Immune color developing reagent for detecting malignant tumor in urine and application thereof

Abstract

The application relates to the field of biological preparations, and particularly discloses an immunochromatographic reagent for detecting malignant tumors in urine and application thereof, which is used for solving the problems of poor recognition of tumor specific epitopes, heavy background interference, spectrum overlap and high subjectivity of artificial judgment in the single staining and color development of acridine orange in the detection of urinary exfoliative cells of bladder cancer. The color development reagent comprises A reagent, B reagent, C reagent and D reagent. The A reagent is provided with an antibody complex for target antigens of bladder tumor cells in urine exfoliation and acridine orange. The B reagent comprises a fluorescently labeled secondary antibody composition corresponding to the A reagent and an enzyme-labeled secondary antibody-tyramide deposition amplification composition. In the application, AI imaging analysis is adopted to calibrate layered recognition labels and acridine orange staining negative and positive labels. Through multi-target immunochromatography, two sets of color separation amplification schemes and AI imaging analysis, the application improves the recognition ability of bladder tumor specific epitopes, the image quality and the image analysis accuracy in the detection of urinary exfoliative cells.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to an immunochromatographic reagent for detecting malignant tumors in urine and its application. Background Technology

[0002] Chinese invention patent application CN119023640A discloses a non-invasive fluorescent chromogenic agent for urinary system tumors and its application method. It uses acridine orange to stain tumor cells and proposes one or more of the following anti-bleaching agents: vitamin C, tris(2-carboxyethyl)phosphine, 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid, cyclooctatetraene, or glutathione. The method combines cell morphology and chemical staining to observe changes in cell morphology and chemifluorescence, overcoming the limitations of traditional Papanicolaou staining in terms of sensitivity and specificity. Since hematuria is a major clinical symptom of urinary system tumors, and hemoglobin exhibits fluorescence inhibition in the proposed reagents and methods, hematuria interference is eliminated. Furthermore, the anti-bleaching agent overcomes the clinical limitation that acridine orange is easily photobleached, hindering long-term observation of cell morphology. Although the above scheme has many advantages, it still has the following shortcomings: (1) Although acridine orange staining can distinguish normal cells and tumor cells in urine exfoliated cells to a certain extent through different color fluorescence reactions, it lacks tumor-specific epitope recognition, especially for tumor cells in the bladder. It is difficult to distinguish reactive and inflammatory cell proliferation from tumor ploidy by relying solely on the total amount of nucleic acid and the deterioration of cell morphology. This leads to an increase in false positives in acridine orange staining positive samples and limited specific recognition, making it difficult to establish hierarchical recognition labels for tumor-related antigens in the bladder at different molecular subtypes. (2) Although the invention eliminates the interference of hematuria, the autofluorescence and absorption of hemoglobin, bilirubin, urine crystals and bacterial film will still cause uneven background and signal masking in the observed image, resulting in fluctuations in the signal-to-noise ratio of the image, making it difficult to identify the boundary of the region, and early and rare tumor cells are easily missed, increasing the re-examination rate; (3) After acridine orange staining, there is a tail spectrum overlap between the bright green fluorescence (about 530nm) and orange-red fluorescence (about 640nm) channels of DNA in the cell nucleus. The cell morphology and fluorescence color that rely on manual interpretation are highly subjective, with both missed detection and mis-picking risks, and the reading accuracy is insufficient and the efficiency is low. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an immunochromatographic reagent for detecting malignant tumors in urine and its application. Through multi-target immunochromatography, two sets of color separation amplification schemes and AI imaging analysis, the ability to identify tumor-specific epitopes in the bladder region, image quality and image analysis accuracy of urine exfoliated cells are improved.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] An immunochromatographic reagent for detecting malignant tumors in urine includes reagent A, reagent B, reagent C, and reagent D. Reagent A comprises an antibody complex targeting tumor cell antigens from the bladder site shed in urine, a fluorescein, a buffer solution, and a blocking and stabilizing composition. The antibody complex comprises 0.5–2.0 μg / mL of 19A211, 0.5–2.0 μg / mL of M344, 0.5–2.0 μg / mL of LDQ10, 0.5–2.0 μg / mL of CK20, and 0.5–2.0 μg / mL of anti-hTERT monoclonal antibody. The fluorescein is 0.001%–0.01% w / v acridine orange. The buffer solution comprises 0.10–0.18 mol / L... A mixture of NaCl and 10–50 mmol / L phosphate; a blocking and stabilizing composition comprising 0.5%–2.0% w / v serum albumin, 0.01%–0.10% v / v Tween-20, and 0.02%–0.10% w / v sodium azide; the pH of reagent A is 6.8–7.6; reagent B comprises a fluorescently labeled secondary antibody composition matched to the antibody complex and an enzyme-labeled secondary antibody-tyramine deposition amplification composition; it is used for colorimetric development and signal amplification of the bound target antigen-antibody, adapted for liquid-based thin-layer cytology slides, and used for fluorescence colorimetric interpretation; reagent C is used for washing after staining with reagent A to remove unbound antibodies and free fluorescein; and reagent D is used for mounting and anti-fluorescence quenching.

[0006] As a further embodiment of the chromogenic reagent of the present invention, in reagent B, the fluorescently labeled secondary antibody composition matched with the antibody complex includes anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse pan-IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750, each with a concentration of 0.5–5.0 µg / mL. It also includes a secondary antibody matrix buffer, which comprises 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, 0.5–2.0 mmol / L EDTA, 0.01–0.10% v / v Tween-20, 1–5 mmol / L Trolox, and 3–10 mmol / L sodium ascorbate.

[0007] As a further embodiment of the chromogenic reagent of the present invention, in reagent B, the enzyme-labeled secondary antibody-tyramine deposition amplification composition matched with the antibody complex includes HRP-anti-mouse IgG1, HRP-anti-mouse IgG2a, HRP-anti-mouse anti-rabbit IgG, and HRP-anti-chicken IgY, each at a concentration of 0.5–5.0 µg / mL, and also includes tyramine-derived fluorescent substrates TSA-405, TSA-555, TSA-610, TSA-647, and TSA-750, each at a concentration of 1–10 µmol / L, containing Tris-HCl 10–50 mmol / L and NaCl. A deposition reaction matrix of 100–150 mmol / L and H2O2 ≤ 0.001% w / v, and a deposition termination composition comprising 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, and 1–5 mmol / L Trolox, wherein the pH of the deposition termination composition is 7.2–7.6.

[0008] As a further embodiment of the colorimetric reagent of the present invention, reagent C comprises 10–50 mmol / L phosphate, 100–150 mmol / L NaCl, 0.03%–0.08% v / v Tween-20, 0.5%–1.0% w / v serum albumin, and 0.5–2.0 mmol / L EDTA, based on the NaH2PO4 / NaH2PO4 pairing. The pH of reagent C is 7.2–7.6.

[0009] As a further embodiment of the colorimetric reagent of the present invention, reagent D comprises 50%–80% v / v glycerol, 10–50 mmol / L phosphate based on NaH2PO4 / Na2HPO4 pairing, and 100–150 mmol / L NaCl, and the pH of reagent D is 7.2–7.6.

[0010] As a further embodiment of the colorimetric reagent of the present invention, reagent D further includes at least one of hydroquinone 0.1-0.5 mmol / L, N-propyl gallate 0.05-0.2 mmol / L, and Trolox 1-5 mmol / L.

[0011] As a further embodiment of the colorimetric reagent of the present invention, reagent D further includes 0.5–2.0 mmol / L of EDTA.

[0012] Application of an immunochromatographic reagent for detecting malignant tumors in urine, wherein the application includes:

[0013] Step 1, Sample collection: Collect 10-50 mL of midstream morning urine from the subject using a disposable sterile urine cup. Process the urine within 2 hours after collection. If temporary storage is required, refrigerate at 2-8℃ for ≤8 hours.

[0014] Step 2, urine sediment preparation: Aliquot the sample collected in Step 1 into 15mL centrifuge tubes. Set the speed of the benchtop low-temperature centrifuge to 300-800rpm × sample mass (sample mass unit is grams), and the centrifugation temperature to 20-25℃. Discard the supernatant and retain 100-300µL of urine sediment.

[0015] Step 3, Fixation: Add an equal volume of 95% methanol or 95% ethanol to the urine sediment, shake gently for 1-3 minutes to obtain the urine sediment fixative and place it in a low-temperature centrifuge. Set the speed parameter to 300 rpm × the mass of the urine sediment fixative. The mass of the urine sediment fixative is grams. After 2 minutes of sedimentation, discard the fixative and retain the urine sediment.

[0016] Step 4, Liquid-based thin-layer preparation: Transfer the urine sediment into the sample chamber of the liquid-based cell slide preparation machine, select the urine sediment or cytology program, and set the target spreading area diameter to 10-13 mm. After the slide is prepared, transfer it to a glass slide. The glass slide is positively charged and has a size of 25 mm × 75 mm.

[0017] Step 5, Drying: Place the glass slide obtained in Step 4 in a constant temperature drying oven and dry it at 37-45℃ for 3-10 minutes, then take it out and cool it to room temperature.

[0018] Step 6, A reagent staining: Add 15-25µL of A reagent to the sample frame using an adjustable pipette to cover the cell spreading area, and place in a humidified box at 20-25℃ for 10-20 minutes in the dark.

[0019] Step 7, Washing: Immerse and wash with reagent C 2-3 times on a shaker at 50-80 rpm, each wash lasting 30-60 seconds. Shake off the liquid at the edges and do not blow dry with air.

[0020] Step 8, Reagent B color separation: This includes direct fluorescent secondary antibody color separation and sequential deposition color separation of enzyme-labeled secondary antibody-tyramine deposition amplification composition;

[0021] Step 9, nuclear contrast staining: If no positive result is found in acridine orange in A, add 0.001% to 0.01% w / v of acridine orange for 20 to 60 seconds, and then wash once quickly with reagent C;

[0022] Step 10, sealing: Add 10-20µL of reagent D to cover an area of ​​18mm×18mm on the coverslip, gently press along the edge to remove air bubbles, and let stand at room temperature for at least 5 minutes.

[0023] Step 11, Colorimetric Imaging: Place the mounted slide on the stage of a fluorescence microscope. Use a fluorescence microscope equipped with excitation modules for 405, 488, 561, 633, and 730 nm for multi-channel acquisition. Select the appropriate objective lens, focus on the monolayer region of the cell, and then sequentially acquire flat-field and dark current correction frames. Subsequently, image in the order of short wavelength to long wavelength. The acridine orange-DNA channel uses 488 nm excitation and 525 / 30 nm emission filters, and the acridine orange-RNA channel uses 561 nm excitation and 650 / 60 nm emission filters. The five immunochromatographic channels used emission filters of 420 / 30, 570 / 20, 620 / 20, 700 / 30, and 775 / 50 nm, respectively. The camera used 12-16 bit sampling, with an exposure time of 50-300 ms, fixed gain, and automatic white balance off. After alignment of each channel in the same field of view, at least three non-overlapping fields of view were acquired. The coordinate images were recorded and saved in a lossless format. The microscope model, objective parameters, excitation / emission combination, exposure time, gain, acquisition date and time, and slide number were also recorded.

[0024] Step 12, Image Processing and Data Organization: Import the original image into the image processing workstation, load the flat field and dark current correction frames, correct each channel one by one, import the channel leakage or compensation matrix established by the monochrome control, and perform spectral unmixing to output multi-channel layers. Then, perform channel registration and stitching, perform batch processing instance segmentation on the image, generate instance labeled masks, and calculate the integral intensity, average intensity, area, perimeter, nuclear / cytoplasmic mask and geometric center coordinates of each channel. Overlay the masks back into the layers to generate an overlay map, and export the channel intensity and geometric features of each instance into a CSV table. Analyze each sample image based on the AI ​​image analysis model, write the positive or negative label for acridine orange detection and the hierarchical identification label. The hierarchical identification label corresponds to 19A211, M344, LDQ10, CK20, and anti-hTERT monoclonal antibody, respectively. Also, write the batch number, field of view number, channel parameters, and timestamp for each image, and archive it together with the original image, correction image, mask image and overlay image to the designated directory.

[0025] As a further application of the present invention, step 8, the process of direct fluorescent secondary antibody color separation includes:

[0026] Step 11: In the sample frame, dilute anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse pan-IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750 to 0.5-5.0 µg / mL with secondary antibody matrix buffer and mix to prepare a secondary antibody mixed working solution.

[0027] Step 12: Add 15-25µL of each sample to the sample frame of the glass slide, incubate in a humidified box at 20-25℃ in the dark for 10-20 minutes, and gently shake on a shaker at 50-80 rpm.

[0028] Step 13: After incubation, wash twice with reagent C, each time for 30-60 seconds;

[0029] Step 14: Add 0.001%–0.01% acridine orange to the sample frame for 20–60 seconds, then wash once with reagent C.

[0030] After completing step 15, proceed to step 9.

[0031] As a further application of the present invention, step 8, the sequential deposition and color separation process of the enzyme-labeled secondary antibody-tyramine deposition amplification composition includes:

[0032] Step 21: Select enzyme-labeled secondary antibody-tyramine that matches the host or subclass of the target primary antibody for each color in the order of 405→555→610→647→750nm, and dilute it with the reaction matrix to 0.5~5.0µg / mL. Add 15~25µL of each sample to the sample frame and incubate in a humidified chamber at 20~25℃ in the dark for 5~10min.

[0033] Step 22: Wash twice with reagent C, 30-60 seconds each time;

[0034] Step 23: Prepare a tyramine fluorescent substrate working solution containing ≤0.001% w / v H2O2 at a concentration of 1–10 µmol / L. The tyramine fluorescent substrate working solution contains one of TSA-405, TSA-555, TSA-610, TSA-647, or TSA-750. Add 15–25 µL to the sample frame and incubate at 20–25°C in the dark for 3–7 min.

[0035] Step 24: After pouring off the working solution, add the deposition termination composition dropwise and let it act for 2-5 minutes, then wash with reagent C 2-3 times, each time for 30-60 seconds;

[0036] Step 25: After completing this round, replace with the next enzyme-labeled secondary antibody - tyramine and the corresponding tyramine fluorescent substrate, and repeat steps 23 to 24 above until all colors are completed;

[0037] Step 26: After all rounds are completed, add 0.001% to 0.01% acridine orange (w / v) to the sample box for 20 to 60 seconds, and wash once with reagent C.

[0038] Step 27, after completion, proceed to step 9.

[0039] The technical effects of this invention are as follows: By introducing a five-antibody complex (19A211, M344, LDQ10, CK20, and anti-hTERT) targeting bladder urothelial tumor-related epitopes into reagent A, and adding a fluorescently labeled secondary antibody composition and an enzyme-labeled secondary antibody-tyramine deposition amplification composition matching the antibody complex into reagent B, this invention not only identifies specific epitopes related to bladder tumors but also colorimetrically develops and amplifies the bound target antigen-antibody combination. This reduces the possibility of false positives from reactive inflammatory cells causing bladder tumor cells to detach from urine during acridine orange staining. Reagent C is used to wash after staining with reagent A to remove unbound antibodies and free acridine orange, and reagent D is used to stabilize the fluorescence at low exposure. Under these conditions, high-intensity, narrow-spectrum deposition signals are obtained, improving the signal-to-noise ratio of the prepared images and reducing background unevenness and occlusion. Multicolor deposition reduces the probability of missed detection of early and low-content positive cells by obtaining clear signals. The bandwidth of acridine orange is avoided in the chromatographic emission window of reagent B, and an appropriate narrow-band filter is configured. Combined with the acquisition or deposition sequence from short-wave to long-wave, the algorithm completely removes crosstalk and performs segmentation and quantization, effectively suppressing crosstalk in color channels, reducing human subjective differences, and improving the accuracy of image reading. The combination of multi-marker multi-channel color display and acridine orange morphological reference detection utilizes AI recognition technology to obtain negative and positive labels and layered recognition labels of acridine orange in the sample, which facilitates the provision of diverse reference data for image reading analysis and improves the efficiency and accuracy of image reading. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0041] This invention proposes an immunochromatographic reagent for detecting malignant tumors in urine, comprising reagent A, reagent B, reagent C, and reagent D. Reagent A comprises an antibody complex targeting tumor cell antigens from the bladder site shed in urine, a fluorescein, a buffer solution, and a blocking and stabilizing composition. The antibody complex comprises 0.5–2.0 μg / mL of 19A211, 0.5–2.0 μg / mL of M344, 0.5–2.0 μg / mL of LDQ10, 0.5–2.0 μg / mL of CK20, and 0.5–2.0 μg / mL of anti-hTERT monoclonal antibody. The fluorescein is 0.001%–0.01% w / v acridine orange. The buffer solution comprises 0.10–0.18 mol / L... A mixture of NaCl and 10–50 mmol / L phosphate; a blocking and stabilizing composition comprising 0.5%–2.0% w / v serum albumin, 0.01%–0.10% v / v Tween-20, and 0.02%–0.10% w / v sodium azide; the pH of reagent A is 6.8–7.6; reagent B comprises a fluorescently labeled secondary antibody composition matched to the antibody complex and an enzyme-labeled secondary antibody-tyramine deposition amplification composition; it is used for colorimetric development and signal amplification of the bound target antigen-antibody, adapted for liquid-based thin-layer cytology slides, and used for fluorescence colorimetric interpretation; reagent C is used for washing after staining with reagent A to remove unbound antibodies and free fluorescein; and reagent D is used for mounting and anti-fluorescence quenching.

[0042] 19A211, M344, and LDQ10 target tumor-associated mucin / glycan epitopes on the surface of bladder urothelial carcinoma cells, sensitively reflecting abnormal mucin expression in superficial lesions and carcinoma in situ; CK20 is a marker of urothelial differentiation lineage, indicating phenotypic changes from normal umbrella cells to abnormal expansion across the entire cell layer, facilitating the differentiation of urothelial-derived lesions; hTERT reflects telomerase activation, capturing the proliferation drive and ploidy abnormalities of malignant clones, thereby establishing complementary image tags at the membrane / glycan, differentiation, and proliferation levels; acridine orange, used at low concentrations as a nucleic acid contrast staining agent, provides morphological and nuclear / cytoplasmic boundary references without overly dominating interpretation; reagent B provides two implementation pathways, one being a multi-channel fluorescent secondary antibody combination, completing five-color separation in one step, facilitating routine experiments. The first method involves rapid setup; the second is sequential deposition of enzyme-labeled secondary antibody-tyramine, which utilizes enzyme-catalyzed deposition to obtain a higher signal-to-noise ratio and a narrower emission spectrum, suitable for low-abundance epitopes and high-background samples. Both methods avoid the 530 / 640nm band region of acridine orange, facilitating subsequent spectral demixing and multi-channel imaging. Reagent C (PB buffer wash) is used after staining with reagent A to wash away unbound antibodies and free acridine orange, reduce non-specific adsorption and metal ion-induced background, and maintain stable ionic strength and pH to protect the bound complex. Reagent D (glycerol-PB mounting solution, containing anti-quenching components) is used to create a constant refractive index and water activity environment before imaging, inhibit photobleaching and fix channel intensity, ensuring consistency and traceability of multi-channel images during long-term acquisition and cross-batch comparison.

[0043] It should be noted that in reagent B, the fluorescently labeled secondary antibody composition matched with the antibody complex includes anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse pan-IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750, each with a concentration of 0.5–5.0 µg / mL. It also includes a secondary antibody matrix buffer, which includes 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, 0.5–2.0 mmol / L EDTA, 0.01–0.10% v / v Tween-20, 1–5 mmol / L Trolox, and 3–10 mmol / L sodium ascorbate.

[0044] The reason for including five types of fluorescent secondary antibodies in reagent B—anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse pan-IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750—is to achieve one-to-one matching with the primary antibodies of different hosts / subclasses in reagent A and to complete the five-color separation. The selected fluorescent secondary antibodies are spaced apart in the emission spectrum and avoid the acridine orange band of approximately 530 / 640 nm, which facilitates simultaneous interpretation and spectral demixing of multiple channels. The secondary antibody matrix buffer uses Tris-HCl to maintain pH and buffer capacity, NaCl to provide isotonicity and ionic strength to reduce non-specific electrostatic adsorption, EDTA to chelate metal ions to inhibit metal-induced background and autofluorescence, Tween-20 to reduce hydrophobic adhesion and improve spreading uniformity, and Trolox and sodium ascorbate as an antioxidant / antibleaching system to stabilize fluorescence intensity, reduce photodamage and signal attenuation during imaging and long-term observation, thereby ensuring the signal-to-noise ratio and batch-to-batch consistency of multicolor separation.

[0045] It should be noted that reagent C includes 10–50 mmol / L phosphate, 100–150 mmol / L NaCl, 0.03%–0.08% v / v Tween-20, 0.5%–1.0% w / v serum albumin, and 0.5–2.0 mmol / L EDTA, calculated as NaH2PO4 / NaH2PO4 pair. The pH of reagent C is 7.2–7.6.

[0046] Reagent C is prepared according to the PB buffer system (phosphate 10–50 mmol / L, NaCl 100–150 mmol / L, pH 7.2–7.6) to gently elute unbound antibodies and free acridine orange in an isotonic, stable pH environment after staining with reagent A. NaCl reduces electrostatic adsorption, Tween-20 (0.03%–0.08%) reduces hydrophobic adhesion and improves spreading, serum albumin BSA (0.5%–1.0%) provides protein blocking and stabilizes bound complexes, and EDTA (0.5–2.0 mmol / L) chelates metal ions to inhibit metal-induced autofluorescence and free radical reactions. This ensures uniform washing conditions, reduces background and channel crosstalk, and improves the signal-to-noise ratio and batch-to-batch consistency of subsequent color separation and imaging with reagent B without disrupting antigen-antibody and nucleic acid-AO binding.

[0047] It should be noted that reagent D includes 50%–80% v / v glycerol, 10–50 mmol / L phosphate (based on NaH2PO4 / Na2HPO4 pairing), and 100–150 mmol / L NaCl. The pH of reagent D is 7.2–7.6.

[0048] A high proportion of glycerol is used to improve viscosity and refractive index matching (closer to the glass slide / coverslip and cell components), reducing evaporation and coverslip drift, stabilizing the focal plane and reducing photobleaching / scintillation; PB buffers a constant neutral microenvironment to prevent fluorophores and acridine orange from being quenched or displaced by pH fluctuations, maintaining cell morphology and antigen-antibody complex conformation; isotonic salinity is used to maintain osmotic pressure, reducing cell shrinkage and membrane structure changes, and reducing nonspecific background; this makes the multi-channel fluorescence signal more stable and has better brightness reproducibility in long-term imaging and cross-slide comparison, with less image distortion and color difference, facilitating subsequent spectral demixing and quantitative analysis.

[0049] It should be noted that reagent D also includes at least one of hydroquinone 0.1–0.5 mmol / L, N-propyl gallate 0.05–0.2 mmol / L, and Trolox 1–5 mmol / L.

[0050] Adding at least one of hydroquinone (0.1–0.5 mmol / L), N-propyl gallate (0.05–0.2 mmol / L), or Trolox (1–5 mmol / L) to reagent D is intended to scavenge reactive oxygen species and free radicals, suppress triplet fall-off and chain oxidation, reduce photobleaching and flickering of fluorophores and acridine orange, stabilize the brightness and ratio of each channel, extend the observation time, and improve the signal-to-noise ratio and batch consistency of multi-channel imaging. Moreover, the above concentration range has little effect on spectral line shift and background autofluorescence.

[0051] It should be noted that reagent D also includes 0.5–2.0 mmol / L of EDTA.

[0052] The purpose of adding EDTA at 0.5–2.0 mmol / L is to chelate residual divalent / trivalent metal ions (such as Ca²⁺, Mg²⁺, Fe²⁺ / Fe³⁺, Cu²⁺) to inhibit metal-catalyzed reactive oxygen species / Fenton reactions, reduce photooxidative bleaching of fluorophores and acridine orange; avoid the formation of precipitates or microcrystals with phosphates, maintain the optical transparency and uniform refractive index of the mounting layer; inhibit residual activity of metal-dependent nucleases / proteases, stabilize nucleic acid and antigen-antibody complexes; and reduce metal-induced autofluorescence and quenching, thereby improving the stability and reproducibility of multichannel signals during imaging. Example

[0053] Unlike Example 1, the composition of reagent B in this example is different, while the rest of the composition is the same as in Example 1.

[0054] In reagent B of this embodiment, the enzyme-labeled secondary antibody-tyramine deposition amplification composition matched with the antibody complex includes HRP-anti-mouse IgG1, HRP-anti-mouse IgG2a, HRP-anti-mouse pan-IgG, HRP-anti-rabbit IgG, and HRP-anti-chicken IgY, each at a concentration of 0.5–5.0 µg / mL; tyramine-derived fluorescent substrates TSA-405, TSA-555, TSA-610, TSA-647, and TSA-750, each at a concentration of 1–10 µmol / L; a deposition reaction matrix containing 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, and H2O2 ≤0.001% w / v; and a deposition termination composition comprising 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, and 1–5 mmol / L Trolox, with a pH of 7.2–7.6.

[0055] HRP-anti-mouse IgG1, HRP-anti-mouse IgG2a, HRP-anti-mouse pan-IgG, HRP-anti-rabbit IgG, and HRP-anti-chicken IgY were selected to precisely match the primary antibodies for different hosts / subclasses in reagent A, ensuring that each target is activated only by its corresponding secondary antibody, achieving specific coupling and cross-channel isolation. TSA-405 / 555 / 610 / 647 / 750 were used as tyramine fluorescent substrates. HRP activates tyramine at low concentrations of H2O2 and covalently deposits it near the antigen site, resulting in high signal-to-noise ratio, narrow spectrum, and bleach-resistant multicolor signals. The emission peaks are staggered from the acridine orange channel, facilitating spectral unmixing and parallel imaging. A deposition termination composition (Tris / NaCl / Trolox, pH 7.2–7.6) was used to rapidly inhibit residual HRP activity, terminate free radical reactions, and stabilize the deposited fluorescence, avoiding cross-color development and time drift in subsequent rounds, ensuring controllable sequential deposition and color separation, and batch-to-batch consistency. Example

[0056] The difference between Embodiment 3 and Embodiments 1 and 2 is that this embodiment introduces the application of an immunochromatographic reagent for detecting malignant tumors in urine.

[0057] This invention proposes an application of an immunochromogenic reagent for detecting malignant tumors in urine, specifically the immunochromogenic reagent for detecting malignant tumors in urine described in Example 1. The application includes:

[0058] Step 1, Sample collection: Collect 10-50 mL of midstream morning urine from the subject using a disposable sterile urine cup. Process the urine within 2 hours after collection. If temporary storage is required, refrigerate at 2-8℃ for ≤8 hours.

[0059] Step 2, urine sediment preparation: Aliquot the sample collected in Step 1 into 15mL centrifuge tubes. Set the speed of the benchtop low-temperature centrifuge to 300-800rpm × sample mass (sample mass unit is grams), and the centrifugation temperature to 20-25℃. Discard the supernatant and retain 100-300µL of urine sediment.

[0060] Step 3, Fixation: Add an equal volume of 95% methanol or 95% ethanol to the urine sediment, shake gently for 1-3 minutes to obtain the urine sediment fixative and place it in a low-temperature centrifuge. Set the speed parameter to 300 rpm × the mass of the urine sediment fixative. The mass of the urine sediment fixative is grams. After 2 minutes of sedimentation, discard the fixative and retain the urine sediment.

[0061] Step 4, Liquid-based thin-layer preparation: Transfer the urine sediment into the sample chamber of the liquid-based cell slide preparation machine, select the urine sediment or cytology program, and set the target spreading area diameter to 10-13 mm. After the slide is prepared, transfer it to a glass slide. The glass slide is positively charged and has a size of 25 mm × 75 mm.

[0062] Step 5, Drying: Place the glass slide obtained in Step 4 in a constant temperature drying oven and dry it at 37-45℃ for 3-10 minutes, then take it out and cool it to room temperature.

[0063] Step 6, A reagent staining: Add 15-25µL of A reagent to the sample frame using an adjustable pipette to cover the cell spreading area, and place in a humidified box at 20-25℃ for 10-20 minutes in the dark.

[0064] Step 7, Washing: Immerse and wash with reagent C 2-3 times on a shaker at 50-80 rpm, each wash lasting 30-60 seconds. Shake off the liquid at the edges and do not blow dry with air.

[0065] Step 8, Reagent B color separation: Direct fluorescent secondary antibody color separation is used;

[0066] Step 9, nuclear contrast staining: If no positive result is found in acridine orange in A, add 0.001% to 0.01% w / v of acridine orange for 20 to 60 seconds, and then wash once quickly with reagent C;

[0067] Step 10, sealing: Add 10-20µL of reagent D to cover an area of ​​18mm×18mm on the coverslip, gently press along the edge to remove air bubbles, and let stand at room temperature for at least 5 minutes.

[0068] Step 11, Colorimetric Imaging: Place the mounted slide on the stage of a fluorescence microscope. Use a fluorescence microscope equipped with excitation modules for 405, 488, 561, 633, and 730 nm for multi-channel acquisition. Select the appropriate objective lens, focus on the monolayer region of the cell, and then sequentially acquire flat-field and dark current correction frames. Subsequently, image in the order of short wavelength to long wavelength. The acridine orange-DNA channel uses 488 nm excitation and 525 / 30 nm emission filters, and the acridine orange-RNA channel uses 561 nm excitation and 650 / 60 nm emission filters. The five immunochromatographic channels used emission filters of 420 / 30, 570 / 20, 620 / 20, 700 / 30, and 775 / 50 nm, respectively. The camera used 12-16 bit sampling, with an exposure time of 50-300 ms, fixed gain, and automatic white balance off. After alignment of each channel in the same field of view, at least three non-overlapping fields of view were acquired. The coordinate images were recorded and saved in a lossless format. The microscope model, objective parameters, excitation / emission combination, exposure time, gain, acquisition date and time, and slide number were also recorded.

[0069] Step 12, Image Processing and Data Organization: Import the original image into the image processing workstation, load the flat field and dark current correction frames, correct each channel one by one, import the channel leakage or compensation matrix established by the monochrome control, and perform spectral unmixing to output multi-channel layers. Then, perform channel registration and stitching, perform batch processing instance segmentation on the image, generate instance labeled masks, and calculate the integral intensity, average intensity, area, perimeter, nuclear / cytoplasmic mask and geometric center coordinates of each channel. Overlay the masks back into the layers to generate an overlay map, and export the channel intensity and geometric features of each instance into a CSV table. Analyze each sample image based on the AI ​​image analysis model, write the positive or negative label for acridine orange detection and the hierarchical identification label. The hierarchical identification label corresponds to 19A211, M344, LDQ10, CK20, and anti-hTERT monoclonal antibody, respectively. Also, write the batch number, field of view number, channel parameters, and timestamp for each image, and archive it together with the original image, correction image, mask image and overlay image to the designated directory.

[0070] This application method employs a standardized acquisition and liquid-based thin-layer slide preparation – alcohol fixation and isothermal drying – multi-target specific staining with reagent A and nucleic acid contrast with low-concentration acridine orange – isotonic buffer elution to remove unbound components – direct fluorescent secondary antibody with a single five-color split (emission peak planning to avoid 530 / 640 nm) – glycerol and PB anti-quenching sealing to stabilize optical conditions – multi-channel microscopy with acquisition and parameter recording from short to long wavelengths – workstation execution of flat field / dark current correction, channel compensation and spectral demixing, instance segmentation and feature export – generation of layered labels corresponding to 19A211 / M344 / LDQ10 / CK20 / hTERT and full archiving in a closed-loop process, achieving high signal-to-noise, low crosstalk, quantifiable and traceable multi-marker detection and rapid slide reading.

[0071] It should be noted that step 8, the direct fluorescent secondary antibody color separation process includes:

[0072] Step 11: In the sample frame, dilute anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse pan-IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750 to 0.5-5.0 µg / mL with secondary antibody matrix buffer and mix to prepare a secondary antibody mixed working solution.

[0073] Step 12: Add 15-25µL of each sample to the sample frame of the glass slide, incubate in a humidified box at 20-25℃ in the dark for 10-20 minutes, and gently shake on a shaker at 50-80 rpm.

[0074] Step 13: After incubation, wash twice with reagent C, each time for 30-60 seconds;

[0075] Step 14: Add 0.001%–0.01% acridine orange to the sample frame for 20–60 seconds, then wash once with reagent C.

[0076] After completing step 15, proceed to step 9.

[0077] This direct fluorescence secondary antibody colorimetric separation workflow is designed for routine laboratory high-throughput scenarios and for samples with medium to high abundance targets, low background, or background controlled by standard washing (such as most bladder urothelial carcinoma follow-up / screening samples). By preparing and incubating five-color secondary antibodies (AF405 / 555 / 610 / 680 / 750) in the sample frame at once, followed by standardized double washing with reagent C and optional short-term acridine orange nucleus contrast, it achieves five-channel colorimetric separation in one step without enzymatic deposition amplification and spectral avoidance of the acridine orange 530 / 640nm band region. This results in a short workflow, high speed, stable signal-to-noise ratio, low inter-channel crosstalk, and good batch-to-batch consistency for multi-marker imaging, making it suitable for batch processing and rapid slide reading. Example

[0078] Unlike Examples 1, 2, and 3, this example is based on the colorimetric reagent proposed in Example 2. In step 8 of its application, an enzyme-labeled secondary antibody-tyramine deposition amplification composition is used for sequential deposition and color separation. The remaining steps are the same as in Example 3.

[0079] It should be noted that step 8, the sequential deposition and color separation process of the enzyme-labeled secondary antibody-tyramine deposition amplification composition, includes:

[0080] Step 21: Select enzyme-labeled secondary antibody-tyramine that matches the host or subclass of the target primary antibody for each color in the order of 405→555→610→647→750nm, and dilute it with the reaction matrix to 0.5~5.0µg / mL. Add 15~25µL of each sample to the sample frame and incubate in a humidified chamber at 20~25℃ in the dark for 5~10min.

[0081] Step 22: Wash twice with reagent C, 30-60 seconds each time;

[0082] Step 23: Prepare a tyramine fluorescent substrate working solution containing ≤0.001% w / v H2O2 at a concentration of 1–10 µmol / L. The tyramine fluorescent substrate working solution contains one of TSA-405, TSA-555, TSA-610, TSA-647, or TSA-750. Add 15–25 µL to the sample frame and incubate at 20–25°C in the dark for 3–7 min.

[0083] Step 24: After pouring off the working solution, add the deposition termination composition dropwise and let it act for 2-5 minutes, then wash with reagent C 2-3 times, each time for 30-60 seconds;

[0084] Step 25: After completing this round, replace with the next enzyme-labeled secondary antibody - tyramine and the corresponding tyramine fluorescent substrate, and repeat steps 23 to 24 above until all colors are completed;

[0085] Step 26: After all rounds are completed, add 0.001% to 0.01% acridine orange (w / v) to the sample box for 20 to 60 seconds, and wash once with reagent C.

[0086] Step 27, after completion, proceed to step 9.

[0087] A monochromatic deposition method using an enzyme-labeled secondary antibody-tyramine deposition amplification composition was employed, with deposition occurring sequentially at 405→555→610→647→750 nm. This ensured that only the HRP-secondary antibody matching the host / subclass of the primary antibody in each round received enzyme activity. Under conditions of H2O2 ≤ 0.001%, TSA was activated and fluorescence was covalently deposited in the antigen neighborhood. Subsequently, residual HRP was rapidly inactivated with a termination composition, followed by thorough washing with C reagent before switching to the next color. This method yielded multi-channel signals with amplification on the order of 10¹–10², high signal-to-noise ratio, narrow spectral density, and temporal and spectral isolation at low excitation doses. This significantly reduced interference and channel crosstalk with the acridine orange ≈530 / ≈640 nm band, improving the detectability and quantitative stability of low-abundance targets and sparse tumor cells. Furthermore, fixed rounds, durations, and buffer conditions enhanced batch-to-batch consistency and resistance to bleaching and traceability during long-term imaging.

[0088] Comparative Example

[0089] This comparative example adopts the technical solution of Example 1 of the non-invasive urinary system urinary tumor fluorescence colorimetric reagent and its usage method disclosed in application publication number CN119023640A.

[0090] The preparation process of its color developer includes:

[0091] To prepare a pH 6.0 phosphate-citrate buffer solution: Dissolve 8g sodium chloride, 8mL glycerol, 0.3g sodium ethylenediaminetetraacetate, and 0.25g Trolox in 992mL of pure water. Use this solution to prepare a 0.18mol / L disodium hydrogen phosphate solution and a 0.08mol / L citric acid solution. After mixing the two solutions, adjust the pH to 6.0 with sodium hydroxide.

[0092] To prepare acridine orange stock solution: Dissolve 1g of acridine orange in 1L of pure water to obtain a 1mg / mL acridine orange stock solution.

[0093] Preparation of pH 6.0 fluorescent staining solution: Dilute 40 mL of 1 mg / mL acridine orange stock solution in 920 mL of pH 6.0 phosphate-citrate buffer to obtain the fluorescent staining solution; then add 40 mL of 1 mg / mL glycerol-sodium chloride stabilizer solution; add 20 mL of 2 mmol / L tris(2-carboxyethyl)phosphine; then add 0.2 g of ethylenediaminetetraacetic acid; this is the non-invasive urinary system urine tumor fluorescent colorimetric reagent.

[0094] The method of use includes: centrifuging 50-200 mL of urine from the subject, smearing 10-15 µL of the bottom cell residue onto a glass slide, adding 15-18 µL of non-invasive urinary system urine tumor fluorescent staining reagent to the slide for staining, covering it with a glass slide, and observing it under a fluorescence microscope. The results are judged based on the following: under the microscope, the stained cytoplasm appears orange-red or flame-colored fluorescence; the nucleus appears yellow-green or yellow fluorescence, and large and irregular cells are judged as bladder cancer tumor cells, otherwise it is normal.

[0095] The chromogenic reagents prepared in Examples 1, 2, and 3 above were used as test groups 1, 2, and 3, respectively, to collect and apply urine samples from 180 individuals from the same population. The chromogenic reagents prepared in Examples 1 and 2 were used according to the procedures and image analysis methods of Examples 3 and 4, respectively. The comparative example adopted the method disclosed in the comparative example. The data records are shown in Table 1.

[0096] Table 1 Test Data Statistics Table

[0097] project Test Group 1 Test Group 2 Test Group 3 Number of enrolled samples (in cases) 180 180 180 True positive / True negative 90 / 90 90 / 90 90 / 90 True positive TP (example) 79 85 65 False negative FN (example) 11 5 25 True negative TN (example) 83 84 72 False positive FP (example) 7 6 18 Sensitivity (%) = TP / (TP + FN) 87.8 94.4 72.2 Specificity (%) = TN / (TN + FP) 92.2 93.3 80.0 Accuracy (%) 90.0 93.9 76.7 Positive predictive value (PPV) (%) 91.9 93.4 78.3 Negative predictive value (NPV) (%) 88.3 94.4 74.2 <![CDATA[Limit of Detection (LOD, tumor cells / 10 4 urinary exfoliated cells)]]> 5–8 1–3 10–15 Image signal-to-noise ratio (SNR) (median) 4.5 8.2 2.1 Channel crosstalk (overlap between immune channels / with AO, %) 6 3 15* <![CDATA[Half-life of photobleaching t1 / 2 (min, after mounting the coverslip)]]> 18 25 8 Interference with hematuria / bilirubin / urine crystals (subjective grading) Mild Mild – Very Mild medium False positive rate (%) for inflammatory / reactive samples 9 8 22 Inter-batch repeatability (total intensity CV, %) 9 6 18 Total process time per chip (min) 90 130 50

[0098] Table 1 shows that test group 2 has the best overall performance, with a sensitivity of 94.4%, specificity of 93.3%, SNR of 8.2, and a minimum detection threshold of 1–3 / 10. 4 The crosstalk was 3%, inter-batch CV was 6%, and the anti-bleaching half-life was 25 min, but the single-chip processing time was relatively long (about 130 min), making it suitable for the validation of low abundance and high background samples; Test group 3 had the second best performance, with a sensitivity of 87.8%, specificity of 92.2%, SNR of 4.5, and LOD of 5–8 / 10. 4 The crosstalk was 6%, CV was 9%, and the antibleaching half-life was 18 min. The procedure time was moderate (about 90 min), suitable for routine batch screening. Test group 1 was the fastest (about 50 min) but had the weakest detection ability and stability, with a sensitivity of 72.2%, specificity of 80.0%, SNR of 2.1, crosstalk of 15%, CV of 18%, and antibleaching half-life of 8 min. It also had a high false positive rate (22%) for inflammatory / reactive samples.

[0099] In summary, this invention introduces a five-antibody complex (19A211, M344, LDQ10, CK20, and anti-hTERT) targeting bladder urothelial tumor-related epitopes into reagent A, and adds a fluorescently labeled secondary antibody composition and an enzyme-labeled secondary antibody-tyramine deposition amplification composition matching the antibody complex into reagent B. This allows for the identification of specific epitopes related to bladder tumors while simultaneously colorimetrically developing and amplifying the bound target antigen-antibody combination. This reduces the possibility of false positives from reactive inflammatory cells causing bladder tumor cells to detach from urine during acridine orange staining. Reagent C is used to wash after staining with reagent A to remove unbound antibodies and free acridine orange, and reagent D is used to stabilize the fluorescence under low-exposure conditions. This method obtains high-intensity, narrow-spectrum deposition signals, improves the signal-to-noise ratio of the prepared images, and reduces background unevenness and occlusion. Multicolor deposition, with its clear signals, reduces the probability of missed detection of early and low-content positive cells. By avoiding the bandwidth of acridine orange in the chromatographic emission window of reagent B and configuring appropriate narrow-band filters, combined with the acquisition or deposition sequence from short-wavelength to long-wavelength, the algorithm completely removes crosstalk and performs segmentation and quantization, effectively suppressing crosstalk in color channels, reducing subjective human differences, and improving the accuracy of image reading. The combined detection of multi-marker multi-channel color display with acridine orange morphological reference, and the use of AI recognition technology to obtain negative and positive labels and layered recognition labels for acridine orange in the sample, facilitates the provision of diverse reference data for image reading analysis, and improves the efficiency and accuracy of image reading.

[0100] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0101] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immunochromatographic reagent for detecting malignant tumors in urine, characterized in that, The reagent comprises reagents A, B, C, and D. Reagent A includes an antibody complex targeting tumor cell antigens in bladder tissue shed in urine, a fluorescein, a buffer solution, and a blocking and stabilizing composition. The antibody complex comprises 0.5–2.0 μg / mL of 19A211, 0.5–2.0 μg / mL of M344, 0.5–2.0 μg / mL of LDQ10, 0.5–2.0 μg / mL of CK20, and 0.5–2.0 μg / mL of anti-hTERT monoclonal antibody. The fluorescein is 0.001%–0.01% w / v acridine orange. The buffer solution contains 0.10–0.18 mol / L... A mixture of NaCl and 10–50 mmol / L phosphate; a blocking and stabilizing composition comprising 0.5%–2.0% w / v serum albumin, 0.01%–0.10% v / v Tween-20, and 0.02%–0.10% w / v sodium azide; the pH of reagent A is 6.8–7.6; reagent B comprises a fluorescently labeled secondary antibody composition matched with the antibody complex and an enzyme-labeled secondary antibody-tyramine deposition amplification composition; it is used to colorimetrically develop and amplify the bound target antigen-antibody, adapt for liquid-based thin-layer cytology slide preparation, and for fluorescence colorimetric interpretation; reagent C is used for washing after staining with reagent A to remove unbound antibodies and free fluorescein; and reagent D is used for mounting and anti-fluorescence quenching. In reagent B, the fluorescently labeled secondary antibody composition matched with the antibody complex includes anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750, each at a concentration of 0.5–5.0 µg / mL. It also includes a secondary antibody matrix buffer, which includes 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, 0.5–2.0 mmol / L EDTA, 0.01–0.10% v / v Tween-20, 1–5 mmol / L Trolox, and 3–10 mmol / L sodium ascorbate. In reagent B, the enzyme-labeled secondary antibody-tyramine deposition amplification composition matched with the antibody complex includes HRP-anti-mouse IgG1, HRP-anti-mouse IgG2a, HRP-anti-mouse IgG, HRP-anti-rabbit IgG, and HRP-anti-chicken IgY, each at a concentration of 0.5–5.0 µg / mL; tyramine-derived fluorescent substrates TSA-405, TSA-555, TSA-610, TSA-647, and TSA-750, each at a concentration of 1–10 µmol / L; a deposition reaction matrix containing 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, and H2O2 ≤0.001% w / v; and a deposition termination composition comprising 10–50 mmol / L Tris-HCl, 100–150 mmol / L NaCl, and 1–5 mmol / L Trolox, with a pH of 7.2–7.

6.

2. The immunochromatographic reagent for detecting malignant tumors in urine according to claim 1, characterized in that, Reagent D consists of 50%–80% v / v glycerol, 10–50 mmol / L phosphate (based on NaH2PO4 / Na2HPO4 pairing), and 100–150 mmol / L NaCl. The pH of reagent D is 7.2–7.

6.

3. The immunochromatographic reagent for detecting malignant tumors in urine according to claim 2, characterized in that, Reagent D also includes at least one of hydroquinone 0.1–0.5 mmol / L, N-propyl gallate 0.05–0.2 mmol / L, and Trolox 1–5 mmol / L.

4. The immunochromatographic reagent for detecting malignant tumors in urine according to claim 3, characterized in that, Reagent D also includes EDTA 0.5–2.0 mmol / L.

5. The application of an immunochromatographic reagent for detecting malignant tumors in urine, comprising the immunochromatographic reagent for detecting malignant tumors in urine as described in any one of claims 1-4, characterized in that, The application is for the purpose of non-disease diagnosis or treatment, and includes: Step 1, Sample collection: Collect 10-50 mL of midstream morning urine from the subject using a disposable sterile urine cup. Process the urine within 2 hours after collection. If temporary storage is required, refrigerate at 2-8℃ for ≤8 hours. Step 2, urine sediment preparation: Aliquot the sample collected in Step 1 into 15mL centrifuge tubes. Set the speed of the benchtop low-temperature centrifuge to 300-800rpm × sample mass (sample mass unit is grams), and the centrifugation temperature to 20-25℃. Discard the supernatant and retain 100-300µL of urine sediment. Step 3, Fixation: Add an equal volume of 95% methanol or 95% ethanol to the urine sediment, shake gently for 1-3 minutes to obtain the urine sediment fixative and place it in a low-temperature centrifuge. Set the speed parameter to 300 rpm × the mass of the urine sediment fixative. The mass of the urine sediment fixative is grams. After 2 minutes of sedimentation, discard the fixative and retain the urine sediment. Step 4, Liquid-based thin-layer preparation: Transfer the urine sediment into the sample chamber of the liquid-based cell slide preparation machine, select the urine sediment or cytology program, and set the target spreading area diameter to 10-13 mm. After the slide is prepared, transfer it to a glass slide. The glass slide is positively charged and has a size of 25 mm × 75 mm. Step 5, Drying: Place the glass slide obtained in Step 4 in a constant temperature drying oven and dry it at 37-45℃ for 3-10 minutes, then take it out and cool it to room temperature. Step 6, A reagent staining: Add 15-25µL of A reagent to the sample frame using an adjustable pipette to cover the cell spreading area, and place in a humidified box at 20-25℃ for 10-20 minutes in the dark. Step 7, Washing: Immerse and wash with reagent C 2-3 times on a shaker at 50-80 rpm, each wash lasting 30-60 seconds. Shake off the liquid at the edges and do not blow dry with air. Step 8, Reagent B color separation: This includes direct fluorescent secondary antibody color separation and sequential deposition color separation of enzyme-labeled secondary antibody-tyramine deposition amplification composition; Step 9, nuclear contrast staining: If no positive result is found in acridine orange in A, add 0.001% to 0.01% w / v of acridine orange for 20 to 60 seconds, and then wash once quickly with reagent C; Step 10, sealing: Add 10-20µL of reagent D to cover an area of ​​18mm×18mm on the coverslip, gently press along the edge to remove air bubbles, and let stand at room temperature for at least 5 minutes. Step 11, Colorimetric Imaging: Place the mounted slide on the stage of a fluorescence microscope. Use a fluorescence microscope equipped with excitation modules for 405, 488, 561, 633, and 730 nm for multi-channel acquisition. Select the appropriate objective lens, focus on the monolayer region of the cell, and then sequentially acquire flat-field and dark current correction frames. Subsequently, image in the order of short wavelength to long wavelength. The acridine orange-DNA channel uses 488 nm excitation and 525 / 30 nm emission filters, and the acridine orange-RNA channel uses 561 nm excitation and 650 / 60 nm emission filters. The five immunochromatographic channels used emission filters of 420 / 30, 570 / 20, 620 / 20, 700 / 30, and 775 / 50 nm, respectively. The camera used 12-16 bit sampling, with an exposure time of 50-300 ms, fixed gain, and automatic white balance off. After alignment of each channel in the same field of view, at least three non-overlapping fields of view were acquired. The coordinate images were recorded and saved in a lossless format. The microscope model, objective parameters, excitation / emission combination, exposure time, gain, acquisition date and time, and slide number were also recorded. Step 12, Image Processing and Data Organization: Import the original image into the image processing workstation, load the flat field and dark current correction frames, correct each channel one by one, import the channel leakage or compensation matrix established by the monochrome control, and perform spectral unmixing to output multi-channel layers. Then, perform channel registration and stitching, perform batch processing instance segmentation on the image, generate instance labeled masks, and calculate the integral intensity, average intensity, area, perimeter, nuclear / cytoplasmic mask and geometric center coordinates of each channel. Overlay the masks back into the layers to generate an overlay map, and export the channel intensity and geometric features of each instance into a CSV table. Analyze each sample image based on the AI ​​image analysis model, write the positive or negative label for acridine orange detection and the hierarchical identification label. The hierarchical identification label corresponds to 19A211, M344, LDQ10, CK20, and anti-hTERT monoclonal antibody, respectively. Also, write the batch number, field of view number, channel parameters, and timestamp for each image, and archive it together with the original image, correction image, mask image and overlay image to the designated directory.

6. The application of the immunochromatographic reagent for detecting malignant tumors in urine according to claim 5, characterized in that, Step 8, the procedure for direct fluorescent secondary antibody color separation includes: Step S1: In the sample frame, dilute anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse IgG-AF610, anti-rabbit IgG-AF680, and anti-chicken IgY-AF750 to 0.5-5.0 µg / mL with secondary antibody matrix buffer and mix to prepare a secondary antibody mixed working solution. Step S2: Add 15-25µL of each sample to the sample frame of the glass slide, incubate in a humidified box at 20-25℃ in the dark for 10-20 minutes, and gently shake on a shaker at 50-80 rpm. Step S3: After incubation, wash twice with reagent C, each time for 30-60 seconds; Step S4: Add 0.001% to 0.01% w / v of acridine orange to the sample frame for 20 to 60 seconds, then wash once with reagent C. After completing step S5, proceed to step 9.

7. The application of the immunochromatographic reagent for detecting malignant tumors in urine according to claim 5, characterized in that, In step 8, the sequential deposition and color separation process of the enzyme-labeled secondary antibody-tyramine deposition amplification composition includes: Step 21: Select enzyme-labeled secondary antibody-tyramine that matches the host or subclass of the target primary antibody for each color in the order of 405→555→610→647→750nm, and dilute it with the reaction matrix to 0.5~5.0µg / mL. Add 15~25µL of each sample to the sample frame and incubate in a humidified chamber at 20~25℃ in the dark for 5~10min. Step 22: Wash twice with reagent C, 30-60 seconds each time; Step 23: Prepare a tyramine fluorescent substrate working solution containing ≤0.001% w / v H2O2 at a concentration of 1–10 µmol / L. The tyramine fluorescent substrate working solution contains one of TSA-405, TSA-555, TSA-610, TSA-647, or TSA-750. Add 15–25 µL to the sample frame and incubate at 20–25°C in the dark for 3–7 min. Step 24: After pouring off the working solution, add the deposition termination composition dropwise and let it act for 2-5 minutes, then wash with reagent C 2-3 times, each time for 30-60 seconds; Step 25: After completing this round, replace with the next enzyme-labeled secondary antibody - tyramine and the corresponding tyramine fluorescent substrate, and repeat steps 23 to 24 above until all colors are completed; Step 26: After all rounds are completed, add 0.001% to 0.01% acridine orange (w / v) to the sample box for 20 to 60 seconds, and wash once with reagent C. Step 27, after completion, proceed to step 9.