylene (15 min / time, 2 times) and hydrated (100% for 3 min, 100% for 3 min, 90% for 3 min, 75% for 3 min, 50% for 3 min), and then washed with 1×PBS for 3 min × 3 times.

[0054] (2) Then place the slides in an autoclave and use Tris-EDTA buffer (pH 9.0) for antigen retrieval for 3 minutes. After cooling to room temperature, rinse the slides with 1×PBS buffer for 3 minutes × 3 times.

[0055] (3) Add 50-100 μL of endogenous peroxidase inhibitor and incubate in a humidified chamber at room temperature for 10 minutes to block endogenous enzyme activity. Rinse again with 1×PBS for 3 minutes × 3 times, dry, add an appropriate amount of normal goat serum blocking solution, block at room temperature for 10 minutes, discard the serum, no PBS rinsing is required;

[0056] (4) Subsequently, the CD4 and CXCR5 antibodies labeled with semiconductor colloidal quantum trap were diluted with 1×PBS according to the ratio of CD4 and CXCR5 antibodies to diluent, respectively, and dropped onto the surface of the slide. The slides were then incubated overnight in a humidified chamber at 4°C.

[0057] (5) After removing the slide the next day, rinse with 1×PBS for 3 minutes × 3 times. After slightly drying, add 10-30 μL of anti-quenching mounting medium containing DAPI, cover with a coverslip, and seal the edges with nail polish to prevent the sample from drying.

[0058] (6) Finally, use a fluorescence microscope or confocal microscope to select an appropriate excitation wavelength to observe and acquire images.

[0059] Similarly, by replacing the antibody with a combination of CD21+CD23+Beclin / LC3 or CD21+CD23+CD80 / HLA-DR, the autophagy and antigen presentation capacity of dendritic cells (DCs) can be detected, and the immune status of lymphoid tissue can be assessed according to the judgment criteria.

[0060] The criteria for determining the immune status of lymphoid tissue are as follows:

[0061] Immune status is determined based on specific immune cell function indicators in lymphoid tissue, including the proportion of Tfh cells, the level of autophagy in dendritic cells (DCs), and their antigen-presenting capacity. These indicators reflect the activity of the immune response and help clarify the state of the immune environment. Specific criteria are as follows:

[0062] Immune active state: When Tfh cells account for a significant proportion of CD4+ in lymphoid tissues + An immune-active state is defined as one where the proportion of T cells is higher than 25%, the autophagy level of dendritic cells is higher than 27%, and the antigen-presenting capacity of dendritic cells is higher than 15%. This state typically indicates that the immune system has already made an initial response to the antigen and is in a relatively active immune surveillance phase.

[0063] Immune activation is defined as a state where the proportion of Tfh cells in lymphoid tissue exceeds 40%, the autophagy level of dendritic cells exceeds 75%, and the antigen-presenting capacity of dendritic cells is higher than 25%. This state reflects a strong immune response and may be associated with excessive immune responses or the development of autoimmune diseases.

[0064] Immunosuppression is defined as a state where the proportion of Tfh cells is below 25%, the level of dendritic cell autophagy is below 27%, and the antigen-presenting capacity of dendritic cells is below 15%. This typically indicates a weakened immune system, which may lead to insufficient immune surveillance and antigen clearance, thereby affecting the defense response against pathogens or being associated with the development of certain immune-related diseases.

[0065] Experiments show that the system of this invention can detect the proportion of Tfh cells, the level of DC autophagy, and antigen presentation ability, with low spectral signal overlap and high spatial resolution, significantly improving the effect compared to traditional multicolor dyes. Compared to traditional multicolor fluorescent staining techniques, this invention utilizes the high sensitivity, high photostability, and low signal crosstalk of semiconductor colloidal quantum wells to significantly improve the signal resolution and image accuracy of multi-target detection, effectively overcoming the problems of signal overlap, fluorescence quenching, and tissue background interference existing in traditional fluorescent probes. This invention has the following outstanding features:

[0066] 1. High optical stability and signal strength: Significantly reduces photobleaching effect and improves long-term imaging capability;

[0067] 2. Precise differentiation of multiple targets: The narrow emission spectrum of the semiconductor colloidal quantum well, combined with highly specific antibodies, can achieve high-resolution co-labeling of 3-5 channels or more;

[0068] 3. High coupling efficiency and biocompatibility: The water-soluble CQW ligand system ensures stable probe dispersion and reduces non-specific adsorption; it also offers broad applicability.

[0069] 4. It is compatible with conventional immunostaining procedures and mainstream confocal microscopy systems, is easy to operate, and has strong applicability. Attached Figure Description

[0070] Figure 1 This is a staining image of Tfh cells with CD4 coupled to a 525nm semiconductor colloidal quantum well (green).

[0071] Figure 2 This is a Tfh cell staining image of CXCR5 coupled to a 625nm semiconductor colloidal quantum well (red).

[0072] Figure 3 This is a DAPI map of Tfh cells stained with.

[0073] Figure 4 This is a Merge plot of Tfh cell staining.

[0074] Figure 5 This is the first set of autophagy staining images of DC cells coupled with CD21 to a 525nm semiconductor colloidal quantum well (green).

[0075] Figure 6 This is the first set of autophagy staining images of DC cells coupled with CD23 and 585nm semiconductor colloidal quantum wells (orange).

[0076] Figure 7 This is the first set of autophagy staining images of DC cells with Beclin-coupled 625nm semiconductor colloidal quantum wells (red).

[0077] Figure 8This is the first DAPI map of autophagy staining in DC cells.

[0078] Figure 9 This is the Merge plot of autophagy staining for the first group of DC cells.

[0079] Figure 10 This is a staining image of autophagy in DC cells of the second group, which are CD21 coupled to a 525nm semiconductor colloidal quantum well (green).

[0080] Figure 11 This is a staining image of autophagy in DC cells of the second group, which are CD23 coupled to a 585nm semiconductor colloidal quantum well (orange).

[0081] Figure 12 This is a staining image of autophagy in DC cells from the second group of Beclin-coupled 625nm semiconductor colloidal quantum wells (red).

[0082] Figure 13 This is a DAPI map of autophagy staining in the second group of DC cells.

[0083] Figure 14 This is a Merge plot of autophagy staining for the second group of DC cells.

[0084] Figure 15 This is a staining diagram of the DC antigen presentation capability of CD21 coupled to a 525nm semiconductor colloidal quantum well (green).

[0085] Figure 16 This is a staining image of the DC antigen presentation capability of a CD23-coupled 585nm semiconductor colloidal quantum well (orange).

[0086] Figure 17 This is a staining image of the DC antigen presentation capability of CD80 or HLA-DR coupled to a 625nm semiconductor colloidal quantum well (red).

[0087] Figure 18 This is a DAPI map showing the ability of DC antigen presentation.

[0088] Figure 19 This is a Merge plot showing the ability of DC antigen presentation. Detailed Implementation

[0089] Example 1

[0090] This embodiment prepares semiconductor colloidal quantum wells with emission wavelengths of 525nm, 585nm, and 625nm.

[0091] CdSe / ZnS-based semiconductor colloidal quantum wells with emission wavelengths of 525 nm (green), 585 nm (yellow), and 625 nm (red) were prepared using a hot-injection method. The reaction was based on the high-boiling solvent ODE (1-Octadecene, 1-octyldecene, boiling point approximately 315 °C) to ensure excellent thermal stability and reaction controllability. Cadmium acetate (Cd(OAc)₂, 0.4 mmol) was selected as the metal precursor to ensure high solubility and reactivity. Cd(OAc)₂ was dissolved in 1 mL of ODE to form a cadmium acetate solution (containing 0.5 mL of oleic acid). Simultaneously, Cd myristate (Cd myristate, 100 mg) was used to precisely control the thickness of the semiconductor colloidal quantum well layer.

[0092] The non-metallic precursors are selenium powder (Se, 40 mg, for 525 nm and 625 nm wavelengths) or sulfur powder (S, 40 mg, for 585 nm wavelength), which are dissolved in 1 mL of trioctylphosphine (TOP) to form TOP-Se or TOP-S injection solutions, or directly dissolved in 2 mL of ODE at 180-200 °C to form a low-activity solution suitable for shell growth.

[0093] The preparation process includes the following steps.

[0094] (1) Add 100 mg Cd laurate, 40 mg Se or S powder and 15 mL ODE (1-octyldecene) to a three-necked flask;

[0095] (2) Heat to 100°C under nitrogen protection and evacuate for 30 minutes to remove moisture and oxygen.

[0096] (3) Under nitrogen protection, the temperature was raised to 230℃, and a preheated and clarified 0.4 mmol cadmium acetate (Cd(OAc)2) solution (dissolved in 1 mL ODE, containing 0.5 mL oleic acid) was rapidly injected. After injection, the system changed from dark to yellow or orange-red within seconds, indicating that the CdSe or CdS nanocrystals rapidly nucleated and entered the two-dimensional growth stage. The reaction times were 10-30 seconds (525 nm wavelength), 30-60 seconds (585 nm wavelength), and (1-3 minutes) at 625 nm wavelength.

[0097] (4) The reaction was terminated by dispersing the product in 10 mL of room temperature n-hexane and cooling it in an ice bath. The reaction product was precipitated by adding ethanol at a volume ratio of 1:3. The supernatant was removed by centrifugation at 8000 rpm for 5 minutes. The dispersion in n-hexane, precipitation in ethanol, and centrifugation were repeated 2-3 times to remove unreacted precursors, free ligands, and byproducts, thus obtaining a pure oil-soluble semiconductor colloidal quantum well.

[0098] The final product (oil-soluble semiconductor colloidal quantum well) dispersed in n-hexane exhibits strong fluorescence with emission wavelengths of 525nm (green light, half-width at half-maximum of about 20-30nm), 585nm (yellow light, half-width at half-maximum of about 25-35nm), and 625nm (red light, half-width at half-maximum of about 30-40nm). It has high photoluminescence efficiency and is suitable for displays and optoelectronic devices.

[0099] (5) To realize the application of CQWs in biolabeling and aqueous phase fields, oil-soluble semiconductor colloidal quantum wells were converted into water-soluble forms by ligand exchange. Thioglycolic acid (TGA, 0.2M) or mercaptopropionic acid (MPA, 0.2M) was dissolved in 10 mL of NaOH aqueous solution at pH 9-10 to obtain a hydrophilic ligand solution, thereby enhancing the dissociation and exchange capacity of carboxyl groups.

[0100] (6) Slowly add 1 mL of the oil-soluble semiconductor colloidal quantum well solution in n-hexane (concentration approximately 2 mg / mL) to the above hydrophilic ligand solution to form an oil-water two-phase system. Vigorously shake or sonicate at room temperature for 30-60 minutes to gradually replace the surface hydrophobic ligand (oleic acid) with TGA or MPA, transferring the semiconductor colloidal quantum well to the aqueous phase. A deepening of the aqueous phase color indicates successful exchange.

[0101] (7) Ethanol was then added at a volume ratio of 1:2 to precipitate the semiconductor colloidal quantum wells. The mixture was centrifuged at 8000 rpm for 5 minutes to remove excess ligands and organic residues. The precipitate was redispersed in deionized water or PBS buffer (5 mL) at pH 7.4. The emission wavelengths of the resulting water-soluble semiconductor colloidal quantum wells (CQWs) showed a slight blue shift (approximately 5-10 nm, i.e., 520 nm, 580 nm, and 620 nm), while the quantum yield remained at 80-90% of the initial value, demonstrating good aqueous dispersibility and fluorescence stability.

[0102] (8) The particle size distribution was determined to be 5-10 nm by dynamic light scattering (DLS), and the Zeta potential was approximately -30 mV, indicating that the surface carries a negative charge and has high stability. This semiconductor colloidal quantum well (CQW) is suitable for applications such as bioimaging, fluorescent probes, and drug delivery.

[0103] By adjusting the reaction conditions, the emission wavelength and optical performance of semiconductor colloidal quantum wells (CQWs) can be further optimized.

[0104] For the 525nm green semiconductor colloidal quantum well, the Se / Cd molar ratio is controlled at 1:1, the injection temperature in step (3) is 230℃, and the reaction time is 10-30 seconds to ensure the formation of a CdSe-based semiconductor colloidal quantum well with a thickness of 3 monolayers (3ML).

[0105] For a 585nm yellow semiconductor colloidal quantum well, an S / Cd ratio of 1:1 is used, the injection temperature in step (3) is 230-250℃, and the reaction time is 30-60 seconds to form a 4ML CdS-based semiconductor colloidal quantum well.

[0106] For a 625nm red semiconductor colloidal quantum well, the implantation temperature in step (3) is 260℃, and the reaction time is extended to 1-3 minutes, or a Se / Cd ratio of 1:1.2 is used to form a 5ML CdSe-based semiconductor colloidal quantum well. The addition of Cd laurate significantly improves the uniformity of the layer thickness, and the full width at half maximum (FWHM) is controlled in the range of 20-40nm.

[0107] Furthermore, by introducing a Se / S mixed precursor (Se:S molar ratio of 0.2:0.8 to 0.8:0.2), CdSSe alloy-type CQWs can be prepared, with continuously tunable emission wavelengths between 450-650 nm, achieving full visible spectrum coverage. Experiments are conducted in a fume hood, and waste liquid is properly treated to reduce the toxicity risk of Cd-based materials. Water-soluble CQWs can be further coated with SiO2 or polymers to enhance biocompatibility. This method achieves a yield of 70-90%, high reproducibility, and is suitable for large-scale production, making it applicable to the field of biomarking.

[0108] Example 2

[0109] The semiconductor colloidal quantum well prepared in Example 1 was coupled with a specific antibody, including the following steps.

[0110] ① The semiconductor colloidal quantum well prepared in Example 1 was diluted to 1.0-2.0 μM and placed in PBS buffer at pH 7.2-7.4 to prepare a semiconductor colloidal quantum well solution.

[0111] ② Slowly add pre-dissolved and deaggregated antibodies dropwise to the semiconductor colloidal quantum well solution, controlling the molar ratio of semiconductor colloidal quantum well to antibody to be 1:(10-200). The antibodies include CD4, CXCR5, CD21, CD23, Beclin, LC3, CD80, and HLA-DR.

[0112] ③ A two-stage coupling reaction is adopted, wherein: in the first stage, EDC and NHS are added to the semiconductor colloidal quantum well solution for carboxyl preactivation, and the reaction is carried out for 30 minutes to generate an active ester intermediate; in the second stage, the antibody solution is slowly added to the activation reaction system, and the mixture is gently rotated and incubated at 20-25℃ for 2-4 hours, and 0.1%-0.5% BSA and 0.01% Tween-20 are added as protective agents and surface stabilizers to reduce non-specific adsorption and aggregation of the semiconductor colloidal quantum well.

[0113] ④ After the reaction is complete, centrifuge at 8000 rpm to remove large particle aggregates, and then perform two rounds of concentration and washing using a 100 kDa ultrafiltration tube. The concentration ratio in each round should not exceed 10 times. The washing buffer is PBS buffer containing 0.01% Tween-20. Purification is performed to achieve a purity of over 90%.

[0114] ⑤ Finally, the volume was adjusted to 50 μL with PBS to obtain a fluorescently stable and sharp signal conjugated antibody semiconductor colloidal quantum well composition.

[0115] Example 3

[0116] This embodiment provides a Tfh cell staining protocol for thymoma tissue.

[0117] 1. Dewaxing and hydration of tissue sections:

[0118] Paraffin-embedded thymoma tissue sections were heated in a 60°C slide oven for 20 minutes. They were then dewaxed twice in xylene (15 minutes each time), followed by hydration with a gradient of 100%, 95%, 90%, 75%, and 50% ethanol (3 minutes each). After dewaxing and hydration, the sections were washed three times with 1×PBS for 3 minutes each. Antigen retrieval was performed using Tris-EDTA buffer (pH 9.0). The sections were placed in an autoclave for heat retrieval for 3 minutes, cooled to room temperature for 20 minutes after retrieval, and then washed three times with PBS to remove residual retrieval solution.

[0119] 2. Staining of tissue sections:

[0120] Add endogenous peroxidase inhibitor (approximately 50 μL / section) and incubate in a humidified chamber at room temperature in the dark for 10 minutes to completely block endogenous enzyme activity and prevent background staining. Rinse three times with PBS buffer, then circulate the surrounding liquid with absorbent paper. Add 10% normal goat serum blocking solution to each section and incubate at room temperature for 10 minutes to block non-specific binding. Discard the serum and do not rinse further. Subsequently, according to the antibody concentrations determined in the preliminary experiment, fluorescently labeled antibodies with semiconductor colloidal quantum wells were prepared: CD4 (525nm semiconductor colloidal quantum well labeling, 1:100 dilution) and CXCR5 (625nm semiconductor colloidal quantum well labeling, 1:500 dilution). Each antibody was diluted with 1×PBS and mixed thoroughly. The CD4 antibody labeled with semiconductor colloidal quantum wells was added, and the slides were placed in a humidified chamber and incubated at 37°C for 1 hour to ensure sufficient antibody binding. The slides were then removed, rinsed three times with PBS buffer, and the surrounding liquid was dried with absorbent paper. The CXCR5 antibody labeled with semiconductor colloidal quantum wells was then added, and the slides were placed in a humidified chamber and incubated at 37°C for 1 hour to ensure sufficient antibody binding.

[0121] 3. Post-staining mounting and image acquisition:

[0122] Remove the slides and wash them thoroughly in PBS three times (3 minutes each time). Gently pat the edges dry, add 10-30 μL of anti-quenching mounting medium containing DAPI (blue nuclear stain), cover with a coverslip, and seal the edges with nail polish to prevent drying. Acquire images of CD4 (green), CXCR5 (red), and DAPI (blue) under a fluorescence microscope or laser confocal microscope, as shown below. Figure 1 , Figure 2 , Figure 3 As shown. Figure 4 Merge plot of Tfh cell staining is given, based on Figure 4 CD4 analysis + CXCR5 + Localization, distribution and expression level of Tfh cells.

[0123] Example 4

[0124] This embodiment provides a staining scheme for detecting the level of DC autophagy in thymoma tissue.

[0125] 1. Dewaxing and hydration of tissue sections:

[0126] Paraffin-embedded thymoma tissue sections were heated in a 60°C slide oven for 20 minutes. They were then dewaxed twice in xylene (15 minutes each time), followed by hydration with a gradient of 100%, 95%, 90%, 75%, and 50% ethanol (3 minutes each). After dewaxing and hydration, the sections were washed three times with 1×PBS for 3 minutes each. Antigen retrieval was performed using Tris-EDTA buffer (pH 9.0). The sections were placed in an autoclave for heat retrieval for 3 minutes, cooled to room temperature for 20 minutes after retrieval, and then washed three times with PBS to remove residual retrieval solution.

[0127] 2. Staining of tissue sections:

[0128] Add endogenous peroxidase inhibitor (approximately 50 μL / section) and incubate in a humidified chamber at room temperature in the dark for 10 minutes to completely block endogenous enzyme activity and prevent background staining. Rinse three times with PBS buffer, then blot dry the surrounding liquid with absorbent paper. Add 10% normal goat serum blocking solution to each section and incubate at room temperature for 10 minutes to block non-specific binding. Discard the serum and do not rinse again. Subsequently, prepare semiconductor colloidal quantum well fluorescently labeled antibodies according to the antibody concentrations determined in the preliminary experiment: CD21 (525 nm, 1:500 dilution), CD23 (585 nm, 1:200 dilution), and Beclin1 (1:100 dilution) or LC3 (1:500 dilution) (625 nm). Dilute each antibody with 1×PBS and mix thoroughly. Add the semiconductor colloidal quantum well labeled CD21 antibody, place the sections in a humidified chamber, and incubate at 37°C for 1 hour to ensure... After the antibody has fully bound, remove the slide, rinse three times with PBS buffer, and then blot dry the surrounding liquid with absorbent paper. Add CD23 antibody labeled with a semiconductor colloidal quantum well, place the slide in a humidified chamber, and incubate at 37°C for 1 hour to ensure full antibody binding. Remove the slide, rinse three times with PBS buffer, and then blot dry the surrounding liquid with absorbent paper. Add Beclin1 or LC3 antibody labeled with a semiconductor colloidal quantum well, place the slide in a humidified chamber, and incubate at 37°C for 1 hour.

[0129] 3. Post-staining mounting and image acquisition:

[0130] Remove the slides and wash them thoroughly in PBS three times (3 minutes each time). Gently pat the edges dry, add 10-30 μL of anti-quenching mounting medium containing DAPI (blue nuclear stain), cover with a coverslip, and seal the edges with nail polish to prevent drying. Acquire two sets of images of CD21 (green), CD23 (orange), Beclin1 or LC3 (red), and DAPI (blue) under a fluorescence microscope or laser confocal microscope. The first set of images is shown below. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, Figure 9 The Merge plot of autophagy staining for the first group of DC cells is presented, based on... Figure 9 Analyze the spatial localization and autophagy level of DCs. The second set of images is shown below. Figure 10 , Figure 11 , Figure 12 and Figure 13 As shown, Figure 14 Merge plots of autophagy staining in the second group of DC cells are shown, based on... Figure 14 Analyze the spatial localization and autophagy level of DCs.

[0131] Example 5

[0132] This embodiment provides a staining scheme for DC antigen presentation capability in thymoma tissue.

[0133] 1. Dewaxing and hydration of tissue sections:

[0134] Paraffin-embedded thymoma tissue sections were heated in a 60°C slide oven for 20 minutes. They were then dewaxed twice in xylene (15 minutes each time), followed by hydration with a gradient of 100%, 95%, 90%, 75%, and 50% ethanol (3 minutes each). After dewaxing and hydration, the sections were washed three times with 1×PBS for 3 minutes each. Antigen retrieval was performed using Tris-EDTA buffer (pH 9.0). The sections were placed in an autoclave for heat retrieval for 3 minutes, cooled to room temperature for 20 minutes after retrieval, and then washed three times with PBS to remove residual retrieval solution.

[0135] 2. Staining of tissue sections:

[0136] Add endogenous peroxidase inhibitor (approximately 50 μL / section) and incubate in a humidified chamber at room temperature in the dark for 10 minutes to completely block endogenous enzyme activity and prevent background staining. Rinse three times with PBS buffer, then blot dry the surrounding liquid with absorbent paper. Add 10% normal goat serum blocking solution to each section and incubate at room temperature for 10 minutes to block non-specific binding. Discard the serum and do not rinse again. Subsequently, prepare semiconductor colloidal quantum well fluorescently labeled antibodies according to the antibody concentrations determined in the preliminary experiment: CD21 (525 nm, 1:500 dilution), CD23 (585 nm, 1:200 dilution), and CD80 (1:200 dilution) or HLA-DR (1:200 dilution) (625 nm). Dilute each antibody with 1×PBS and mix thoroughly. Add the semiconductor colloidal quantum well labeled CD21 antibody, place the sections in a humidified chamber, and incubate at 37°C for 1 hour to ensure... After the antibody has fully bound, remove the slide, rinse three times with PBS buffer, and then blot dry the surrounding liquid with absorbent paper. Add CD23 antibody labeled with a semiconductor colloidal quantum well, place the slide in a humidified chamber, and incubate at 37°C for 1 hour to ensure full antibody binding. Remove the slide, rinse three times with PBS buffer, and then blot dry the surrounding liquid with absorbent paper. Add CD80 or HLA-DR antibody labeled with a semiconductor colloidal quantum well, place the slide in a humidified chamber, and incubate at 37°C for 1 hour.

[0137] 3. Post-staining mounting and image acquisition:

[0138] Remove the slides and wash them thoroughly in PBS three times (3 minutes each time). Gently pat the edges dry, add 10-30 μL of anti-quenching mounting medium containing DAPI (blue nuclear stain), cover with a coverslip, and seal the edges with nail polish to prevent drying. Acquire images of CD21 (green), CD23 (orange), CD80 or HLA-DR (red), and DAPI (blue) under a fluorescence microscope or laser confocal microscope, as shown below. Figure 15 , Figure 16 , Figure 17 , Figure 18 As shown, Figure 19 Merge plots of DC antigen presentation ability staining are given, based on Figure 19 Analyzing the colocalization of CD80 or HLA-DR with CD21 / CD23 reflects the maturation status of dendritic cells (DCs) and their antigen presentation ability.

[0139] Example 6

[0140] This embodiment assesses the immune status in thymoma tissue.

[0141] Samples were collected from 30 pathologically confirmed paraffin-embedded thymoma tissue specimens. Sections were 4 μm thick and subjected to multiplex fluorescence staining. The staining method was the same as above, using a fluorescence microscope. Five 400× fields were randomly selected from each section, and cell counting and proportion analysis were performed using ImageJ and the Cell Counter plugin. Immune status was classified according to Tfh cells (>40% activated, 25-40% active, <25% inhibited), DC autophagy level (>75% activated, 27-75% active, <27% inhibited), and antigen presentation capacity (>25% activated, 15-25% active, <15% inhibited).

[0142] In 30 thymoma samples, the immune status distribution was as follows through multiplex labeling and quantitative analysis: Immune activation status (10 cases, CD4+) + CXCR5 + Average 45.6%, Beclin / LC3 + Average 82.3%, CD80 / HLA-DR + Average 29.1%); Immune active status (12 cases, CD4 + CXCR5 + Average 31.8%, Beclin / LC3 + Average 51.7%, CD80 / HLA-DR + (Average 19.7%); Immunosuppressed state (8 cases, CD4) + CXCR5 + Average 18.3%, Beclin / LC3 +The average percentage was 16.4%, and the average percentage of CD80 / HLA-DR+ was 8.9%. Activated samples were more common in patients with myasthenia gravis, with significantly upregulated Tfh cell and DC activity; suppressed samples were often accompanied by less lymphoid infiltration, suggesting possible immune escape or tumor immunosuppression mechanisms.

Claims

1. An application of an antibody composition based on a semiconductor colloidal quantum well, characterized in that, This method is used for multi-label staining of paraffin sections of lymphoid tissue to achieve multi-target detection and spatial localization of Tfh cell proportion, DC autophagy level, and antigen presentation capacity in lymphoid tissue sections. It also enables spatial localization and quantitative analysis of three cell subsets and evaluation of lymphoid tissue immune status based on judgment criteria. The application is for non-disease diagnosis purposes. The antibody composition based on a semiconductor colloidal quantum well includes a semiconductor colloidal quantum well and a specific antibody, wherein the semiconductor colloidal quantum well is coupled to at least one specific antibody to form a semiconductor colloidal quantum well antibody conjugate, and the specific antibody selectively recognizes the following target antibodies: CD4, CXCR5, CD21, CD23, Beclin, LC3, CD80 and HLA-DR. The emission wavelength of the semiconductor colloidal quantum well is 450-630 nm; The semiconductor colloidal quantum well antibody conjugate comprises: a 525nm wavelength semiconductor colloidal quantum well with a CD4 antibody conjugate, a 625nm wavelength semiconductor colloidal quantum well with a CXCR5 antibody conjugate, a 525nm wavelength semiconductor colloidal quantum well with a CD21 antibody conjugate, a 585nm wavelength semiconductor colloidal quantum well with a CD23 antibody conjugate, a 625nm wavelength semiconductor colloidal quantum well with a Beclin or LC3 antibody conjugate, and a 625nm wavelength semiconductor colloidal quantum well with a CD80 or HLA-DR antibody conjugate; wherein: (1) The following materials were used for Tfh cell detection: 525nm wavelength semiconductor colloidal quantum well with CD4 antibody conjugate and 625nm wavelength semiconductor colloidal quantum well with CXCR5 antibody conjugate; staining was performed in the order of CD4 first and then CXCR5. (2) The following are used for detecting DC autophagy levels: 525nm wavelength semiconductor colloidal quantum well with CD21 antibody conjugate, 585nm wavelength semiconductor colloidal quantum well with CD23 antibody conjugate, and 625nm wavelength semiconductor colloidal quantum well with Beclin or LC3 antibody conjugate; staining is performed in the order of CD21, CD23 and Beclin or LC3. (3) The following are used for detecting the ability of DC antigen presentation: 525nm wavelength semiconductor colloidal quantum well with CD21 antibody conjugate, 585nm wavelength semiconductor colloidal quantum well with CD23 antibody conjugate, and 625nm wavelength semiconductor colloidal quantum well with CD80 or HLA-DR antibody conjugate; staining is performed in the order of CD21, CD23, CD80 or HLA-DR.

2. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 1, characterized in that, The semiconductor colloidal quantum well includes CdSe, CdSe / ZnS, CdSe / CdS, CdZnSe / ZnS, and CdSe / CdZnS and their derived structures.

3. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 1, characterized in that, The method for preparing the antibody composition based on semiconductor colloidal quantum well includes the following steps: (1) Synthesis of colloidal quantum wells: Two-dimensional colloidal quantum wells were prepared in the oil phase by the hot injection method to obtain oil phase colloidal quantum wells; (2) Use ligand exchange strategy to perform aqueous phase transfer and surface functionalization in order to achieve stable dispersion and biocompatibility of CQWs in the aquatic environment; (3) The semiconductor colloidal quantum well synthesized in step (1) is coupled with a specific antibody, and the target antibody is stably grafted onto the surface of the colloidal quantum well to form a semiconductor colloidal quantum well antibody conjugate. (4) The obtained semiconductor colloidal quantum trap antibody conjugate was purified to obtain a stable water-soluble fluorescent label, which is the colloidal quantum trap antibody composition.

4. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 3, characterized in that, The specific process of step (2) is as follows: ① Resuspend the oil phase colloidal quantum trap in chloroform or hexane, and slowly add an alkaline aqueous solution containing mercaptopropionic acid or mercaptoacetic acid. The pH of the alkaline aqueous solution is 10-11. ②Irrigate or sonicate at room temperature for 30-60 minutes until the fluorescence signal is transferred to the aqueous phase; ③ By purifying and removing free ligands, a water-soluble colloidal quantum well with carboxyl groups on its surface is obtained.

5. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 3, characterized in that, The process of coupling the semiconductor colloidal quantum well with the specific antibody in step (3) includes the following steps: ① Dilute the semiconductor colloidal quantum well to 1.0-2.0 μM and place it in PBS buffer at pH 7.2-7.4; ② Add pre-dissolved and deaggregated antibody dropwise to the semiconductor colloidal quantum well solution, controlling the molar ratio of semiconductor colloidal quantum well to antibody to be 1:(10-200); ③ A two-stage coupling reaction is adopted, wherein: in the first stage, EDC and NHS are added to the semiconductor colloidal quantum well solution for carboxyl preactivation to generate an active ester intermediate; in the second stage, the antibody solution is slowly added to the activation reaction system and gently rotated and incubated at 20-25℃ for 2-4 hours, and 0.1%-0.5% BSA and 0.01% Tween-20 are added as protective agents and surface stabilizers to reduce non-specific adsorption and aggregation of the semiconductor colloidal quantum well. ④ After the reaction is complete, centrifuge to remove large particle aggregates, and perform two rounds of concentration and washing through a 100kDa ultrafiltration tube. The concentration ratio in each round shall not exceed 10 times. The washing solution is PBS buffer containing 0.01% Tween-20. ⑤ Finally, the volume was adjusted to 50 μL with PBS to obtain a fluorescently stable and sharp signal conjugate antibody semiconductor colloidal quantum well composition.

6. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 1, characterized in that, The multi-marker staining includes the following steps: ① Lymphoid tissue sections were dewaxed with xylene, hydrated, subjected to Tris-EDTA antigen heat retrieval for 3 minutes, blocked with peroxidase for 10 minutes, and blocked with goat serum for 10 minutes; ② Add the semiconductor colloidal quantum trap-antibody conjugate, incubate overnight at 4°C, wash three times with PBS, add DAPI for anti-quenching and mounting; ③ Use a fluorescence microscope or confocal microscope to image and observe and record the results.

7. The application of the antibody composition based on a semiconductor colloidal quantum well according to claim 1, characterized in that, The judgment criteria are as follows: Immune active state: When Tfh cells account for a significant proportion of CD4+ in lymphoid tissues + An immune-active state is defined as having a T cell ratio greater than 25%, an autophagy level of dendritic cells greater than 27%, and an antigen-presenting capacity of dendritic cells greater than 15%. Immune activation state: When the proportion of Tfh cells in lymphoid tissue exceeds 40%, the autophagy level of dendritic cells exceeds 75%, and the antigen presentation capacity of dendritic cells is higher than 25%, it is determined to be an immune activation state. Immunosuppressive state: When the proportion of Tfh cells is less than 25%, the level of autophagy of dendritic cells is less than 27%, and the antigen presentation capacity of dendritic cells is less than 15%, it is considered an immunosuppressive state.

Images