Lung cancer immunofluorescence detection kit and immunofluorescence staining method

Abstract

The invention relates to the technical field of biological detection, in particular to a lung cancer immunofluorescence detection kit and an immunofluorescence staining method, and the lung cancer immunofluorescence detection kit comprises a functionalized glass slide, a fluorescence labeling probe group, a confining liquid, a washing buffer solution and a sample diluent. According to the method, multi-target synchronous detection is realized through sample pretreatment, antigen capture, non-specific binding site closing, fluorescence labeling and signal detection. By optimizing the reagent formula and the operation process, the detection efficiency, the sensitivity and the result reliability are remarkably improved, and the clinical requirements for efficient and accurate lung cancer diagnosis are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to a lung cancer immunofluorescence detection kit and an immunofluorescence staining method. Background Art

[0002] With the increasing demand for early diagnosis of lung cancer, immunofluorescence detection technology has gradually become a research hotspot in the field of lung cancer diagnosis due to its characteristics of high sensitivity, high specificity and visualization. However, there are still some deficiencies in the existing lung cancer detection kits and detection methods when realizing immunofluorescence staining, which affect the detection efficiency and the reliability of the results.

[0003] After retrieval, a small cell lung cancer detection kit with the Chinese patent publication number CN111234023B was published on September 1, 2020. This patent relates to a detection kit based on specific binding antibodies of NSE, which is mainly used for early screening diagnosis, efficacy detection and prognosis judgment of small cell lung cancer. However, this technical solution mainly relies on the specific binding of a single antibody and lacks the ability of multiplex labeling, making it difficult to simultaneously detect multiple lung cancer-related markers. In addition, this kit does not involve immunofluorescence staining technology, and there are certain limitations in the intuitiveness and quantitative analysis of the detection results, and its ability to meet the needs of modern clinical multi-target detection is limited.

[0004] In addition, a lung cancer-related microRNA detection kit with the Chinese patent publication number CN106636317B was published on June 2, 2020. This patent achieved high-sensitivity detection of lung cancer-related microRNA by designing multi-stage signal amplification probes and capture probes. However, this technical solution mainly focuses on the detection of microRNA and does not cover the detection range of proteins or other biomarkers. At the same time, the detection method of this kit requires multiple hybridization reactions and signal amplification steps, and the operation is relatively complex and time-consuming. In addition, this solution does not involve immunofluorescence staining technology and cannot provide intuitive fluorescence imaging results, which forms a certain limitation to its popularization in rapid diagnosis and clinical applications.

[0005] The above problems indicate that there is still room for improvement in the multi-target detection ability, operation convenience and application of immunofluorescence staining technology of the existing lung cancer detection kits. Therefore, the present invention provides a lung cancer immunofluorescence detection kit and an immunofluorescence staining method, aiming to realize the simultaneous detection of multiple lung cancer-related markers, optimize the detection process, and provide intuitive and reliable detection results through immunofluorescence staining technology, so as to meet the clinical needs for efficient and accurate lung cancer diagnosis. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide a lung cancer immunofluorescence detection kit and an immunofluorescence staining method. Through the multi-target synchronous detection design, optimized operation process and high-sensitivity fluorescence signal output mechanism, the kit solves the deficiencies of the prior art in terms of multi-target detection ability, operation convenience and result intuitiveness. At the same time, by introducing specific functionalized carrier materials and new labeling probes, the detection efficiency and result reliability are significantly improved.

[0007] The present invention solves its technical problems by adopting the following technical solutions: The present invention provides a lung cancer immunofluorescence detection kit, which includes the following components: Functionalized glass slide: A glass substrate modified with aminosilane on the surface is used as a solid-phase carrier, and specific capture antibodies for lung cancer-related markers are covalently fixed on the surface of the substrate; Fluorescent labeling probe group: Composed of monoclonal antibodies against different lung cancer markers, each antibody is conjugated with a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488nm, 561nm and 633nm respectively; Blocking solution: A phosphate buffer solution containing 1%-5% (by volume) bovine serum albumin and 0.01%-0.05% sodium azide; Washing buffer: A phosphate buffer solution containing 0.05%-0.1% (by mass) Tween-20 and 0.1mol / L sodium chloride; Sample diluent: A phosphate buffer solution containing 0.5%-2% (by mass) polyethylene glycol and 0.1%-0.5% glycine.

[0008] In addition, the present invention also provides an immunofluorescence staining method based on the above kit, which includes the following steps: S1. Sample pretreatment: Take the sample to be tested, add the sample diluent, dilute it at a volume ratio of 1:10, mix well and let it stand for 10min - 30min; S2. Antigen capture: Drop the diluted sample onto the surface of the functionalized glass slide, with a coverage area of 1cm² - 2cm², place it in a wet box, and incubate at 37°C for 1h - 2h; S3. Block non-specific binding sites: Remove the sample solution, wash the surface of the glass slide with the washing buffer 3 times, each washing time is 2min - 5min; then add the blocking solution to cover the entire surface of the glass slide and incubate at room temperature for 30min - 60min; S4. Fluorescent labeling: Remove the blocking solution, wash the surface of the glass slide with the washing buffer 3 times, each washing time is 2min - 5min; then add the fluorescent labeling probe group, with a coverage area of 1cm² - 2cm², place it in a wet box, and incubate at 37°C for 1h - 2h; S5, Signal Detection: Remove the fluorescently labeled probe set, wash the surface of the glass slide 3 times with the washing buffer, with each washing time being 2 min - 5 min; subsequently, place the glass slide under a fluorescence microscope, first scan it using an excitation light source with an excitation wavelength of 488 nm, then scan it using an excitation light source with an excitation wavelength of 561 nm, and finally scan it using an excitation light source with an excitation wavelength of 633 nm, and record the fluorescence signal intensity of each channel.

[0009] Preferably, the method for preparing the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in a sodium hydroxide solution with a volume fraction of 5% - 10% for 1 h - 2 h, then rinse it with deionized water until neutral, and dry it at 80°C - 100°C for 1 h - 2 h; (2) Immerse the dried glass substrate in a 3-aminopropyltriethoxysilane ethanol solution with a volume fraction of 2% - 5% and react at room temperature for 2 h - 4 h; then rinse it 3 times with absolute ethanol and dry it at 100°C - 120°C for 30 min - 60 min; (3) Immerse the amino-functionalized glass substrate in a glutaraldehyde phosphate buffer solution with a mass concentration of 0.1% - 0.5% and react for 1 h - 2 h; then rinse it 3 times with the phosphate buffer and dry it at room temperature for 10 min - 30 min; (4) Dissolve the lung cancer-related marker-specific capture antibody in the phosphate buffer to prepare a solution with a mass concentration of 0.1 mg / ml - 0.5 mg / ml; drop the solution onto the surface of the glass substrate, with the covered area being 1 cm² - 2 cm², place it in a wet box, and incubate it at 4°C for 12 h - 24 h; (5) Remove the antibody solution, wash the surface of the glass substrate 3 times with the phosphate buffer, with each washing time being 2 min - 5 min; then place the glass substrate in a phosphate buffer solution containing bovine serum albumin with a mass concentration of 0.1% - 0.5% and incubate it at room temperature for 1 h - 2 h; (6) Remove the blocking solution, wash the surface of the glass substrate 3 times with the phosphate buffer, with each washing time being 2 min - 5 min; then place the glass substrate in a vacuum drying oven and dry it at room temperature for 30 min - 60 min.

[0010] Preferably, the fluorescent dyes in the fluorescently labeled probe set are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:1.

[0011] In the present invention, the molecular weight of polyethylene glycol in the sample diluent is 2000 - 6000.

[0012] In the present invention, the pH value of the washing buffer is 7.2 - 7.6.

[0013] In the present invention, the scanning parameters of the fluorescence microscope are set as follows: the excitation light intensity is 50%-80%, the exposure time is 200 ms - 500 ms, and the gain value is 10 - 20.

[0014] Preferably, the lung cancer-related markers include, but are not limited to, carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and squamous cell carcinoma antigen (SCC).

[0015] The beneficial effects of the present invention compared with the prior art are as follows: (1) In the present invention, the functionalized glass slide immobilizes the capture antibody by chemical bonding, ensuring the stability and specific binding ability of the antibody, and greatly improving the accuracy of detection; (2) By introducing a fluorescently labeled probe set, multi-target detection is achieved. At the same time, the combination of different fluorescent dyes avoids the problem of signal crosstalk; (3) The present invention optimizes the formulations of the sample diluent and washing buffer, improving the sensitivity and accuracy of detection; (4) In addition, the standardized operation steps simplify the experimental process and reduce the influence of human error, thus meeting the clinical requirements for efficient and accurate lung cancer diagnosis. Description of the Drawings

[0016] Figure 1 It is a detection result diagram of using the lung cancer immunofluorescence detection kit in Example 1. Detailed Embodiments

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] The present invention relates to a lung cancer immunofluorescence detection kit, which includes the following components: Functionalized glass slide: A glass substrate modified with amino-silane on the surface is used as a solid-phase carrier, and a specific capture antibody for lung cancer-related markers is immobilized on the surface of the substrate in a covalent bond form; Fluorescently labeled probe set: Composed of multiple monoclonal antibodies against different lung cancer markers, each antibody is conjugated with a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively; Blocking solution: Phosphate buffer containing 1%-5% (by volume) bovine serum albumin and 0.01%-0.05% sodium azide; used to block non-specific binding sites on the surface of functionalized glass slides.

[0019] Washing buffer: Phosphate buffer containing 0.05%-0.1% (by mass) Tween-20 and 0.1 mol / L sodium chloride; the pH value of the washing buffer is 7.2-7.6.

[0020] Sample diluent: Phosphate buffer containing 0.5%-2% (by mass) polyethylene glycol and 0.1%-0.5% glycine, used to clean residual impurities on the surface of glass slides.

[0021] Among them, the molecular weight of polyethylene glycol in the sample diluent is 2000-6000, used to dilute the sample to be tested to optimize the antigen capture effect.

[0022] In addition, in the present invention, the preparation method of the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in a sodium hydroxide solution with a volume fraction of 5%-10% for 1 h-2 h, then rinse with deionized water until neutral, and dry at 80°C-100°C for 1 h-2 h; the glass substrate, as a solid-phase carrier, needs to be treated with alkali solution to clean the surface and increase the reaction activity; (2) Immerse the dried glass substrate in a 3-aminopropyltriethoxysilane ethanol solution with a volume fraction of 2%-5% and react at room temperature for 2 h-4 h, then rinse 3 times with absolute ethanol, and dry at 100°C-120°C for 30 min-60 min; (3) Immerse the amino-functionalized glass substrate in a glutaraldehyde phosphate buffer with a mass concentration of 0.1%-0.5% and react for 1 h-2 h, then rinse 3 times with phosphate buffer, and dry at room temperature for 10 min-30 min; (4) Dissolve the lung cancer-related biomarker-specific capture antibody in phosphate buffer to prepare a solution with a mass concentration of 0.1 mg / ml-0.5 mg / ml, drop it onto the surface of the glass substrate, with a coverage area of 1 cm²-2 cm², place it in a wet box, and incubate at 4°C for 12 h-24 h; (5) Remove the antibody solution, wash the surface of the glass substrate 3 times with phosphate buffer, with each washing time being 2 min-5 min, then place the glass substrate in a phosphate buffer containing 0.1%-0.5% (by mass) bovine serum albumin and incubate at room temperature for 1 h-2 h; (6) Remove the blocking solution, wash the surface of the glass substrate 3 times with phosphate buffer solution, with each washing time being 2 min - 5 min. Subsequently, place the glass substrate in a vacuum drying oven and dry it at room temperature for 30 min - 60 min.

[0023] Thus configured, the fluorescently labeled probe set consists of multiple monoclonal antibodies against different lung cancer markers, and each antibody is conjugated with a different fluorescent dye respectively.

[0024] In the present invention, the fluorescent dyes are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:1. The excitation wavelengths of these fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively, which can avoid the problem of signal crosstalk.

[0025] It should be understood that the preparation of the fluorescently labeled probe set requires strict control of the conjugation ratio of the fluorescent dye to the antibody to ensure the stability of the labeling efficiency and the fluorescence signal intensity. During the specific operation process, the monoclonal antibody and the fluorescent dye are mixed at a molar ratio of 1:1, reacted at room temperature for 2 h - 4 h, and then the unbound fluorescent dye is removed by dialysis, finally obtaining the labeled probe set.

[0026] In addition, the present invention also provides an immunofluorescent staining method for a kit, including the following steps: S1. Sample pretreatment: Take the sample to be tested, add the sample diluent, dilute it at a volume ratio of 1:10, mix well and let it stand for 10 min - 30 min; S2. Antigen capture: Drop the diluted sample onto the surface of the functionalized glass slide, with the coverage area being 1 cm² - 2 cm², place it in a wet box, and incubate it at 37 °C for 1 h - 2 h; S3. Block non-specific binding sites: Remove the sample solution, wash the surface of the glass slide 3 times with the washing buffer solution, with each washing time being 2 min - 5 min. Subsequently, add the blocking solution to cover the entire surface of the glass slide and incubate it at room temperature for 30 min - 60 min; S4. Fluorescent labeling: Remove the blocking solution, wash the surface of the glass slide 3 times with the washing buffer solution, with each washing time being 2 min - 5 min. Subsequently, add the fluorescently labeled probe set, with the coverage area being 1 cm² - 2 cm², place it in a wet box, and incubate it at 37 °C for 1 h - 2 h; S5. Signal detection: Remove the fluorescently labeled probe set, wash the surface of the glass slide 3 times with the washing buffer solution, with each washing time being 2 min - 5 min. Subsequently, place the glass slide under a fluorescence microscope, first scan it with an excitation light source with an excitation wavelength of 488 nm, then scan it with an excitation light source with an excitation wavelength of 561 nm, and finally scan it with an excitation light source with an excitation wavelength of 633 nm, and record the fluorescence signal intensity of each channel.

[0027] Among them, the scanning parameters of the fluorescence microscope are set as follows: the excitation light intensity is 50%-80%, the exposure time is 200 ms - 500 ms, and the gain value is 10 - 20.

[0028] In the above method, the lung cancer-related markers include one of carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen.

[0029] It should be understood that during the preparation of the functionalized glass slide, the mass concentration of the glutaraldehyde solution used to activate the surface of the glass substrate is 0.1% - 0.5%.

[0030] In addition, the mass concentration of sodium azide in the blocking solution is 0.01% - 0.05%.

[0031] It should be noted that this kit can be used to detect lung cancer-related markers, including but not limited to carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen. Through standardized operation steps, the influence of human error is reduced, meeting the clinical demand for efficient and accurate lung cancer diagnosis. For example, when detecting carcinoembryonic antigen, first dilute the sample to be tested and drop it onto the surface of the functionalized glass slide. After incubation and washing, add the fluorescence-labeled probe set specific to carcinoembryonic antigen, and the corresponding fluorescence signal intensity can be obtained through fluorescence microscope scanning. Similarly, the detection of other markers can be operated according to the same steps.

[0032] Example 1: A lung cancer immunofluorescence detection kit, including the following components: Functionalized glass slide: Using a glass substrate surface-modified with aminosilane as a solid-phase carrier, the lung cancer-related marker-specific capture antibody is covalently fixed on the substrate surface; Fluorescence-labeled probe set: Composed of multiple monoclonal antibodies (purchased on the market, common antibodies) specific to different lung cancer markers, such as carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen; each antibody is conjugated with a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively; the fluorescent dyes are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:1. Mix the monoclonal antibody and the fluorescent dye according to a molar ratio of 1:1, react at room temperature for 2 h - 4 h, and then remove the unbound fluorescent dye by dialysis to finally obtain the labeled probe set.

[0033] Blocking solution: A phosphate buffer solution containing 1% (v / v) bovine serum albumin and 0.01% sodium azide; used to block non-specific binding sites on the surface of the functionalized glass slide.

[0034] Washing buffer: a phosphate buffer containing 0.05% (mass concentration) of Tween-20 and 0.1 mol / L of sodium chloride; the pH value of the washing buffer is 7.2 - 7.6.

[0035] Sample diluent: a phosphate buffer containing 0.5% (mass concentration) of polyethylene glycol and 0.1% (mass concentration) of glycine, used to clean the impurities remaining on the surface of the glass slide; the molecular weight of polyethylene glycol in the sample diluent is 2000.

[0036] In addition, in the present invention, the method for preparing the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in a 5% (volume fraction) sodium hydroxide solution and react for 1 h, then rinse with deionized water until neutral, and dry at 80°C - 100°C for 1 h; the glass substrate, as a solid-phase carrier, needs to be treated with an alkaline solution to clean the surface and increase the reaction activity; (2) Immerse the dried glass substrate in a 2% (volume fraction) ethanol solution of 3-aminopropyltriethoxysilane and react at room temperature for 2 h, then rinse 3 times with absolute ethanol, and dry at 100°C - 120°C for 30 min; (3) Immerse the amino-functionalized glass substrate in a phosphate buffer containing 0.1% (mass concentration) of glutaraldehyde and react for 1 h, then rinse 3 times with the phosphate buffer, and dry at room temperature for 10 min; (4) Dissolve the lung cancer-related marker specific capture antibody in a phosphate buffer to prepare a solution with a mass concentration of 0.1 mg / ml, drop it onto the surface of the glass substrate, with a coverage area of 1 cm², place it in a wet box, and incubate at 4°C for 12 h; the lung cancer-related markers include carcinoembryonic antigen; (5) Remove the antibody solution, wash the surface of the glass substrate 3 times with the phosphate buffer, with each washing time being 2 min, then place the glass substrate in a phosphate buffer containing 0.1% (mass concentration) of bovine serum albumin and incubate at room temperature for 1 h; (6) Remove the blocking solution, wash the surface of the glass substrate 3 times with the phosphate buffer, with each washing time being 2 min, then place the glass substrate in a vacuum drying oven and dry at room temperature for 30 min.

[0037] In addition, the present invention also provides an immunofluorescence staining method for a kit, including the following steps: S1. Sample pretreatment: Take the test sample, add the sample diluent, dilute it at a volume ratio of 1:10, mix well and let it stand for 30 min; S2. Antigen capture: Drop the diluted sample onto the surface of the functionalized glass slide, with a coverage area of 2 cm², place it in a wet box, and incubate at 37°C for 2 h; S3. Block non-specific binding sites: Remove the sample solution, wash the surface of the glass slide with the washing buffer three times, with each washing time being 5 min. Subsequently, add the blocking solution to cover the entire surface of the glass slide and incubate at room temperature for 60 min; S4. Fluorescent labeling: Remove the blocking solution, wash the surface of the glass slide with the washing buffer three times, with each washing time being 5 min. Subsequently, add the fluorescent labeling probe set, with the covered area being 1 cm² - 2 cm², place it in a wet box, and incubate at 37 °C for 2 h; S5. Signal detection: Remove the fluorescent labeling probe set, wash the surface of the glass slide with the washing buffer three times, with each washing time being 5 min. Subsequently, place the glass slide under a fluorescence microscope, and set the scanning parameters of the fluorescence microscope as follows: the excitation light intensity is 80%, the exposure time is 450 ms, and the gain value is 20; First, scan using an excitation light source with an excitation wavelength of 488 nm, then scan using an excitation light source with an excitation wavelength of 561 nm, and finally scan using an excitation light source with an excitation wavelength of 633 nm, and record the fluorescence signal intensities of each channel.

[0038] Test results: The scanning results are as Figure 1 , with cytokeratin (b) and programmed death ligand 1 (c) being non-lung cancer cell references, and the staining result in sample (a) is correct. The kit of the present application can accurately detect lung cancer samples.

[0039] Example 2: A lung cancer immunofluorescence detection kit, comprising the following components: Functionalized glass slide: Use a glass substrate surface-modified with aminosilane as the solid-phase carrier, and covalently immobilize the lung cancer-related marker-specific capture antibody on the surface of the substrate; Fluorescent labeling probe set: Composed of multiple monoclonal antibodies (purchased on the market, common antibodies) against different lung cancer markers, such as carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen; Each antibody is conjugated with a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively; The fluorescent dyes are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:1. Mix the monoclonal antibody and the fluorescent dye according to a molar ratio of 1:1, react at room temperature for 4 h, and then remove the unbound fluorescent dye by dialysis to finally obtain the labeled probe set.

[0040] Blocking solution: A phosphate buffer solution containing 5% (v / v) bovine serum albumin and 0.05% sodium azide; Used to block non-specific binding sites on the surface of the functionalized glass slide.

[0041] Washing buffer: A phosphate buffer solution containing 0.1% (w / v) Tween-20 and 0.1 mol / L sodium chloride; The pH value of the washing buffer is 7.2 - 7.6.

[0042] Sample diluent: A phosphate buffer solution containing 2% polyethylene glycol and 0.5% glycine by mass concentration, used to clean the impurities remaining on the surface of the glass slide; the molecular weight of the polyethylene glycol in the sample diluent is 2000 - 6000.

[0043] In addition, in the present invention, the method for preparing the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in a 10% sodium hydroxide solution by volume fraction for 2 h, then rinse with deionized water until neutral, and dry at 80°C - 100°C for 2 h; the glass substrate, as a solid-phase carrier, needs to be treated with an alkaline solution to clean the surface and increase the reaction activity; (2) Immerse the dried glass substrate in a 5% 3-aminopropyltriethoxysilane ethanol solution by volume fraction, react at room temperature for 4 h, then rinse 3 times with absolute ethanol, and dry at 100°C - 120°C for 60 min; (3) Immerse the amino-functionalized glass substrate in a phosphate buffer solution containing 0.5% glutaraldehyde by mass concentration, react for 2 h, then rinse 3 times with the phosphate buffer solution, and dry at room temperature for 30 min; (4) Dissolve the specific capture antibody for lung cancer-related markers in a phosphate buffer solution to prepare a solution with a mass concentration of 0.5 mg / ml, drop it onto the surface of the glass slide, with a coverage area of 2 cm², place it in a wet box, and incubate at 4°C for 24 h; the lung cancer-related markers include cytokeratin 19 fragment; (5) Remove the antibody solution, wash the surface of the glass slide 3 times with the phosphate buffer solution, with each washing time being 5 min, then place the glass slide in a phosphate buffer solution containing 0.5% bovine serum albumin and incubate at room temperature for 2 h; (6) Remove the blocking solution, wash the surface of the glass slide 3 times with the phosphate buffer solution, with each washing time being 5 min, then place the glass slide in a vacuum drying oven and dry at room temperature for 60 min.

[0044] The present invention also provides an immunofluorescence staining method for a kit, as shown in Example 1.

[0045] Example 3: A lung cancer immunofluorescence detection kit, including the following components: Functionalized glass slide: Using a glass substrate modified with amino-functionalized silane as a solid-phase carrier, the specific capture antibody for lung cancer-related markers is covalently fixed on the surface of the substrate; Fluorescently labeled probe set: Composed of multiple monoclonal antibodies (purchased commercially, commonly used antibodies) targeting different lung cancer markers, such as carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen; each antibody is conjugated to a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively; the fluorescent dyes are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:1. Mix the monoclonal antibody and the fluorescent dye according to a molar ratio of 1:1, react at room temperature for 2 h - 4 h, and then remove the unbound fluorescent dye by dialysis to finally obtain the labeled probe set.

[0046] Blocking solution: Phosphate buffer containing 3% (v / v) bovine serum albumin and 0.03% sodium azide; used to block non-specific binding sites on the surface of the functionalized glass slide.

[0047] Washing buffer: Phosphate buffer containing 0.08% (w / v) Tween - 20 and 0.1 mol / L sodium chloride; the pH value of the washing buffer is 7.2 - 7.6.

[0048] Sample diluent: Phosphate buffer containing 0.8% (w / v) polyethylene glycol and 0.3% glycine, used to clean residual impurities on the surface of the glass slide; the molecular weight of polyethylene glycol in the sample diluent is 5000.

[0049] In addition, in the present invention, the preparation method of the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in an 8% (v / v) sodium hydroxide solution and react for 1 h, then rinse with deionized water until neutral and dry at 80 °C - 100 °C for 1 h; the glass substrate, as a solid-phase carrier, needs to be treated with an alkaline solution to clean the surface and increase the reaction activity; (2) Immerse the dried glass substrate in a 3% (v / v) 3-aminopropyltriethoxysilane ethanol solution and react at room temperature for 3 h, then rinse 3 times with absolute ethanol and dry at 100 °C - 120 °C for 30 min; (3) Immerse the aminated glass substrate in a phosphate buffer containing 0.3% (w / v) glutaraldehyde and react for 1 h, then rinse 3 times with phosphate buffer and dry at room temperature for 10 min; (4) Dissolve the lung cancer-related marker-specific capture antibody in phosphate buffer to prepare a solution with a mass concentration of 0.3 mg / ml, drop it onto the surface of the glass substrate, with a coverage area of 1 cm², place it in a wet box, and incubate at 4 °C for 24 h; the lung cancer-related markers include squamous cell carcinoma antigen. (5) Remove the antibody solution and wash the surface of the glass substrate 3 times with phosphate buffer solution, with each washing time being 3 min. Subsequently, place the glass substrate in phosphate buffer solution containing 0.3% (mass concentration) bovine serum albumin and incubate at room temperature for 1 h; (6) Remove the blocking solution and wash the surface of the glass substrate 3 times with phosphate buffer solution, with each washing time being 2 min. Subsequently, place the glass substrate in a vacuum drying oven and dry at room temperature for 60 min.

[0050] The present invention also provides an immunofluorescence staining method for a kit, as shown in Example 1.

[0051] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0052] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0053] The above describes the present invention and its embodiments. Such description is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural forms and embodiments without creative efforts without departing from the gist of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A lung cancer immunofluorescence detection kit, characterized in that: It includes the following components: Functionalized glass slide: A glass substrate modified with amino-silane on its surface is used as a solid-phase carrier, and specific capture antibodies for lung cancer-related markers are immobilized on the substrate surface in a covalent bond form. Fluorescently labeled probe set: Composed of multiple monoclonal antibodies against different lung cancer markers, each antibody is conjugated with a different fluorescent dye, and the excitation wavelengths of the fluorescent dyes are 488 nm, 561 nm, and 633 nm respectively. Blocking solution: A phosphate buffer solution containing bovine serum albumin and sodium azide. Washing buffer: A phosphate buffer solution containing 0.05% - 0.1% (mass concentration) of Tween-20 and 0.1 mol / L of sodium chloride. Sample diluent: A phosphate buffer solution containing 0.5% - 2% (mass concentration) of polyethylene glycol and 0.1% - 0.5% (mass concentration) of glycine.

2. The lung cancer immunofluorescence detection kit according to claim 1, wherein: The molecular weight of polyethylene glycol in the sample diluent is 2000 - 6000.

3. The lung cancer immunofluorescence detection kit according to claim 1, characterized in that: The pH value of the washing buffer is 7.2 - 7.

6.

4. The lung cancer immunofluorescence detection kit according to claim 1, wherein: The lung cancer-related markers include one or more of carcinoembryonic antigen, cytokeratin 19 fragment, and squamous cell carcinoma antigen.

5. The lung cancer immunofluorescence detection kit according to claim 1, wherein: The mass concentration of sodium azide in the blocking solution is 0.01% - 0.05%, and the protein volume fraction of bovine serum albumin is 1% - 5%.

6. The lung cancer immunofluorescence detection kit according to claim 1, wherein: The fluorescent dyes in the fluorescently labeled probe set are FITC, Cy3, and Cy5 respectively, and their molar ratio is 1:1:

1.

7. The lung cancer immunofluorescence detection kit according to claim 1, characterized in that: The preparation method of the functionalized glass slide includes the following steps: (1) Immerse the glass substrate in a sodium hydroxide solution with a volume fraction of 5% - 10% for 1 h - 2 h, then rinse it with deionized water until neutral, and dry it at 80°C - 100°C for 1 h - 2 h. (2) Immerse the dried glass substrate in a 3-aminopropyltriethoxysilane ethanol solution with a volume fraction of 2% - 5% and react at room temperature for 2 h - 4 h, then rinse it with absolute ethanol three times and dry it at 100°C - 120°C for 30 min - 60 min. (3) Immerse the amino-functionalized glass substrate in a glutaraldehyde phosphate buffer solution and react for 1 h - 2 h, then rinse it with a phosphate buffer solution three times and dry it at room temperature for 10 min - 30 min. (4) Dissolve the specific capture antibody for lung cancer-related markers in a phosphate buffer solution to prepare a solution with a mass concentration of 0.1 mg / ml - 0.5 mg / ml, drop it onto the surface of the glass substrate, with a coverage area of 1 cm² - 2 cm², place it in a wet box, and incubate at 4°C for 12 h - 24 h. (5) Remove the antibody solution, wash the surface of the glass substrate with a phosphate buffer solution three times, with each washing time being 2 min - 5 min, then place the glass substrate in a phosphate buffer solution containing 0.1% - 0.5% (mass concentration) of bovine serum albumin and incubate at room temperature for 1 h - 2 h. (6) Remove the blocking solution, wash the surface of the glass substrate with a phosphate buffer solution three times, with each washing time being 2 min - 5 min, then place the glass substrate in a vacuum drying oven and dry it at room temperature for 30 min - 60 min.

8. The lung cancer immunofluorescence detection kit according to claim 7, characterized in that: During the preparation of the functionalized glass slide, the mass concentration of the glutaraldehyde solution used to activate the surface of the glass substrate is 0.1% - 0.5%.

9. An immunofluorescence staining method using the kit according to claim 1, characterized in that: It includes the following steps: S1. Sample pretreatment: Take the sample to be tested, add the sample diluent, and dilute it at a volume ratio of 1:

10. After mixing evenly, let it stand for 10 min - 30 min; S2. Antigen capture: Drop the diluted sample onto the surface of the functionalized glass slide, with a coverage area of 1 cm² - 2 cm². Place it in a wet box and incubate at 37°C for 1 h - 2 h; S3. Block non-specific binding sites: Remove the sample solution, wash the surface of the glass slide with the washing buffer 3 times, with each washing time being 2 min - 5 min. Then add the blocking solution to cover the entire surface of the glass slide and incubate at room temperature for 30 min - 60 min; S4. Fluorescent labeling: Remove the blocking solution, wash the surface of the glass slide with the washing buffer 3 times, with each washing time being 2 min - 5 min. Then add the fluorescent labeling probe set, with a coverage area of 1 cm² - 2 cm². Place it in a wet box and incubate at 37°C for 1 h - 2 h; S5. Signal detection: Remove the fluorescent labeling probe set, wash the surface of the glass slide with the washing buffer 3 times, with each washing time being 2 min - 5 min. Then place the glass slide under a fluorescence microscope. First, scan it with an excitation light source with an excitation wavelength of 488 nm, then scan it with an excitation light source with an excitation wavelength of 561 nm, and finally scan it with an excitation light source with an excitation wavelength of 633 nm, and record the fluorescence signal intensity of each channel.

10. The immunofluorescence staining method according to claim 9, characterized in that: The scanning parameters of the fluorescence microscope are set as follows: the excitation light intensity is 50% - 80%, the exposure time is 200 ms - 500 ms, and the gain value is 10 - 20.

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