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CN-121679025-B - Immune chromogenic reagent for detecting malignant tumor in urine and application thereof

CN121679025BCN 121679025 BCN121679025 BCN 121679025BCN-121679025-B

Abstract

The invention relates to the field of biological agents, and particularly discloses an immune chromogenic reagent for detecting malignant tumors in urine and application thereof, which are used for solving the problems of poor identification of a single-staining chromogenic image of acridine orange on a tumor specific epitope of a bladder part in detection of bladder cancer urine shedding cells, heavy background interference, spectrum overlapping and high subjectivity of manual interpretation, wherein the chromogenic reagent comprises a reagent A, a reagent B, a reagent C and a reagent D, an antibody complex aiming at a tumor cell target antigen of the bladder part in urine shedding is arranged in the reagent A, acridine orange, the reagent B comprises a fluorescent marker secondary antibody composition corresponding to the reagent A and an enzyme-labeled secondary antibody-tyramine deposition amplification composition, and AI imaging analysis is adopted in application, and a layering identification label and an acridine orange staining negative and positive label are marked; according to the invention, through multi-target immune color development, two sets of color separation amplification schemes and AI imaging analysis, the bladder tumor specific epitope recognition capability, image quality and image analysis accuracy of urine abscission cell detection are improved.

Inventors

  • YANG XIAOFANG
  • GUO TIANHUAN

Assignees

  • 浙江丹晖生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260211

Claims (7)

  1. 1. An immune chromogenic reagent for detecting malignant tumor in urine is characterized by comprising an A reagent, a B reagent, a C reagent and a D reagent, wherein the A reagent comprises an antibody compound aiming at tumor cell target antigens at bladder positions in urine shedding, fluorescein, a buffer solution and a sealing stable composition, the antibody compound comprises 19A211 of 0.5-2.0 mu g/mL, M344 of 0.5-2.0 mu g/mL, LDQ10 of 0.5-2.0 mu g/mL, CK20 of 0.5-2.0 mu g/mL, anti-hTERT monoclonal antibodies of 0.5-2.0 mu g/mL, fluorescein is acridine orange of 0.001-0.01%w/v, the buffer solution comprises a mixed solution of NaCl of 0.10-0.18 mol/L and phosphate of 10-50 mmol/L, the sealing stable composition comprises serum albumin of 0.5-2.0.0%w/v, tween-20.02%w/v and sodium of 0.10%w/v, and a dye-6-7-B reagent is used for preparing a contrast medium and amplifying a dye-free antibody to a dye-free dye-labeled antibody, and a dye-free antibody is used for removing the dye-labeled antibody, and a dye-free antibody is used for a dye-labeled reagent, and a dye-free dye-labeled antibody-free dye-pair reagent is prepared by the dye-labeled reagent; In the reagent B, a fluorescence-labeled secondary antibody composition matched with the antibody complex comprises anti-mouse IgG1-AF405, anti-mouse IgG2a-AF555, anti-mouse IgG-AF610, anti-rabbit IgG-AF680 and anti-chicken IgY-AF750 which are all at the concentration of 0.5-5.0 mug/mL, and a secondary antibody matrix buffer solution which comprises 10-50 mmol/L Tris-HCl, 100150mmol/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 the reagent B, the enzyme-labeled secondary anti-tyramine deposition amplification composition matched with the antibody complex comprises HRP-anti-mouse IgG1, HRP-anti-mouse IgG2a, HRP-anti-mouse IgG, HRP-anti-rabbit IgG and HRP-anti-chicken IgY with concentration of 0.5-5.0 mug/mL, further comprises tyramine derivative fluorogenic substrates TSA-405, TSA-555, TSA-610, TSA-647 and TSA-750 with concentration of 1-10 mug/L, a deposition reaction matrix containing 10-50 mmol/L, naCl-150 mmol/L of Tris-HCl and H 2 O 2 less than or equal to 0.001%w/v, and a deposition termination composition, wherein the deposition termination composition comprises 10-50 mmol/L of Tris-HCl, 100-150 mmol/L of NaCl and 1-5 mmol/L of Trolox, and the pH of the deposition termination composition is 7.2-7.6.
  2. 2. The immunochromatographic reagent for detecting malignant tumors in urine according to claim 1, wherein the reagent D comprises 50% -80% v/v of glycerol, 10% -50 mmol/L of phosphate based on NaH 2 PO 4 /Na 2 HPO 4 pairs, 100% -150 mmol/L of NaCl, and the pH of the reagent D is 7.2% -7.6.
  3. 3. The immunochromatography reagent for detecting malignant tumors in urine according to claim 2, wherein the reagent D further comprises at least one of 0.1-0.5 mmol/L, N-propyl gallate and 0.05-0.2 mmol/L, trolox-5 mmol/L of hydroquinone.
  4. 4. The immunochromatographic reagent for detecting malignant tumors in urine according to claim 3, wherein the reagent D further comprises EDTA in an amount of 0.5-2.0 mmol/L.
  5. 5. Use of an immunochromogenic reagent for detecting malignancy in urine according to any one of claims 1-4, wherein the use is for diagnosis or treatment of a non-disease, the use comprising: Step1, collecting a sample, namely using a disposable sterile urine cup to leave 10-50 mL of morning urine middle section of a subject, collecting the sample, processing the sample within 2 hours, and if temporary storage is needed, refrigerating the sample at 2-8 ℃ for less than or equal to 8 hours; Step 2, preparing urinary sediment, namely subpackaging the sample acquired in the step 1 into a 15mL centrifuge tube, setting the rotating speed of a table type low-temperature centrifuge to be 300-800 rpm multiplied by sample mass, wherein the unit of the sample mass is gram, the centrifugation temperature is 20-25 ℃, discarding the supernatant, and reserving 100-300 mu L of urinary sediment; Step3, fixing, namely adding 95% methanol or 95% ethanol with equal volume into the urinary sediment, mixing the mixture for 1 to 3 minutes by shaking, obtaining a urinary sediment fixing solution, placing the urinary sediment fixing solution in a low-temperature centrifuge, setting the rotating speed to be 300rpm multiplied by the mass of the urinary sediment fixing solution, setting the mass of the urinary sediment fixing solution to be gram, and discarding the fixing solution after sedimentation for 2 minutes to retain the urinary sediment; Step 4, liquid-based thin-layer flaking, namely transferring urinary sediment into a sample bin of a liquid-based cell flaking machine, selecting urinary sediment or cytology procedure, wherein the diameter of a target spreading area is 10-13 mm, transferring the urinary sediment to a glass slide after flaking, and treating the glass slide by positive charges, wherein the size of the glass slide is 25mm multiplied by 75mm; Step 5, drying, namely placing the glass slide obtained in the step 4 in a constant-temperature drying oven, keeping the temperature at 37-45 ℃ and drying for 3-10 min, and taking out and cooling to room temperature; Step 6, A reagent staining, namely dripping 15-25 mu L of A reagent into a sample frame by using an adjustable pipette, covering a cell spreading area, and placing a wet box for 10-20 min at 20-25 ℃ in a dark place; Step 7, washing, namely immersing and washing the table with a reagent C for 2-3 times under the condition of 50-80 rpm, washing for 30-60 seconds each time, spin-drying the edge liquid, and drying the edge liquid without air flow; step 8, reagent B color separation, which comprises direct fluorescence secondary antibody color separation and enzyme-labeled secondary antibody-tyramine deposition amplification composition sequential deposition color separation; Step 9, checking and comparing, namely if the acridine orange in the A is not collected positive, dripping 0.001% -0.01% w/v of acridine orange for 20-60 seconds, and then rapidly washing for 1 time by using a reagent C; Step 10, sealing, namely dripping 10-20 mu L of reagent D, covering the range of 18mm multiplied by 18mm of a cover glass, slightly pressing and discharging bubbles along the edge, and standing at room temperature for more than 5 minutes; step 11, color development imaging, namely placing a sealed glass slide on a fluorescent microscope stage, carrying out multi-channel acquisition by adopting a fluorescent microscope provided with excitation modules of 405, 488, 561, 633 and 730nm, selecting an adaptive objective lens, sequentially completing flat field and dark current correction frame acquisition after focusing on a cell monolayer area, then carrying out imaging according to a short wave to long wave sequence, using 488nm excitation and 525/30nm emission filter sheets for an acridine orange-DNA channel, using 561nm excitation and 650/60nm emission filter sheets for an acridine orange-RNA channel, using 420/30, 570/20, 620/20, 700/30 and 775/50nm emission filter sheets for an immune five-color channel respectively, sampling by adopting 12-16 bit cameras, carrying out exposure time of 50-300 ms, gain fixing and automatic white balance closing, acquiring at least 3 mutually non-overlapping visual fields after each channel finishes alignment in the same visual field, recording coordinate images and storing the images in a nondestructive format, and simultaneously recording microscope model, objective lens parameters, excitation/emission combination, exposure time, gain, acquisition date and glass slide number; Step 12, image processing and data arrangement, namely, an original image is imported into an image processing workstation, flat field and dark current correction frames are loaded, channels are corrected one by one, channel leakage or compensation matrixes established by monochromatic contrast are imported, spectral unmixing is carried out, a multi-channel image layer is output, channel registration and splicing are carried out, batch processing example segmentation is carried out on the image, an example labeling mask is generated, the integral intensity, average intensity, area, circumference, nuclear/cytoplasmic mask and geometric center coordinates of each channel are calculated, the mask is overlapped back to the image layer to generate an overlapped graph, the channel intensity and geometric features of each example are exported to be a CSV table, each sample image is analyzed based on an AI image analysis model, positive or negative labels detected by acridine orange and layered identification labels are written, the layered identification labels respectively correspond to 19A211, M344, LDQ10, CK20 and anti-hTERT monoclonal antibodies, and batch numbers, field numbers, channel parameters and time stamps are written for each image, and the original graph, the corrected graph, the mask graph and the overlapped graph are filed to a specified directory.
  6. 6. The use of an immunochromatographic reagent for detecting malignant tumors in urine according to claim 5, wherein in step 8, the procedure of direct fluorescence secondary antibody color separation comprises: S1, respectively diluting an anti-mouse IgG1-AF405, an anti-mouse IgG2a-AF555, an anti-mouse IgG-AF610, an anti-rabbit IgG-AF680 and an anti-chicken IgY-AF750 to 0.5-5.0 mug/mL in a sample frame by using a secondary antibody matrix buffer solution, and mixing to prepare a secondary antibody mixed working solution; Step S2, dripping 15-25 mu L of each piece into a slide sample frame, placing a 20-25 ℃ wet box, incubating for 10-20 min in a dark place, and shaking the slide glass in a shaking table at 50-80 rpm; Step S3, washing with the reagent C for 2 times after incubation, and 30-60S each time; S4, dropwise adding 0.001% -0.01% w/v of acridine orange into a sample frame, and washing with a reagent C for 1 time after 20-60 seconds; step S5, after the completion, the process goes to step 9.
  7. 7. The use of an immunochromatographic reagent for detecting malignant tumors in urine according to claim 5, wherein in step 8, the procedure of sequential deposition and color separation of the enzyme-labeled secondary antibody-tyramine deposition amplification composition comprises: step 21, sequentially selecting enzyme-labeled secondary antibody-tyramine matched with a primary antibody host or subclass of the round of targets for each color according to the sequence of 405-555-610-647-750 nm, diluting to 0.5-5.0 mug/mL by a reaction matrix, dripping 15-25 mug of each tablet into a sample frame, and incubating the wet box at 20-25 ℃ for 5-10 min in a dark place; step 22, washing for 2 times with a reagent C for 30-60 s each time; Step 23, preparing tyramine fluorescence substrate working solution containing H 2 O 2 -0.001% w/v according to 1-10 mu mol/L, wherein the tyramine fluorescence substrate working solution contains one of TSA-405, TSA-555, TSA-610, TSA-647 or TSA-750, and dropwise adding 15-25 mu L into a sample frame, and incubating for 3-7 min at 20-25 ℃ in a dark place; Step 24, after pouring the working solution, dropwise adding a deposition termination composition to act for 2-5 minutes, and then washing for 2-3 times with a reagent C for 30-60 seconds each time; step 25, replacing the enzyme-labeled secondary antibody-tyramine and the corresponding tyramine fluorogenic substrate with the next enzyme-labeled secondary antibody-tyramine after the round is completed, and repeating the steps 23 to 24 until all colors are completed; Step 26, after all rounds are finished, dripping 0.001% -0.01% w/v 20-60 s of acridine orange into a sample frame, and washing for 1 time by using a reagent C; step 27, after completion, the process proceeds to step 9.

Description

Immune chromogenic reagent for detecting malignant tumor in urine and application thereof Technical Field The invention relates to the technical field of biological agents, in particular to an immune chromogenic reagent for detecting malignant tumors in urine and application thereof. Background The invention patent of China with the application publication number of CN119023640A discloses a fluorescent color developing agent for a urine tumor of a non-invasive urinary system and a use method thereof, acridine orange is used for staining tumor cells, one or more of vitamin C, tri (2-carboxyethyl) phosphine, 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid, cyclooctatetraene or glutathione is provided as an anti-bleaching agent, and a cell morphology and chemical combination method is adopted to observe the change of cell morphology and chemical fluorescence, so that the defect of sensitivity and specificity of traditional Babbitt cytology is overcome, the main clinical symptoms of the tumor of the urinary system are haematuria, hemoglobin has fluorescence inhibition in the proposed agent and the quoted method, the interference of haematuria is eliminated, and the anti-bleaching agent makes up the clinical limitation that the acridine orange is easy to be photo-bleached and is unfavorable for observing the cell morphology for a long time. Although the acridine orange staining can distinguish normal cells and tumor cells in urine shedding cells to a certain extent through different color fluorescence reactions, the defects of lack of tumor specific epitope recognition, especially insufficient tumor cell specific epitope recognition aiming at bladder sites, are difficult to distinguish reactivity and inflammatory cell proliferation and tumor ploidy abnormality only by virtue of poor nucleic acid total quantity and cell morphology, and cause false positive elevation and limited specific recognition in acridine orange staining positive samples, and difficult to establish bladder site tumor related antigen layering recognition labels of different molecular subtypes, (2) the invention eliminates blood urine interference, but autofluorescence and absorption of hemoglobin, bilirubin, urine crystals, bacterial membranes and the like still cause partial background unevenness of observation images, signal shielding, so that the boundary of a recognition area is difficult to judge, early and rare tumor cells are easy to be missed, the recheck rate is increased, (3) DNA in the cell nuclei after acridine orange staining is bright green fluorescent (about 530 nm) and orange fluorescent light (about 640 nm) overlaps with human tail part fluorescent light, and has low error-reading accuracy and low subjective interpretation accuracy and low-grade fluorescence interpretation channel quality. Disclosure of Invention The invention aims to solve the technical problem of providing an immune chromogenic reagent for detecting malignant tumors in urine and application thereof, and the bladder tumor specific epitope recognition capability, image quality and image analysis accuracy of urine abscission cell detection are improved through multi-target immune chromogenic, two sets of color separation amplification schemes and AI imaging analysis. In order to achieve the above purpose, the present invention provides the following technical solutions: An immune chromogenic reagent for detecting malignant tumor in urine comprises an A reagent, a B reagent, a C reagent and a D reagent, wherein the A reagent comprises an antibody complex aiming at a tumor cell target antigen of a bladder part in urine shedding, fluorescein, a buffer solution and a closed stable composition, the antibody complex comprises 19A211 of 0.5-2.0 mu g/mL, M344 of 0.5-2.0 mu g/mL, LDQ10 of 0.5-2.0 mu g/mL, CK20 of 0.5-2.0 mu g/mL, an anti-hTERT monoclonal antibody of 0.5-2.0 mu g/mL, acridine orange of 0.001% -0.01% w/v, the buffer solution is a mixed solution comprising NaCl of 0.10-0.18 mol/L and phosphate of 10-50 mmol/L, the blocking stable composition comprises 0.5% -2.0% w/v of serum albumin, 0.01% -0.10% v/v of Tween-20 and 0.02% -0.10% w/v of sodium azide, the pH of reagent A is 6.8% -7.6, reagent B comprises a fluorescent marked secondary antibody composition matched with the antibody complex and an enzyme marked secondary antibody-tyramine deposition amplifying composition, color separation and color development are carried out on the combined target antigen-antibody, signals are amplified, liquid-based thin layer cytology is adapted for fluorescent color development interpretation, reagent C is used for washing after the reagent A is dyed to remove unbound antibody and free fluorescein, and reagent D is used for sealing and anti-fluorescence quenching. As a further scheme of the developing reagent, in the reagent B, the fluorescent marked secondary antibody composition matched with the antibody complex comprises anti-mouse IgG1-AF405, anti-mouse IgG