ELISPOT analysis method for detecting IFN-gamma and IL-2 secretion and detection kit thereof

Abstract

The invention provides an ELISPOT (Enzyme-Linked Immunospot Assay) analysis method for detecting IFN-gamma and IL-2 secretion, which comprises the following steps: activating an ELISPOT plate, washing, adding IFN-gamma and IL-2 capture antibody coating with specific concentration, and staying overnight at 4 DEG C; recovering and counting target sample cells, resuspending the target sample cells according to a certain concentration, and adding the target sample cells into coated plate holes; incubating for more than 16 hours in an environment of 37 DEG C and 5% COC; washing the incubated plate, adding IFN-HRP and IL-2-ALP detection antibodies, and incubating at room temperature for 2 hours; after washing, adding a blue developing solution for developing for 10-15 minutes; adding a red developing solution, incubating for 10-15 minutes, and stopping developing; according to the method, T cell subset identification and multifunctional analysis can be carried out, in the early stage of vaccine research and development, due to the fact that target positive population is small, the number of obtained PBMC cells is small, two-factor detection can detect different cell factor reaction frequencies and locate different cell populations and subtypes at the same time, and comparison of two-factor data is beneficial to finding out population immune differences; and scientific research and vaccine development are promoted.

Description

Technical Field

[0001] This invention relates to an ELISPOT analytical method and a detection kit for detecting IFN-γ and IL-2 secretion. Background Technology

[0002] Cellular immunity refers to the immune response of T cells (CD4+ or CD8+) by releasing lymphokines. Specifically, when T cells are stimulated by antigens (such as viruses or bacteria), they undergo proliferation and differentiation, transforming into sensitized T cells (also known as effector T cells). When the same antigen invades the body's cells again, sensitized T cells (effector T cells) directly kill the antigen, and the cytokines released by the sensitized T cells also work synergistically to kill the antigen.

[0003] For detecting cellular immune responses, the current mainstream methods are flow cytometry and ELISPOT assays. Flow cytometry has certain limitations: it has low throughput, requires a high number of cells, has moderate sensitivity, and requires blocking treatment to accumulate secretion over 4-6 hours, making it relatively expensive. In contrast, ELISPOT assays are superior to flow cytometry in all these aspects, requiring no blocking treatment and more accurately reflecting the secretion volume accumulated over 16 hours. Therefore, the ELISPOT method is internationally recognized as the standard tool for detecting and evaluating vaccine cellular immune levels, and it is easier to implement quality control and standardization. ELISPOT is a highly sensitive assay capable of detecting multiple cytokines or cytotoxic substances at the single-cell level. Among them, interferon-γ (IFN-γ) is produced in large quantities in cells and is easily detected, thus becoming the most commonly used cytokine for analyzing T lymphocyte and NK cell function. ELISpot has two significant advantages: first, in addition to performing qualitative functional testing, it can also serve as a quantitative method for accurately monitoring treatment efficacy; second, in terms of sensitivity, ELISpot is 10-200 times higher than enzyme-linked immunosorbent assay (ELISA), enabling the detection of low levels of cytokines. Furthermore, while ELISA primarily quantifies the concentration of target proteins / antibodies in the sample solution, ELISpot dynamically assesses the immune status of live cells in the sample.

[0004] Currently, most commercially available reagent kits (such as those from Mabtech, CTL, U-CyTech, BD, and Biolegend) focus on single-factor detection, with only 2-3 domestic manufacturers developing such kits (such as Dakota and Sinocare). To simultaneously detect multiple cytokines secreted by single cells in the same well, saving time and sample volume, and better ensuring batch-to-batch consistency, dual-factor detection is crucial. This method can detect not only the frequency of cells secreting only a single factor (IFN-γ or IL-2) but also the frequency of cells secreting two factors. Combining this with multi-factor secretion data allows for more accurate identification of cell subpopulations, enabling multifunctional analysis of cell populations.

[0005] Given the lack of commercially available two-factor ELISPOT kits in China, and the fact that fewer than ten manufacturers worldwide are developing such kits (Mabtech, CTL, U-CyTech, Diaclone, etc.), with most focusing on fluorescent two-factor kits (Mabtech, CTL, U-CyTech), dual-fluorescence kits are expensive and highly dependent on commercially available, mature reagents, instruments, and counting software. Only one or two dual-enzyme system products exist (CTL, Diaclone, the latter's manufacturer is suspected of discontinuing production). Commercially available kits impose numerous limitations on usage conditions and generally suffer from long and unstable lead times and high prices, making them unsuitable for demanding scientific research and other R&D work. Furthermore, dual-fluorescence kits suffer from lower sensitivity, higher cost, and strong dependency. This invention, based on existing feasible technologies, develops a diversified, flexible, and low-cost dual-enzyme, dual-factor ELISPOT detection and analysis method and approach to meet diverse detection and R&D needs.

[0006] In the fields of vaccine development and clinical evaluation, ELISPOT technology is a standard tool for detecting and assessing the responsiveness of activated specific T cells, such as evaluating vaccine efficacy by detecting IFN-γ secretion. Furthermore, T-cell ELISPOT is also a commonly used primary tool for diagnosing and assessing the efficacy of treatments in the clinical diagnosis and treatment of infectious diseases. This technology can also be applied to research on autoimmune diseases, allergies, gene and cell therapy, and basic immunological research. The National Institutes for Food and Drug Control (NIFDC) of China clearly states in its regulations, including the "Technical Guidelines for Preclinical Research of DNA Vaccines for Prevention" and the "Technical Indicators for Live Vaccine Preparations Using Viruses as Vectors for Prevention," that the ELISPOT method is an effective method for detecting and evaluating the cellular immune titer of vaccines. As a more sensitive method for detecting T / B cell-specific secretion, ELISPOT technology is particularly important for detecting a small number of antigen-specific memory T cells and has been widely used in the development and evaluation of preventive and therapeutic vaccines, such as HIV vaccines, hepatitis B vaccines, influenza vaccines, and various tumor vaccines. Summary of the Invention

[0007] The purpose of this invention is to provide an ELISPOT analysis method for detecting IFN-γ and IL-2 secretion, enabling T cell subset identification and multifunctional analysis of T cell populations. In the early stages of vaccine development, the target positive population is small, and the amount of PBMCs available in these populations is limited. Separate factor detection would significantly increase development costs and difficulty. Therefore, dual-factor detection has its advantages, as it can simultaneously detect different cytokine response frequencies, locating different cell populations and subtypes. Simultaneous comparison of dual-factor data further helps identify immune differences in populations, further promoting scientific research and vaccine development. This invention better assists in the progress of vaccines or other products with similar needs. Furthermore, this detection and analysis method is superior to other detection methods that can only detect viral nucleic acid levels and antibody levels. It can be used throughout the entire drug development stage to study the infection, recovery, and memory levels of patients or vaccinated populations, achieving a single standard to evaluate the entire T cell immune response process, completely simulating the T cell immune response process in vitro—an advantage not found in other detection methods.

[0008] The technical solution adopted by this invention to solve its technical problem is: An ELISPOT assay for detecting IFN-γ and IL-2 secretion includes the following steps: Use an ELISPOT plate and wash after activation treatment; A specific concentration of IFN-γ and IL-2 capture antibody was added for coating, and the coating reaction was carried out overnight at 4°C. Wash the coated plates, add a sealing agent to seal them, and the sealing reaction time is 30 minutes; Prepare a target-specific antigen peptide solution and add a positive stimulant. Set up unstimulated and duplicate wells as controls. The target sample cells were revived and counted, resuspended at a certain concentration, and added to the wells of the coated plate; Incubate at 37°C and 5% CO2 for more than 16 hours; After washing and incubating the plate, add the detection antibodies for IFN-HRP and IL-2-ALP, and incubate at room temperature for 2 hours; After washing, add blue color developing solution for color development and incubate for 10 to 15 minutes; After stopping the color development, add ultrapure water to terminate the process, and wash three times. Discard the solution. Add red developing solution and incubate for 10 to 15 minutes. Stop developing the color after the color has developed. After washing and drying the plate, take a picture and process the data.

[0009] Preferably, the IFN-γ and IL-2 capture antibodies, detection antibodies, and chromogenic solutions are all outsourced for production, and the antibody conjugates are conjugated using HRP and ALP, with the conjugation process completed within a few hours.

[0010] Preferably, the color development process uses blue and red color developing solutions, and the color development is terminated by stopping the reaction after each color development, with the number of color spots serving as the basis for analysis.

[0011] As a preferred method, CTL enzyme-linked spot analyzer is used for photography and data processing. Photos are taken according to the set program, and spot analysis and counting are performed using two-color spot analysis software.

[0012] Preferably, the two-color spot analysis software includes single-factor spot control adjustment parameters to ensure the reliability of the software analysis results.

[0013] As a preferred method, the number of spots for IFN-γ and IL-2 single factors and the mean number of spots for two factors are counted, and after deducting negative control spots, the spot units are calculated as an indicator of immune response.

[0014] Preferably, the target sample is peripheral blood mononuclear cells, which are added to the well plate at a set concentration after resuscitation for reaction.

[0015] Preferably, the concentrations, incubation times, and temperatures of the IFN-γ and IL-2 capture antibodies and detection antibodies have been optimized to ensure the sensitivity and specificity of the detection.

[0016] Another technical problem to be solved by the present invention is to provide an ELISPOT detection plate for IFN-γ and IL-2 secretion, comprising multiple reaction wells, each reaction well having a pre-coated bottom with IFN-γ and IL-2 capture antibodies. This plate is suitable for the ELISPOT analysis method for detecting IFN-γ and IL-2 secretion as described above.

[0017] Another technical problem to be solved by the present invention is to provide a dual-enzyme, dual-factor ELISPOT assay kit that uses the ELISPOT assay method described in any one of the above-mentioned methods for detecting IFN-γ and IL-2 secretion.

[0018] Another technical problem to be solved by the present invention is to provide an application of an adhesive for electronic products as a drug carrier for tumor treatment, in which an external light source is used to activate a photosensitizer in the local area of ​​the tumor to promote drug release and achieve a therapeutic effect.

[0019] Another technical problem to be solved by the present invention is to provide an application of an adhesive for electronic products in wound healing, tissue repair and regenerative medicine, which promotes cell proliferation and wound healing by activating photosensitizers through local light irradiation.

[0020] Another technical problem to be solved by the present invention is to provide an application of an adhesive for electronic products as a treatment method for photodynamic therapy, in which an external light source irradiates a photosensitizer to activate its phototoxicity, generate oxygen free radicals, and kill tumor cells.

[0021] The beneficial effects of this invention are: There are no commercially available Elispot two-factor assay kits in China, and only 2-3 similar two-enzyme two-factor kits were found abroad. These kits (10-plate pack) abroad typically cost 23,000-25,000 RMB, while this patented kit costs only 7,000-8,000 RMB. Compared to current market kits, it saves approximately 2 hours of testing time. Compared to single-factor assays which require separate testing, the two-factor kit reduces the required sample volume, significantly decreases processing time, and improves comparability. Compared to fluorescent two-factor kits, the two-enzyme kit enhances the detection's reliance on enzyme-linked immunospot (ELISA) amplification. ELISA is more sensitive than fluorescent spot assays, capable of detecting trace amounts of analytes secreted by individual cells, while the latter does not involve enzyme-linked signal amplification. It avoids the common problems associated with imported reagents: long lead times, instability, and susceptibility to various factors. Attached Figure Description

[0022] Figure 1 This is a roadmap for single-factor testing; Figure 2 This is a schematic diagram of the dual-enzyme dual-factor diagram displayed on the CTL company's official website; Figure 3 A schematic diagram of a dual-enzyme, dual-factor array presented by Diaclone. Figure 4 This is a schematic diagram of the dual-enzyme dual-factor method of this patent. Specific implementation methods The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0024] Example 1 An ELISPOT assay for detecting IFN-γ and IL-2 secretion includes the following steps: Use an ELISPOT plate and wash after activation treatment; A specific concentration of IFN-γ and IL-2 capture antibody was added for coating, and the coating reaction was carried out overnight at 4°C. Wash the coated plates, add a sealing agent to seal them, and the sealing reaction time is 30 minutes; Prepare a target-specific antigen peptide solution and add a positive stimulant. Set up unstimulated and duplicate wells as controls. The target sample cells were revived and counted, resuspended at a certain concentration, and added to the wells of the coated plate; Incubate at 37°C and 5% CO2 for more than 16 hours; After washing and incubating the plate, add the detection antibodies for IFN-HRP and IL-2-ALP, and incubate at room temperature for 2 hours; After washing, add blue color developing solution for color development and incubate for 10 to 15 minutes; After stopping the color development, add ultrapure water to terminate the process, and wash three times. Discard the solution. Add red developing solution and incubate for 10 to 15 minutes. Stop developing the color after the color has developed. After washing and drying the plate, take a picture and process the data.

[0025] For the detection pathway of this method (taking a single factor as an example), please refer to [link / reference]. Figure 1 As shown, see reference Figure 2 The image shown is a schematic diagram of the dual-enzyme dual-factor (IFN / IL-2) approach displayed on the CTL company's official website. (See attached image.) Figure 3 The image shown is a schematic diagram of the dual-enzyme dual-factor (IFN / IL-2) assay demonstrated by Diaclone. (See attached image.) Figure 4 The diagram shown is a schematic of the dual-enzyme dual-factor (IFN / IL-2) of this patent, where the left side represents the NC without the stimulation and the right side represents the result of CD3 monoclonal antibody stimulation.

[0026] By using specific concentrations of IFN-γ and IL-2 capture antibodies, target secreted factors can be precisely captured and quantified. This precise antibody coating ensures high specificity for the target molecule, avoiding interference from non-specific binding, thereby greatly improving the sensitivity and accuracy of the detection.

[0027] The dual colorimetric method (blue and red chromogenic solutions) in the protocol allows for the simultaneous detection of IFN-γ and IL-2 secretion in the same experiment. This simultaneous detection strategy not only saves experimental time and costs but also enables researchers to compare the interactions between different factors in a single experiment, enhancing the comparability of data and the depth of analysis.

[0028] Every step, such as cell resuspending, colorimetric reaction time, and incubation temperature, is precisely controlled and optimized to ensure the consistency and reproducibility of the experiment. Furthermore, the use of outsourced antibodies and colorimetric solutions guarantees the quality stability of the reagents, further improving the reliability and standardization of experimental results. By employing a coating technology for IFN-γ and IL-2 specific capture antibodies optimized with specific concentration gradients, target secreted factors in samples can be identified and bound efficiently and accurately. This rigorously screened antibody combination not only possesses extremely high affinity but also significantly reduces non-specific binding to other non-target molecules through steric hindrance, thereby achieving highly selective capture and quantitative detection of IFN-γ and IL-2 at the molecular level, greatly improving the sensitivity and accuracy of the entire detection system.

[0029] The experimental protocol innovatively employed a dual-color labeling system (blue and red chromogenic solutions). Through carefully designed substrate pairing, it enabled the simultaneous detection of the secretion dynamics of two cytokines, IFN-γ and IL-2, within the same experimental system. This dual-detection technique not only significantly saved experimental time and reduced reagent costs, but more importantly, it provided researchers with the unique advantage of directly comparing the expression levels of the two factors at the same time point. This greatly enhanced the spatial correlation and temporal comparability of the experimental data, providing more comprehensive experimental evidence for in-depth analysis of the cytokine network regulatory mechanisms.

[0030] Throughout the entire experimental process, key steps, including controlling cell resuspension density, determining the optimal time window for colorimetric reactions, and precisely controlling incubation temperature, underwent systematic condition optimization and multiple rounds of validation. This meticulous control of experimental parameters ensured a high degree of consistency in experimental conditions across different batches, effectively guaranteeing the reproducibility of experimental results. Simultaneously, all critical reagents were standardized antibody and colorimetric solutions produced by internationally renowned manufacturers. Their rigorous quality control system and stable batch-to-batch variations further enhanced the reliability and comparability of experimental data, resulting in better inter-laboratory reproducibility and clinical translational value.

[0031] The IFN-γ and IL-2 capture antibodies, detection antibodies, and chromogenic solutions are all outsourced for production. The antibody conjugates are conjugated using HRP and ALP, and the conjugation process is completed within several hours. The chromogenic process uses blue and red chromogenic solutions, and the chromogenic process is terminated by stopping the reaction after each chromogenic process. The number of chromogenic spots is used as the basis for analysis.

[0032] Because key reagents such as the capture antibodies, detection antibodies, and chromogenic solutions for IFN-γ and IL-2 are manufactured by specialized outsourcing companies, all reagents and chemicals used are subject to a rigorous quality control system throughout the entire production process. From raw material selection to manufacturing processes, each step undergoes multiple quality tests and verifications to ensure the stability and consistency of reagents across different batches. This stringent quality management significantly reduces batch-to-batch variability, thereby guaranteeing the reliability and reproducibility of experimental results and providing strong support for the accuracy of research data.

[0033] In our experiments, we used two highly efficient enzyme markers, HRP (horseradish peroxidase) and ALP (alkaline phosphatase), for antibody conjugation. These two enzyme markers not only possess excellent catalytic activity, enabling highly sensitive detection, but their conjugation process is also simple and time-efficient. The rapid conjugation characteristics of HRP and ALP significantly shortened the entire experimental cycle, greatly improving experimental efficiency and allowing researchers to obtain reliable experimental results in a shorter time.

[0034] To achieve visual detection of IFN-γ and IL-2 secretion levels, we specifically selected blue and red chromogenic solutions for labeling. This dual-color labeling system allows the secretion of different cytokines to be visually presented through the number of chromogenic spots. After the chromogenic reaction was completed, we promptly added a stop solution to stop the reaction; this crucial step effectively prevented over-development and significantly reduced background noise interference. Through this precisely controlled chromogenic process, researchers were able to obtain clearly distinguishable chromogenic spots, providing an accurate and reliable data foundation for subsequent quantitative analysis and greatly enhancing the scientific validity and credibility of the experimental results.

[0035] Photography and data processing were performed using a CTL enzyme-linked spot analyzer. Photos were taken according to the set program, and spot analysis and counting were performed using two-color spot analysis software. The two-color spot analysis software includes single-factor spot control adjustment parameters to ensure the reliability of the software analysis results.

[0036] Using a CTL enzyme-linked spot analyzer combined with dual-color spot analysis software, the automated photo-taking and analysis process significantly reduces manual intervention and improves data processing efficiency. This not only speeds up the experimental process but also avoids errors from manual counting, thus improving the accuracy of the results.

[0037] The two-color spot analysis software includes single-factor spot control adjustment parameters, which can precisely adjust the spot recognition criteria during the analysis process. This allows for consistent comparison of spot counts under different experimental conditions, enhancing the reliability of experimental data and ensuring more accurate quantitative analysis of the results.

[0038] By using standardized analysis software and control parameters, errors caused by human factors are reduced. Maintaining consistency in the speckle analysis process and standards across each experiment helps ensure the reproducibility of results across different experimental cycles and personnel, making the data more reliable.

[0039] By statistically analyzing the number of spots for IFN-γ and IL-2 single factors and the mean number of spots for dual factors, and after deducting negative control spots, the spot units were calculated as an indicator of the immune response. The target sample was peripheral blood mononuclear cells, which were added to the well plate at a set concentration after resuscitation for reaction. The concentrations, incubation times, and temperatures of the IFN-γ and IL-2 capture antibodies and detection antibodies were optimized to ensure the sensitivity and specificity of the detection.

[0040] By counting the number of spots in single-factor and two-factor analyses, and calculating spot units after subtracting negative control spots, the intensity of the immune response can be accurately quantified. This method eliminates background noise interference, making the final immune response indicators more accurate and reliable, thus providing clearer data for subsequent immunological analyses.

[0041] By optimizing the concentration, incubation time, and temperature of resuscitated peripheral blood mononuclear cells, it is possible to ensure that the cells participate in the reaction under optimal conditions. This helps to maximize the activation of the cells' immune response, improve the sensitivity and specificity of the experiment, and avoid errors caused by poor cell condition.

[0042] Optimization of IFN-γ and IL-2 capture antibody and detection antibody concentrations, incubation times, and temperatures ensured efficient antibody binding to target molecules. This fine-tuning not only improved detection sensitivity, enabling accurate detection even at low concentrations of target molecules, but also increased experimental specificity, reducing the risk of non-specific binding or cross-reactions, thereby enhancing the reliability and accuracy of the data.

[0043] An ELISPOT assay plate for IFN-γ and IL-2 secretion includes multiple reaction wells, each well having a bottom pre-coated with IFN-γ and IL-2 capture antibodies. The plate is suitable for the ELISPOT assay method for detecting IFN-γ and IL-2 secretion as described above.

[0044] A dual-enzyme, dual-factor ELISPOT assay kit employing any one of the above-described ELISPOT assay methods for detecting IFN-γ and IL-2 secretion.

[0045] The above embodiments of the present invention are not intended to limit the scope of protection of the present invention. The implementation of the present invention is not limited thereto. All other modifications, substitutions or alterations made to the above structure of the present invention based on the above content of the present invention, in accordance with ordinary technical knowledge and common practice in the field, without departing from the basic technical idea of ​​the present invention, shall fall within the scope of protection of the present invention.

Claims

1. An ELISPOT analytical method for detecting IFN-γ and IL-2 secretion, characterized in that, Includes the following steps: Use an ELISPOT plate and wash after activation treatment; A specific concentration of IFN-γ and IL-2 capture antibody was added for coating, and the coating reaction was carried out overnight at 4°C. Wash the coated plates, add a sealing agent to seal them, and the sealing reaction time is 30 minutes; Prepare a target-specific antigen peptide solution and add a positive stimulant. Set up unstimulated and duplicate wells as controls. The target sample cells were revived and counted, resuspended at a certain concentration, and added to the wells of the coated plate; Incubate at 37°C and 5% CO2 for more than 16 hours; After washing and incubating the plate, add the detection antibodies for IFN-HRP and IL-2-ALP, and incubate at room temperature for 2 hours; After washing, add blue color developing solution for color development and incubate for 10 to 15 minutes; After stopping the color development, add ultrapure water to terminate the process, and wash three times. Discard the solution. Add red developing solution and incubate for 10 to 15 minutes. Stop developing the color after the color has developed. After washing and drying the plate, take a picture and process the data.

2. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 1, characterized in that, The IFN-γ and IL-2 capture antibodies, detection antibodies, and chromogenic solutions are all outsourced for production, and the antibody conjugates are conjugated using HRP and ALP, with the conjugation process completed within a few hours.

3. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 1, characterized in that, The color development process uses blue and red color developing solutions, and the color development is terminated by stopping the reaction after each color development. The number of color spots is used as the basis for analysis.

4. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to any one of claims 1-3, characterized in that, The CTL enzyme-linked spot analyzer was used for photography and data processing. Photos were taken according to the set program, and spot analysis and counting were performed using two-color spot analysis software.

5. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 4, characterized in that, The two-color spot analysis software includes single-factor spot control adjustment parameters to ensure the reliability of the software analysis results.

6. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 5, characterized in that, By statistically analyzing the number of spots for IFN-γ and IL-2 single factors, as well as the mean number of spots for two factors, and after subtracting negative control spots, spot units were calculated as an indicator of immune response.

7. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 6, characterized in that, The target sample is peripheral blood mononuclear cells, which are added to the well plate at a set concentration after revival for reaction.

8. The ELISPOT analytical method for detecting IFN-γ and IL-2 secretion according to claim 7, characterized in that, The concentrations, incubation times, and temperatures of the IFN-γ and IL-2 capture antibodies and detection antibodies have been optimized to ensure the sensitivity and specificity of the detection.

9. An ELISPOT assay plate for IFN-γ and IL-2 secretion, comprising a plurality of reaction wells, each well having a bottom pre-coated with capture antibodies against IFN-γ and IL-2, the plate being suitable for the method of any one of claims 1-8.

10. A dual-enzyme, dual-factor ELISPOT assay kit employing the ELISPOT assay method for detecting IFN-γ and IL-2 secretion according to any one of claims 1-8.

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