CN117143830A - Hybridoma cell, preparation method thereof, monoclonal antibody and kit - Google Patents

Abstract

The application relates to a hybridoma cell, a preparation method thereof, a monoclonal antibody and a kit, wherein the preservation number of the hybridoma cell is GDMCC No:63628. the monoclonal antibody is prepared from the monoclonal antibody with the accession number of GDMCC No:63628 hybridoma cells secrete. The application provides a hybridoma cell with specific binding with FDP antigen, the secreted antibody is a monoclonal antibody prepared aiming at specific determinants on specific antigen, and the monoclonal antibody has no cross-reactivity with other antigens, strong specificity, high sensitivity and high titer, and can be used for developing a detection kit for bladder cancer.

Description

Hybridoma cell, preparation method thereof, monoclonal antibody and kit

Technical Field

The application belongs to the technical field of biomedical detection, and particularly relates to a hybridoma cell, a preparation method thereof, a monoclonal antibody and a kit.

Background

Cystoscopy and uroabscisic cytology remain the primary means of detecting bladder cancer in clinical work, but cystoscopy requires invasive sampling and uroabscisic cytology has low sensitivity to in situ cancer diagnosis. The detection methods such as the commonly used ELISA method, the latex agglutination method and the like need manual operation, are easy to cause detection errors, and have long detection time in the existing detection means.

Therefore, there is an urgent need to find a detection kit with high sensitivity, which can be applied to bladder cancer detection in a non-invasive way.

Disclosure of Invention

Accordingly, there is a need for a hybridoma cell that can secrete anti-FDP monoclonal antibodies with high titers, which can increase the sensitivity of detection of FDP antigen concentration, and can be used for noninvasive diagnosis of the bladder.

The specific technical scheme is as follows:

a hybridoma cell having a deposit number of GDMCC No:63628 or said hybridoma cells are deposited with GDMCC No:63631.

a monoclonal antibody is secreted by the hybridoma cells.

The preparation method of the hybridoma cell comprises the following steps:

immunizing an animal with an immunogen to obtain spleen cells of the immunized animal;

after fusing the spleen cells and myeloma cells, screening to prepare positive fusion cells; and

Cloning the positive fusion cells to obtain the hybridoma cells.

The hybridoma cells or the monoclonal antibodies are applied to the preparation of products for diagnosing bladder cancer or assisting diagnosis or early screening or recrudescence detection.

A bladder cancer detection kit comprising the monoclonal antibody.

In one embodiment, the bladder cancer detection kit comprises a capture antibody and a detection antibody, wherein the capture antibody and the detection antibody specifically bind to different epitopes of FDP protein respectively, and one of the capture antibody and the detection antibody is the monoclonal antibody.

In one embodiment, the detection antibody is selected from the group consisting of the antibodies having accession numbers GDMCC No:63628 monoclonal antibody secreted by hybridoma cells and deposited under accession number GDMCC No: one of the monoclonal antibodies secreted by the hybridoma cells of 63631; the capture antibody is selected from the group consisting of the antibodies having accession number GDMCC No:63628 monoclonal antibody secreted by hybridoma cells and GDMCC No:63631, and another of the monoclonal antibodies secreted by the hybridoma cells.

In one embodiment, the detection antibody is a polypeptide consisting of the polypeptide of accession number GDMCC No:63628, the capture antibody is a monoclonal antibody secreted by the hybridoma cell deposited under accession number GDMCC No:63631 hybridoma cells.

In one embodiment, the capture antibody is capable of being attached to a solid support.

In one embodiment, the solid support comprises magnetic particles.

Optionally, the capture antibody and the solid support are linked by a biotin-streptavidin system.

In one embodiment, the detection antibody is linked to a label, and the label is one of ruthenium complex, acridinium ester, horseradish peroxidase, pyruvate kinase, alkaline phosphatase, glucose oxidase, fluorescein, and colloidal gold. Optionally, the label is a ruthenium complex.

In one embodiment, the bladder cancer detection kit further comprises an antibody diluent. Optionally, the antibody diluent comprises 10 mM-200 mM phosphate-citrate buffer, 1% (v/v) -50% (v/v) sounding stabilizer, 1% (w/w) -5% (w/w) trehalose, 0.5% (w/w) -5% (w/w) BSA, 0.05M-0.35M sodium chloride, 1% (v/v) -10% (v/v) glycerol, 1 μg/mL-20 μg/mL aprotinin, 1 μg/mL-20 μg/mL leupeptin, 50 μg/mL-1000 μg/mL ABESF,1% (v/v) -20% (v/v) Stabilzyme@selector, 0.05% (v/v) to 0.5% (v/v) PC300,1 μg/mL-300 μg/mL gentamycin, 0.01% (v/v) to 0.5% (v/v) EEN-20% (v) Triton 0.01% (v/v) 100.5% (v/v).

Compared with the prior art, the application has the following beneficial effects:

The application provides a hybridoma cell with specific binding with FDP antigen, the secreted antibody is a monoclonal antibody prepared aiming at a specific determinant on the specific antigen, and the monoclonal antibody has no cross-reactivity with other antigens, has strong specificity, high sensitivity and high titer, and can be used for developing a detection kit for bladder cancer.

Detailed Description

The following detailed description of the present application will provide further details in order to make the above-mentioned objects, features and advantages of the present application more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the application, whereby the application is not limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

As used herein,% (w/w) refers to mass percent,% (v/v) refers to volume percent.

In one embodiment, the application provides a hybridoma cell deposited with the cantonese province microbiological bacterial collection center (GDMCC) at 2023, month 07, address: building 5, no. 59, of Guangzhou Md.A. Xian Zhonglu 100, accession number is GDMCC No:63631. the hybridoma cell is named as follows: FDP Mus musculus hybridoma 2G5-E3-H1, proposed taxonomic names are: FDP Mus musculus hybridoma 2G5-E3-H1. The hybridoma cell can secrete high-titer and high-affinity anti-FDP monoclonal antibody, can be applied to preparation of the anti-FDP monoclonal antibody, can be specifically combined with FDP antigen, and can be used for detecting FDP antigen concentration with high sensitivity so as to prepare a bladder cancer detection kit.

In one embodiment, the application also provides a hybridoma cell deposited with the cantonese province microbiological bacterial collection center (GDMCC) at 2023, month 07, address: building 5, no. 59, of Guangzhou Md.A. Xian Zhonglu 100, accession number is GDMCC No:63628. the hybridoma cell is named as follows: FDP Mus musculus hybridoma 3D1-C1-H5, proposed taxonomic names are: FDP Mus musculus hybridoma 3D1-C1-H5. The hybridoma cell can secrete high-titer and high-affinity anti-FDP monoclonal antibody, can be applied to preparation of the anti-FDP monoclonal antibody, can be specifically combined with FDP antigen, and can be used for detecting FDP antigen concentration with high sensitivity so as to prepare a bladder cancer detection kit.

fibrin/Fibrinogen Degradation Products (FDPs) are urine markers approved for clinical use by the U.S. Food and Drug Administration (FDA). When the clotting factors released by bladder tumor cells convert fibrinogen to fibrin deposits, plasmin breaks down the fibrin deposits into fibrin/fibrinogen degradation products. Classical FDP detection methods are latex agglutination and ELISA.

In one embodiment, the present application also provides a monoclonal antibody comprising the polypeptide of the disclosure as set forth above under accession number GDMCC No:63628, and/or the monoclonal antibody is secreted by the hybridoma cell having deposit number GDMCC No: the monoclonal antibody can be specifically combined with FDP, has strong affinity, good specificity, high sensitivity and high potency, and can be used for early screening, auxiliary diagnosis and recurrence inspection of bladder cancer.

In one embodiment, the application also provides a hybridoma cell deposited with the cantonese province microbiological bacterial collection center (GDMCC) at 2023, month 07, address: building 5, no. 59, of Guangzhou Md.A. Xian Zhonglu 100, accession number is GDMCC No:63630. the hybridoma cell is named as follows: cyfra21-1 Mus musculus hybridoma 5E3-A8-F4, proposed taxonomic names are: cyfra21-1 Mus musculus hybridoma 5E3-A8-F4. The hybridoma cell can secrete high-titer and high-affinity anti-Cyfra 21-1 monoclonal antibody, can be applied to preparation of the anti-Cyfra 21-1 monoclonal antibody, can be specifically combined with a Cyfra21-1 antigen, can detect the concentration of the Cyfra21-1 antigen with high sensitivity, and can be used for preparing a bladder cancer detection kit.

In one embodiment, the application also provides a hybridoma cell deposited with the cantonese province microbiological bacterial collection center (GDMCC) at 2023, month 07, address: building 5, no. 59, of Guangzhou Md.A. Xian Zhonglu 100, accession number is GDMCC No:63629. the hybridoma cell is named as follows: cyfra21-1 Mus musculus hybridoma 3F2-B8-C6, proposed taxonomic names are: cyfra21-1 Mus musculus hybridoma 3F2-B8-C6. The hybridoma cell can secrete high-titer and high-affinity anti-Cyfra 21-1 monoclonal antibody, can be applied to preparation of the anti-Cyfra 21-1 monoclonal antibody, can be specifically combined with a Cyfra21-1 antigen, can detect the concentration of the Cyfra21-1 antigen with high sensitivity, and can be used for preparing a bladder cancer detection kit.

The cytokeratin 19 fragment (Cyfra 21-1) is a soluble fragment of cytokeratin 19 and is normally present in very low amounts, and when bladder cancer occurs, the keratin 19 content increases and the cancer cells shed and necrose, releasing it as a dissolved fragment. The method is commonly used for observing curative effects and monitoring recurrence of the non-small cell lung cancer clinically at present.

In one embodiment, the present application also provides another monoclonal antibody comprising the polypeptide of the application as set forth in the accession number GDMCC No:63629, and/or the monoclonal antibody is secreted by the hybridoma cell having deposit number GDMCC No:63630 hybridoma cells secrete. The monoclonal antibody can be specifically combined with Cyfra21-1, has strong affinity, good specificity, high sensitivity and high potency, and can be used for early screening, auxiliary diagnosis and recurrence inspection of bladder cancer.

In addition, an embodiment of the present application also provides a method for preparing the hybridoma cell according to any one of the above, wherein the method comprises steps a to b, specifically:

step a: the animals are immunized with the immunogen to obtain spleen cells of the immunized animals.

Specifically, the immunized animal is prepared by immunizing the animal with the immunogen a plurality of times, and spleen cells of the immunized animal are then taken. The immunogen is any one of Cyfra21-1 protein and FDP protein, and can be prepared by genetic engineering technology or artificial synthesis and other conventional technical means in the field, or can be commercially available.

In one specific example, the immunogen is mixed with an immunoadjuvant to immunize an animal, and spleen cells of the immunized animal are prepared.

Specifically, the animal is immunized by the immunogen emulsified by Freund's complete adjuvant, and then the immunogen emulsified by Freund's incomplete adjuvant is continuously and repeatedly boosted to prepare the immunized animal. Further, the immunized animal was a mouse, and the volume ratio of Freund's complete adjuvant to immunogen was 1:1, the volume ratio of Freund's incomplete adjuvant to immunogen is 1:1, a step of; in the immunization stage of emulsifying the immunogen by Freund's complete adjuvant, the immunization dose of the immunized animal is 25 mu g/dose to 50 mu g/dose; in the immunization stage of emulsifying the immunogen with Freund's incomplete adjuvant, the immunization dose of the immunized animal is 25-50 mug/animal.

In particular, the immunization is by subcutaneous injection.

Step b: and (3) fusing spleen cells with myeloma cells, and screening to obtain hybridoma cells.

Specifically, positive fusion cells are obtained after screening, and cloning is carried out on the positive fusion cells to obtain hybridoma cells.

The preparation method of the hybridoma is simple and easy to operate.

An embodiment of the present application also provides the use of a monoclonal antibody according to any one of the above in the preparation of a product for diagnosis or auxiliary diagnosis or early screening or recurrence check of bladder cancer.

In particular, the product comprises a reagent, a kit, a system, an apparatus or a device.

The anti-Cyfra 21-1 monoclonal antibody and the anti-FDP monoclonal antibody are used for respectively detecting the content of Cyfra21-1 and the content of FDP in a sample and are used for preparing a bladder cancer detection kit, and the kit has high accuracy, high sensitivity, strong specificity and good stability.

The application also provides a bladder cancer detection kit, which comprises a kit consisting of a fluorescent dye with the accession number of GDMCC No:63631, and/or, by a hybridoma cell having accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628. The monoclonal antibody of the kit can be specifically combined with FDP antigen, and the concentration of FDP antigen can be detected with high sensitivity.

In a specific example, the kit is at least one of a colloidal gold immunoassay kit, an electrochemiluminescence immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit, a microfluidic chip kit, or a fluorescence immunoassay kit.

In one specific example, the kit includes reagents for detecting the concentration of FDP antigen using a double antibody sandwich method.

In a preferred example, the kit is an electrochemiluminescence kit, more preferably a double-antibody sandwich electrochemiluminescence kit, and the application of the kit and the electrochemiluminescence platform greatly shortens the detection time, reduces the generation of manual detection errors, can complete the detection of a single sample within 9min, and realizes the automation and standardization of the detection. The method is simple in operation, short in detection time, capable of reducing the generation of manual operation errors and realizing the automation and standardization of detection.

In a specific example, the kit comprises a capture antibody and a detection antibody, wherein the capture antibody and the detection antibody specifically bind to different epitopes of the FDP protein, respectively, optionally the capture antibody is a protein consisting of a protein having the accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No:63628, and/or the detection antibody is a monoclonal antibody secreted by the hybridoma cell deposited under accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628.

In a specific example, the capture antibody can be attached to a solid support, optionally a magnetic particle. Alternatively, the capture antibody and the solid support are linked by a biotin-streptavidin system.

In a specific example, the detection antibody has a label attached thereto, the label being one of ruthenium complex, acridinium ester, horseradish peroxidase, pyruvate kinase, alkaline phosphatase, glucose oxidase, fluorescein, and colloidal gold, optionally the label being ruthenium complex.

In a preferred example, the capture antibody is a heavy chain antibody consisting of the polypeptide deposited under accession number GDMCC No:63631, the detection antibody is a monoclonal antibody secreted by hybridoma cells deposited under accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628. The kit has higher sensitivity and stronger specificity in detecting FDP antigen.

In a specific example, the kit further comprises one or more of a magnetic bead solution, a calibrator, a quality control, and an antibody diluent.

Specifically, the magnetic bead solution contains magnetic beads for connection with the capture antibody, and specifically, the magnetic beads are streptavidin-coated magnetic beads.

In a specific example, each of the above antibodies was diluted separately using an antibody diluent to prepare an antibody solution, optionally diluted to a final concentration of 1.0mg/L as a working concentration.

In one specific example, the antibody dilution comprises 10 mM-200 mM phosphate-citrate buffer, 1% (v/v) -50% (v/v) sounding stabilizer, 1% (w/w) -5% (w/w) trehalose, 0.5% (w/w) -5% (w/w) BSA, 0.05-0.35M sodium chloride, 1% (v/v) -10% (v/v) glycerol, 1 μg/mL-20 μg/mL aprotinin, 1 μg/mL-20 μg/mL leupeptin, 50 μg/mL-1000 μg/mL ABESF,1% (v/v) -20% (v/v) Stabilzyme@selector, 0.05% (v/v) -0.5% (v/v) PC300,1 μg/mL-300 μg/mL gentamycin, 0.01% (v/v) -0.5% (v/v) Tween-20.01% (v/v) -100.5% (v/v). Alternatively, the pH is 7.2 to 7.4.

Alternatively, the antibody dilutions include 30 mM-70 mM phosphate-citrate buffer, 3% (v/v) -8% (v/v) sounding stabilizer, 1% (w/w) -3% (w/w) trehalose, 1% (w/w) -3% (w/w) BSA, 0.1-0.3M sodium chloride, 2% (v/v) -8% (v/v) glycerol, 2 μg/mL-10 μg/mL aprotinin, 2 μg/mL-10 μg/mL leupeptin, 50 μg/mL-150 μg/mL ABESF,2% (v/v) -10% (v/v) StabilZyme@select stabilizer,0.08% (v/v) -0.3% (v/v) PC300, 10 μg/mL-50 μg/mL gentamicin, 0.01% (v/v) -0.2% (Tween-20, 0.01% (v/v) -100.5% (v/v).

Further alternatively, the antibody dilution comprises 50 mM-100 mM phosphate-citrate buffer, 5% (v/v) -20% (v/v) sounding stabilizer, 2% (w/w) -5% (w/w) trehalose, 2% (w/w) -5% (w/w) BSA, 0.15M-0.35M sodium chloride, 5% (v/v) -8% (v/v) glycerol, 5 μg/mL-15 μg/mL aprotinin, 5 μg/mL-15 μg/mL leupeptin, 100 μg/mL-400 μg/mL ABESF,5% (v/v) -10% (v/v) StabilZyme selection stabilizer,0.1% (v/v) -0.3% (v/v) PC300, 30 μg/mL-100 μg/mL gentamicin, 0.05% (v/v) -0.3% (v/v) Tween-20,0.05% (v/v) 0.05% (v-100.5% Triv/v).

Wherein, the sounding stabilizer and the stabilzyme@select stabilizer protein stabilizer are protein protectants, and the protein is protected to be stable in structure. Aprotinin, leupeptin and AEBSF are protease inhibitors, which inhibit protease activity and prevent antibody degradation. Tween-20 and Triton X-100 are nonionic surfactants, and the nonionic surfactants are non-ionized in water, have stable properties, have good emulsifying, wetting, dispersing and dissolving assisting properties, and have renaturation proteins and the effect of improving antibody specific recognition. The application greatly improves the sensitivity and stability of the reagent through the combination of the special antibody protecting agent, aprotinin and surfactant. It was further found that the combination of the nonionic surfactants Tween-20 and Triton X-100 provides a synergistic effect.

The kit provided by the application has high sensitivity and high specificity, and can accurately detect the content of FDP in a urine sample. Urine sampling has the advantages of rapidness and painless, is an ideal non-invasive tumor marker source, and is an ideal index for assisting in diagnosing bladder cancer.

In a specific example, the sample used by the kit is urine, so that noninvasive sampling is realized, the kit can be generally accepted and matched by a subject, noninvasive examination of colony screening is facilitated, and reliable clinical reference values are provided for timely discovery, clinical prognosis evaluation and recurrence monitoring of bladder cancer. It should be noted that, besides urine samples, the detection samples of the kit may also be other samples, such as blood, urine, cells and tissues.

The application also provides a bladder cancer detection kit, which comprises a kit comprising a fluorescent dye consisting of a fluorescent dye with the accession number GDMCC No:63629, and/or, by the hybridoma cell line deposited under accession number GDMCC No:63630 hybridoma cells. The monoclonal antibody of the kit can be specifically combined with the Cyfra21-1 antigen, and can detect the concentration of the Cyfra21-1 antigen with high sensitivity.

In a specific example, the kit is at least one of a colloidal gold immunoassay kit, an electrochemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit, a microfluidic chip kit, or a fluorescence immunoassay kit.

In one specific example, the kit comprises reagents for detecting the amount of Cyfra21-1 antigen using a double antibody sandwich method.

In a preferred example, the kit is an electrochemiluminescence kit, more preferably a double-antibody sandwich electrochemiluminescence kit, and the application of the kit and the electrochemiluminescence platform greatly shortens the detection time, reduces the generation of manual detection errors, can complete the detection of a single sample within 9min, and realizes the automation and standardization of the detection. The method is simple in operation, short in detection time, capable of reducing the generation of manual operation errors and realizing the automation and standardization of detection.

Specifically, the bladder cancer detection kit comprises a first capture antibody and a first detection antibody, wherein the first capture antibody and the first detection antibody are respectively specifically combined with different epitopes of Cyfra21-1 protein. Optionally, the first capture antibody is a polypeptide consisting of the polypeptide having accession number GDMCC No:63630 or by the hybridoma cell under accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63629; and/or the first detection antibody is a polypeptide consisting of the polypeptide having accession number GDMCC No:63630 or by the hybridoma cell under accession number GDMCC No: 63629.

In a specific example, the first capture antibody can be attached to a first solid support. Optionally, the first solid support is a magnetic particle. Optionally, the first capture antibody and the first solid support are linked by a biotin-streptavidin system.

In a specific example, the first detection antibody has a label attached thereto, the label being one of ruthenium complex, acridinium ester, horseradish peroxidase, pyruvate kinase, alkaline phosphatase, glucose oxidase, fluorescein, and colloidal gold. Alternatively, the label is a ruthenium complex.

In a preferred example, the first capture antibody is a heavy chain antibody consisting of the polypeptide having accession number GDMCC No:63630, the first detection antibody is a monoclonal antibody secreted by hybridoma cells deposited under accession number GDMCC No: 63629. The kit has higher sensitivity and stronger specificity in detecting the Cyfra21-1 antigen.

In order to further improve the diagnostic performance of bladder cancer, the kit also comprises a reagent for detecting the concentration of FDP antigen by adopting a double antibody sandwich method. Reagents for detecting the concentration of FDP antigen include a second capture antibody and a second detection antibody. Wherein the second capture antibody and the second detection antibody specifically bind to different epitopes of the FDP protein, respectively. Optionally, the second capture antibody is a polypeptide consisting of the polypeptide having accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628; and/or the second detection antibody is a polypeptide consisting of the polypeptide having accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628.

The first capture antibody may be a commercially available monoclonal antibody against Cyfra21-1, and the second capture antibody may be a commercially available monoclonal antibody against FDP.

In a further preferred example, the second capture antibody is a heavy chain antibody consisting of the polypeptide deposited under accession number GDMCC No:63631, the second test antibody is a monoclonal antibody secreted by hybridoma cells deposited under accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628. The kit has higher sensitivity and stronger specificity in detecting FDP antigen. Thereby improving the sensitivity and specificity of the diagnosis of bladder cancer.

The application relates to a Cyfra21-1 and FDP combined detection electrochemiluminescence immunoassay kit for bladder cancer diagnosis, which adopts a double-antibody sandwich electrochemiluminescence method, a sample and a biotin-marked Cyfra21-1 antibody and a ruthenium complex-marked Cyfra21-1 antibody form an antigen-antibody sandwich complex, the sample and a biotin-marked FDP and ruthenium complex-marked FDP antibody form an antigen-antibody sandwich complex, the two complexes are transferred into a measuring tank and fixed on the surface of an electrode after being combined with magnetic particles coated by streptavidin, unbound substances are washed off, chemiluminescence is generated after the electrode is electrified, a generated optical signal is measured by a photomultiplier tube, and the concentration of the Cyfra21-1 and the concentration of the FDP in the sample are obtained through calculation of a calibration curve by instrument treatment. The kit is simple and convenient to operate, quick, low in cost, high in sensitivity and strong in specificity, and can be used for assisting in diagnosing bladder cancer and monitoring recurrence of bladder cancer.

The performance evaluation of the FDP and Cyfra21-1 combined detection electrochemiluminescence immunoassay kit for bladder cancer auxiliary diagnosis is as follows:

performance evaluation of the Cyfra21-1 assay: (1) sensitivity: the lowest detection limit of Cyfra21-1 is less than or equal to 0.01ng/mL; (2) linear range: 0.007 ng/mL-500.0 ng/mL; (3) accuracy: preparing a 37.07ng/mL high-value sample and a 3.047ng/mL low-value sample, wherein each sample is tested for 3 times, the relative deviation between the detection result of the high-value sample and the target value is 1.33%, and the relative deviation between the detection result of the low-value sample and the target value is 2.23%; (4) stability: the stability is good; (5) precision: the same batch of reagent is used for testing a high-value sample with the precision of 45.05ng/mL and a low-value sample with the precision of 4.021ng/mL, each sample is continuously tested for 10 times, the variation coefficient of the measured result of the high-value sample is 1.47%, and the variation coefficient of the measured result of the low-value sample is 1.28%.

Performance evaluation for FDP detection: (1) sensitivity: FDP minimum detection limit is less than or equal to 1.0 mug/mL; (2) linear range: 0.083 μg/mL-100 μg/mL; (3) accuracy: preparing a 53.01 mug/mL high-value sample and a 7.015 mug/mL low-value sample, wherein each sample is tested for 3 times, the relative deviation between the detection result of the high-value sample and the target value is 0.21%, and the relative deviation between the detection result of the low-value sample and the target value is-0.27%; (4) stability: the stability is good; (5) precision: the same batch of reagent was used to test 49.02. Mu.g/mL precision high value samples and 7.032. Mu.g/mL precision low value samples, each sample was tested 10 times in succession, the coefficient of variation measured for the high value samples was 2.14%, and the coefficient of variation measured for the low value samples was 0.50%.

In a specific example, the kit further comprises one or more of a magnetic bead solution, a calibrator, a quality control, and an antibody diluent.

Specifically, the magnetic bead solution contains magnetic beads for connection with the capture antibody, and optionally, the magnetic beads are streptavidin-coated magnetic beads. Optionally, the capture antibody is linked to biotin.

In a specific example, each of the above antibodies is diluted separately using an antibody diluent, optionally at a final concentration of 0.8 to 1.5mg/L as a working concentration, optionally at a final concentration of 1.0mg/L as a working concentration.

In one specific example, the antibody dilution comprises 10 mM-200 mM phosphate-citrate buffer, 1% (v/v) -50% (v/v) sounding stabilizer, 1% (w/w) -5% (w/w) trehalose, 0.5% (w/w) -5% (w/w) BSA, 0.05-0.35M sodium chloride, 1% (v/v) -10% (v/v) glycerol, 1 μg/mL-20 μg/mL aprotinin, 1 μg/mL-20 μg/mL leupeptin, 50 μg/mL-1000 μg/mL ABESF,1% (v/v) -20% (v/v) Stabilzyme@selector, 0.05% (v/v) -0.5% (v/v) PC300,1 μg/mL-300 μg/mL gentamycin, 0.01% (v/v) -0.5% (v/v) Tween-20.01% (v/v) -100.5% (v/v). Alternatively, the pH is 7.2 to 7.4.

Alternatively, the antibody dilutions include 30 mM-70 mM phosphate-citrate buffer, 3% (v/v) -8% (v/v) sounding stabilizer, 1% (w/w) -3% (w/w) trehalose, 1% (w/w) -3% (w/w) BSA, 0.1-0.3M sodium chloride, 2% (v/v) -8% (v/v) glycerol, 2 μg/mL-10 μg/mL aprotinin, 2 μg/mL-10 μg/mL leupeptin, 50 μg/mL-150 μg/mL ABESF,2% (v/v) -10% (v/v) StabilZyme@select stabilizer,0.08% (v/v) -0.3% (v/v) PC300, 10 μg/mL-50 μg/mL gentamicin, 0.01% (v/v) -0.2% (Tween-20, 0.01% (v/v) -100.5% (v/v).

Further alternatively, the antibody dilution comprises 50 mM-100 mM phosphate-citrate buffer, 5% (v/v) -20% (v/v) sounding stabilizer, 2% (w/w) -5% (w/w) trehalose, 2% (w/w) -5% (w/w) BSA, 0.15M-0.35M sodium chloride, 5% (v/v) -8% (v/v) glycerol, 5 μg/mL-15 μg/mL aprotinin, 5 μg/mL-15 μg/mL leupeptin, 100 μg/mL-400 μg/mL ABESF,5% (v/v) -10% (v/v) StabilZyme selection stabilizer,0.1% (v/v) -0.3% (v/v) PC300, 30 μg/mL-100 μg/mL gentamicin, 0.05% (v/v) -0.3% (v/v) Tween-20,0.05% (v/v) 0.05% (v-100.5% Triv/v).

Wherein, the sounding stabilizer and the stabilzyme@select stabilizer protein stabilizer are protein protectants, and the protein is protected to be stable in structure. Aprotinin, leupeptin and AEBSF are protease inhibitors, which inhibit protease activity and prevent antibody degradation. Tween-20 and Triton X-100 are nonionic surfactants, and the nonionic surfactants are non-ionized in water, have stable properties, have good emulsifying, wetting, dispersing and dissolving assisting properties, and have renaturation proteins and the effect of improving antibody specific recognition. The application greatly improves the sensitivity and stability of the reagent through the combination of the special antibody protecting agent, aprotinin and surfactant. It was further found that the combination of the nonionic surfactants Tween-20 and Triton X-100 provides a synergistic effect.

The kit has high sensitivity and high specificity, and can accurately detect the content of Cyfra21-1 and FDP in urine samples. Urine sampling has the advantages of rapidness and painless, is an ideal non-invasive tumor marker source, and is an ideal index for assisting in diagnosing bladder cancer.

In a specific example, the sample used by the kit is urine, so that noninvasive sampling is realized, the kit can be generally accepted and matched by a subject, noninvasive examination of colony screening is facilitated, and reliable clinical reference values are provided for timely discovery, clinical prognosis evaluation and recurrence monitoring of bladder cancer. It should be noted that, besides urine samples, the detection samples of the kit may also be other samples, such as blood, urine, cells and tissues.

The application also provides a bladder cancer detection kit, which comprises a kit consisting of a fluorescent dye with the accession number of GDMCC No:63631, and/or, by a hybridoma cell having accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628. The monoclonal antibody of the kit can be specifically combined with FDP antigen, and the concentration of FDP antigen can be detected with high sensitivity.

In one specific example, the kit includes reagents for detecting the concentration of FDP antigen using a double antibody sandwich method.

In a specific example, the kit comprises a second capture antibody and a second detection antibody, wherein the second capture antibody and the second detection antibody specifically bind to different epitopes of the FDP protein, respectively, optionally the second capture antibody is a polypeptide consisting of a polypeptide having the accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No:63628, and/or the second detection antibody is a monoclonal antibody secreted by the hybridoma cell deposited under accession number GDMCC No:63631 or by a hybridoma cell deposited under the accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628.

In a specific example, the second capture antibody can be attached to a second solid support, optionally, a magnetic particle. Optionally, the second capture antibody and the second solid support are linked by a biotin-streptavidin system.

In a specific example, the second detection antibody has a label attached thereto, the label being one of ruthenium complex, acridinium ester, horseradish peroxidase, pyruvate kinase, alkaline phosphatase, glucose oxidase, fluorescein, and colloidal gold, optionally the label being ruthenium complex.

In a preferred example, the second capture antibody is a heavy chain antibody consisting of the polypeptide of accession number GDMCC No:63631, the second test antibody is a monoclonal antibody secreted by hybridoma cells deposited under accession number GDMCC No: a monoclonal antibody secreted by the hybridoma cells of 63628. The kit has higher sensitivity and stronger specificity in detecting FDP antigen.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

EXAMPLE 1 screening, preparation and potency determination of anti-Cyfra 21-1 monoclonal antibodies

1. Preparation of myeloma cells

The myeloma cells SP2/0 are recovered 7 days before cell fusion, so that the cells are in the logarithmic growth phase during fusion, the survival rate is over 95 percent, and the activity is best.

2. Preparation of feeder cells

The day before fusion, BALB/c mice with 6-8 weeks age are killed by neck breaking, the mice are soaked in 75% alcohol for 5 minutes for sterilization and then transferred to an ultra-clean workbench, abdominal skin is cut off, peritoneum is cut off, 1-5mL of serum-free culture medium is sucked into abdominal cavity for flushing for 2 times, and the mice are sucked into a centrifuge tube. Centrifugation at 1500rpm for 5 minutes, discarding the supernatant, and transferring the mouse peritoneal macrophages into a proper amount of complete medium for uniform mixing to prepare 5 trophoblast plates. The feeder cell suspension was added to a 96-well plate at 100 μl per well.

3. Preparation of fusion agent and stop solution

1.5mL of PEG and 20mL of serum-free RPMI-1640 were placed in a 37℃incubator for preheating.

4. Cell fusion

Spleen cells obtained from mice immunized with Cyfra21-1 antigen were collected and cultured in vitro for myeloma cells, preferably SP2/0. With RPMI-1640 baseWashing cells 3 times with basal medium, mixing spleen cells and tumor cells at a ratio of 10:1, centrifuging and washing once again, removing supernatant, fully dispersing precipitate, adding 1mL 50% PEG1450 to the precipitate, gently stirring while adding, finishing the addition within 60 seconds, and stopping the action of PEG with RPMI-1640 medium. After centrifugation of the supernatant, the cells were resuspended in the appropriate volume of HAT medium and the fused cells were added at 100. Mu.L/well to 96-well plates with feeder cells spread, and placed at 37℃in 5% CO ₂ Culturing in a carbon dioxide incubator with concentration, and half-changing HAT medium on the fourth day.

5. Screening of Positive cell lines

Hybridoma cell lines specifically binding to the Cyfra21-1 antigen, designated as Cyfra21-1Mus musculus hybridoma 5E3-A8-F4 and Cyfra21-1Mus musculus hybridoma 3F2-B8-C6, respectively, were screened using an indirect ELISA assay.

6. Monoclonal antibody ascites preparation and purification

The method comprises the steps of adopting a syngeneic mouse in-vivo induction method to produce a large amount of monoclonal antibodies, injecting certain hybridoma cells into the abdominal cavity of the mouse, and injecting 0.5mL of paraffin/pristane oil into the abdominal cavity of the mouse two weeks before injection. Thus, the hybridoma cells were propagated in the abdominal cavity of the mouse, the antibody was secreted in large amounts, the ascites was collected once or more times after 1 week, and the solid fraction was removed by centrifugation, and the supernatant thereof was the monoclonal antibody. The monoclonal antibodies were further purified by caprylic acid-ammonium sulfate precipitation, protein G affinity chromatography.

7. Determination of anti-Cyfra 21-1 monoclonal antibody titers

(1) And (3) coating an ELISA plate: the titers of anti-Cyfra 21-1 monoclonal antibodies were determined by indirect ELISA, and Cyfra21-1 antigen was diluted to 1. Mu.g/mL with 0.05M coating solution pH9.6, 100. Mu.L per well was added to the ELISA plate, and the plate was kept overnight at 4 ℃. The ELISA plate is washed 3 times by PBST, antigen which is not combined with the ELISA plate is washed off, 5% BSA blocking solution is taken, 200 mu L/hole is added into the ELISA plate, the ELISA plate is incubated for 3 hours at room temperature, the plate is washed 3 times, and antigen coating and blocking are completed.

(2) Adding a sample: collected mouse blood was centrifuged at 12000r/min at 4℃for 5 minutes, and serum was aspirated and stored at-20 ℃. A small amount of serum was serially diluted with PBST from 500-fold and 1-well diluent control was established, diluted serum was added to the coated ELISA plate at 100. Mu.L per well, incubated for 60 min at 37℃and plates were washed 3 times with PBST.

(3) Adding enzyme-labeled secondary antibodies: the second enzyme-labeled antibody was diluted to working concentration with PBST, 100. Mu.L per well was added to the enzyme-labeled plate, incubated at 37℃for 30 minutes, and the plate was washed 3 times with PBST.

(4) And (3) color development of the substrate: 100 mu L H was added per well ₂ O ₂ TMB was developed as a substrate, the reaction was terminated with hydrochloric acid, and the absorbance at 450nm was measured to give a titer of 1:241000000 for the anti-Cyfra 21-1 monoclonal antibody (secreted by the Cyfra21-1 Mus musculus hybridoma 3F2-B8-C6 hybridoma cell line having accession number GDMCC No. 63629) and 1:254000000 for the Cyfra21-1 monoclonal antibody (secreted by the Cyfra21-1 Mus musculus hybridoma 5E3-A8-F4 hybridoma cell line having accession number GDMCC No. 63630).

Example 2 screening, preparation and potency determination of anti-FDP monoclonal antibodies

1. Preparation of myeloma cells

The myeloma cells SP2/0 are recovered 7 days before cell fusion, so that the cells are in the logarithmic growth phase during fusion, the survival rate is over 95 percent, and the activity is best.

2. Preparation of feeder cells

The day before fusion, BALB/c mice with 6-8 weeks age are killed by neck breaking, the mice are soaked in 75% alcohol for 5 minutes for sterilization and then transferred to an ultra-clean workbench, abdominal skin is cut off, peritoneum is cut off, 1-5mL of serum-free culture medium is sucked into abdominal cavity for flushing for 2 times, and the mice are sucked into a centrifuge tube. Centrifugation at 1500rpm for 5 minutes, discarding the supernatant, and transferring the mouse peritoneal macrophages into a proper amount of complete medium for uniform mixing to prepare 5 trophoblast plates. The feeder cell suspension was added to a 96-well plate at 100 μl per well.

3. Preparation of fusion agent and stop solution

1.5mL of PEG and 20mL of serum-free RPMI-1640 were placed in a 37℃incubator for preheating.

4. Cell fusion

Spleen cells obtained by immunizing mice with FDP antigen are collected and cultured in vitro to obtain myeloma cells, and the myeloma cells are excellentSP2/0 is selected. Washing cells 3 times with RPMI-1640 basal medium, mixing spleen cells and tumor cells at a ratio of 10:1, centrifuging and washing once again, removing supernatant, fully dispersing precipitate, adding 1mL 50% PEG1450 into the precipitate, stirring gently while adding, finishing the addition within 60 seconds, and stopping the action of PEG with RPMI-1640 medium. After centrifugation of the supernatant, the cells were resuspended in the appropriate volume of HAT medium and the fused cells were added at 100. Mu.L/well to 96-well plates with feeder cells spread, and placed at 37℃in 5% CO ₂ Culturing in a carbon dioxide incubator with concentration, and half-changing HAT medium on the fourth day.

5. Screening of Positive cell lines

Hybridoma cell lines, designated FDP Mus musculus hybridoma G5-E3-H1 and FDP Mus musculus hybridoma D1-C1-H5, respectively, with specific binding to FDP antigen were screened using an indirect ELISA assay.

6. Monoclonal antibody ascites preparation and purification

The method comprises the steps of adopting a syngeneic mouse in-vivo induction method to produce a large amount of monoclonal antibodies, injecting certain hybridoma cells into the abdominal cavity of the mouse, and injecting 0.5mL of paraffin/pristane oil into the abdominal cavity of the mouse two weeks before injection. Thus, the hybridoma cells were propagated in the abdominal cavity of the mouse, the antibody was secreted in large amounts, the ascites was collected once or more times after 1 week, and the solid fraction was removed by centrifugation, and the supernatant thereof was the monoclonal antibody. The monoclonal antibodies were further purified by caprylic acid-ammonium sulfate precipitation, protein G affinity chromatography.

7. Determination of anti-FDP monoclonal antibody titers

(1) And (3) coating an ELISA plate: the titers of anti-FDP monoclonal antibodies were determined by indirect ELISA, FDP antigen was diluted to 1. Mu.g/mL with 0.05M coating solution pH9.6, 100. Mu.L per well was added to the ELISA plate and placed in a refrigerator at 4℃overnight. The ELISA plate is washed 3 times by PBST, antigen which is not combined with the ELISA plate is washed off, 5% BSA blocking solution is taken, 200 mu L/hole is added into the ELISA plate, the ELISA plate is incubated for 3 hours at room temperature, the plate is washed 3 times, and antigen coating and blocking are completed.

(2) Adding a sample: collected mouse blood was centrifuged at 12000r/min at 4℃for 5 minutes, and serum was aspirated and stored at-20 ℃. A small amount of serum was serially diluted with PBST from 500-fold and 1-well diluent control was established, diluted serum was added to the coated ELISA plate at 100. Mu.L per well, incubated for 60 min at 37℃and plates were washed 3 times with PBST.

(3) Adding enzyme-labeled secondary antibodies: the second enzyme-labeled antibody was diluted to working concentration with PBST, 100. Mu.L per well was added to the enzyme-labeled plate, incubated at 37℃for 30 minutes, and the plate was washed 3 times with PBST.

(4) And (3) color development of the substrate: 100 mu L H was added per well ₂ O ₂ TMB as substrate for color development, after termination of the reaction with hydrochloric acid, the absorbance at 450nm was measured to give the FDP monoclonal antibody (secreted by the FDP Mus musculus hybridoma 3D1-C1-H5 hybridoma cell line having accession number GDMCC No. 63628) a titer of 1:32500 the titer of the FDP monoclonal antibody (secreted by the FDP Mus musculus hybridoma G5-E3-H1 hybridoma cell having accession number GDMCC No. 63631) was 1:40100.

example 3 composition of kit and preparation and use thereof

The present example provides five kits, the composition of which is as follows:

(1) Electrochemiluminescence immunoassay kit for combined detection of Cyfra21-1 and FDP

Magnetic bead solution (MB): streptavidin-coated magnetic particles 0.45mg/mL, proClin 300;

Biotin tag 1 (RB 1): biotin-labeled Cyfra21-1 monoclonal antibody (secreted by hybridoma cell line deposited as GDMCC No. 63630) at 1.0mg/L, antibody dilution;

biotin label 2 (RB 2): biotin-labeled FDP monoclonal antibody (secreted by hybridoma cell line deposited under accession number GDMCC No. 63631) 1.0mg/L, antibody dilution;

ruthenium marker 1 (RA 1): 1.0mg/L of a ruthenium (Ru) complex-labeled Cyfra21-1 monoclonal antibody secreted by the hybridoma cell line having accession number GDMCC No. 63629, antibody dilution;

ruthenium label 2 (RA 2): 1.0mg/L of ruthenium complex-labeled FDP monoclonal antibody (secreted by hybridoma cell with accession number GDMCCNo: 63628), antibody dilution;

calibration material: cyfra21-1 antigen, FDP antigen; lyophilized bovine serum-containing product, proClin 300;

quality control product: cyfra21-1 antigen, FDP antigen, lyophilized bovine serum containing, proClin 300.

(2) Cyfra21-1 electrochemiluminescence immunoassay kit

a. Cyfra21-1 electrochemiluminescence immunoassay kit A:

magnetic bead solution (MB): streptavidin-coated magnetic particles 0.45mg/mL, proClin 300;

biotin label (RB): biotin-labeled Cyfra21-1 monoclonal antibody 1.0mg/L, antibody dilution, wherein the biotin-labeled Cyfra21-1 monoclonal antibody was identified by accession number GDMCC No:63630 hybridoma cell line secretion;

Ruthenium label (RA): 1.0mg/L of the ruthenium complex-labeled Cyfra21-1 monoclonal antibody and an antibody dilution, wherein the ruthenium complex-labeled Cyfra21-1 monoclonal antibody is prepared from the polypeptide with the accession number GDMCC No:63629 hybridoma cell line secretion;

calibration material: cyfra21-1 antigen, lyophilized product containing bovine serum, proClin 300;

quality control product: cyfra21-1 antigen, lyophilized product containing bovine serum, proClin 300.

b. Cyfra21-1 electrochemiluminescence immunoassay kit B:

the component A of the electrochemiluminescence immunoassay kit is basically the same as that of the Cyfra21-1, and the difference is that:

biotin label (RB): biotin-labeled Cyfra21-1 monoclonal antibody 1.0mg/L, antibody dilution, wherein the biotin-labeled Cyfra21-1 monoclonal antibody is a commercially available antibody.

(3) FDP electrochemiluminescence immunoassay kit

a. FDP electrochemiluminescence immunoassay kit A:

magnetic bead solution (MB): streptavidin-coated magnetic particles 0.45mg/mL, proClin 300;

biotin label (RB): biotin-labeled FDP monoclonal antibody 1.0mg/L, antibody dilution, wherein the biotin-labeled FDP monoclonal antibody was purified from the antibodies under accession number GDMCC No:63631 hybridoma cell line secretion;

Ruthenium label (RA): 1.0mg/L of ruthenium complex-labeled FDP monoclonal antibody, and an antibody diluent, wherein the ruthenium complex-labeled FDP monoclonal antibody is prepared from the following components with the accession number GDMCC No:63628 hybridoma cell line secretion;

calibration material: FDP antigen, lyophilized bovine serum-containing, proClin 300;

quality control product: FDP antigen, lyophilized bovine serum-containing, proClin 300.

b. FDP electrochemiluminescence immunoassay kit B:

the component A is basically the same as that of the FDP electrochemiluminescence immunoassay kit, and the difference is that:

biotin label (RB): biotin-labeled FDP monoclonal antibody 1.0mg/L, antibody dilution, wherein the biotin-labeled FDP monoclonal antibody is a commercially available antibody.

2. Preparation of the kit

Taking the preparation of the electrochemiluminescence immunoassay kit for the combined detection of Cyfra21-1 and FDP as an example, the method comprises the following steps:

(1) Preparation of antibody dilutions

The following components were mixed uniformly to obtain an antibody dilution consisting of 50mM phosphate-citrate buffer, 5% (v/v) sounding stabilizer, 2% (w/w) trehalose, 2% (w/w) BSA,0.15M sodium chloride, 5% (v/v) glycerol, 5. Mu.g/mL aprotinin, 5. Mu.g/mL leupeptin, 100. Mu.g/mL ABESF,5% (v/v) Stabilzyme@select stabizer, 0.1% (v/v) PC300, 30. Mu.g/mL gentamicin, 0.05% (v/v) Tween-20,0.05% (v/v) Triton X-100, pH7.4.

Wherein, the kyropoulos stabilizer is purchased from kyropoulos organisms, and the product number is QM08, and is a commercial protein stabilizer. Stabilzyme@select stabilizer protein stabilizer, available from SurModics, cat: SZ03-1000. Aprotinin, leupeptin and AEBSF (all 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride) are protease inhibitors. Tween-20 and Triton X-100 are nonionic surfactant inhibitors.

(2) Biotin labelling of antibodies

Taking an ultrafiltration tube, adding 1mg of Cyfra21-1 monoclonal antibody into the inner tube, performing ultrafiltration centrifugation for 3 times by using a pH 8.025mM Tris-HCl buffer solution, centrifuging at 14000rpm for 5min each time, centrifuging at 1000rpm for 2min after centrifugation, transferring the concentrated Cyfra21-1 monoclonal antibody from the ultrafiltration tube to a collection tube, and measuring the concentration of the antibody for subsequent reaction. The labeling ratio of Cyfra21-1 monoclonal antibody to biotin was designated 1:20, biotin was added to the Cyfra21-1 monoclonal antibody and reacted at room temperature for 30min. After the reaction is finished, the reaction product is put into an ultrafiltration centrifuge tube which is pretreated in advance, and the reaction product is centrifuged for three times (namely, buffer solution is replaced twice) to purify the reaction product, so as to obtain the biotin-labeled Cyfra21-1 monoclonal antibody. The same labeling method was used to obtain biotin-labeled FDP monoclonal antibodies.

(3) Ruthenium labelling of antibodies

Taking an ultrafiltration tube, adding 1mg of Cyfra21-1 monoclonal antibody into the inner tube, performing ultrafiltration centrifugation for 3 times by using PBS buffer solution with pH of 7.525mM, centrifuging at 14000rpm for 5min each time, centrifuging at 1000rpm for 2min after centrifugation, transferring the concentrated Cyfra21-1 monoclonal antibody from the ultrafiltration tube to a collection tube, and measuring the concentration of the antibody for subsequent reaction. The labeling ratio of Cyfra21-1 monoclonal antibody to ruthenium was designated as 1:30, ruthenium was added to Cyfra21-1 monoclonal antibody and reacted at room temperature for 30min. After the reaction is finished, the reaction product is put into an ultrafiltration centrifuge tube which is pretreated in advance, and is centrifuged for three times (namely buffer is replaced twice), and the reaction product is purified to obtain the ruthenium marked Cyfra21-1 monoclonal antibody. The same labeling method was used to obtain ruthenium-labeled FDP monoclonal antibodies.

(4) Preparation of Biotin Label 1 (RB 1)

The biotin-labeled Cyfra21-1 monoclonal antibody was diluted to a concentration of 1.0mg/L using the antibody dilution prepared in this example, to obtain biotin-labeled 1 (RB 1).

(5) Preparation of Biotin Label 2 (RB 2)

The biotin-labeled FDP monoclonal antibody was diluted to a concentration of 1.0mg/L using the antibody dilution prepared in this example, to give biotin-labeled 2 (RB 2).

(6) Preparation of ruthenium marker 1 (RA 1)

The ruthenium-labeled Cyfra21-1 monoclonal antibody and the ruthenium-labeled FDP monoclonal antibody were each diluted to a concentration of 1.0mg/L using the antibody dilution prepared in this example, to obtain ruthenium label 1 (RA 1).

(7) Preparation of ruthenium marker 2 (RA 2)

The ruthenium-labeled Cyfra21-1 monoclonal antibody and the ruthenium-labeled FDP monoclonal antibody were each diluted to a concentration of 1.0mg/L using the antibody dilution prepared in this example, to obtain ruthenium label 2 (RA 2).

(8) Preparation of calibrator and quality control product

First, a matrix solution at pH 7.4, consisting of Tris buffer containing 10% bovine serum albumin, 0.05% proclin 300, was prepared. Cyfra21-1 antigens with different concentrations are respectively added into the matrix liquid, and FDP antigens are prepared into high-low point working liquid of a calibrator and high-low point working liquid of a quality control product. Subpackaging the prepared calibrator working solution and quality control working solution at a rate of 1.0 mL/branch, and freeze-drying the subpackaged calibrator working solution and quality control working solution in a freeze dryer, wherein the freeze-drying curves are shown in Table 1:

table 1 lyophilization profile

Stage(s)	Time	Temperature (temperature)	Vacuum degree
				Pre-freezing	5h	-45℃	/
Primary drying	15h	-10C	50Pa
				Secondary drying	7h	20C	10Pa

Through the steps, the preparation of the electrochemiluminescence immunoassay kit for combined detection of Cyfra21-1 and FDP is completed.

3. Using method of electrochemical luminescence immunoassay kit for combined detection of Cyfra21-1 and FDP

Sample pretreatment: taking urine of a person, and centrifuging at 5000rpm at 4 ℃ for 5min for later use.

Before the reagent is used, the reagent is put into a full-automatic electrochemical luminescence tester of Primen company eCL8000 for 30 minutes in advance to automatically stir the magnetic bead particles so as to be in a suspension state.

Calibration and quality control of the kit: adding 1.0mL of ultrapure water into the matched freeze-dried calibrator and quality control product respectively, standing for 10min, then inverting and standing for 10min, fully mixing the calibrator and the quality control product, obtaining a calibration curve of the current system through a test result of the calibrator, and determining the accuracy of the current system through a quality control result.

Testing of samples:

cyfra21-1 and FDP concentrations in the sample are detected by the combined detection electrochemiluminescence immunoassay kit of Cyfra21-1 and FDP respectively. In particular, the method comprises the steps of,

(1) After the user applies for the test, the system automatically absorbs 70 mu L of biotinylated Cyfra21-1 monoclonal antibody, 70 mu L of ruthenium (Ru) complex labeled Cyfra21-1 monoclonal antibody, 35 mu L of streptavidin coated magnetic particles and 10 mu L of sample to be tested and adds the mixture into a reaction cup; the system automatically aspirates 70. Mu.L of biotinylated FDP monoclonal antibody, 70. Mu.L of ruthenium (Ru) complex labeled FDP monoclonal antibody, 35. Mu.L of streptavidin-coated magnetic particles, and 10. Mu.L of the sample to be tested together into another reaction cup. The antigen-antibody sandwich complex is formed by automatic incubation for 9 minutes in a 37 ℃ environment, and then the whole complex is bound to the magnetic particles under the interaction of biotin and streptavidin.

(2) After incubation, the system automatically absorbs the reaction mixture into the measuring pool, magnetic particles are adsorbed on the electrode through the magnet, unbound substances are washed away, chemiluminescence is generated after the electrode is electrified, the generated optical signals are detected through the photomultiplier, and the detection result is automatically detected from the calibration curve by the instrument (the calibration curve is obtained by the instrument through two-point calibration on the main calibration curve obtained by scanning).

Example 4

This example provides an antibody dilution consisting of 50mM phosphate-citrate buffer, 1% (v/v) sounding stabilizer, 2% (w/w) trehalose, 2% (w/w) BSA,0.15M sodium chloride, 5% (v/v) glycerol, 20. Mu.g/mL aprotinin, 20. Mu.g/mL leupeptin, 50. Mu.g/mL ABESF,1% (v/v) Stabilzyme@select stabilizer,0.1% (v/v) PC300, 30. Mu.g/mL gentamicin, 0.01% (v/v) Tween-20,0.01% (v/v) Triton X-100, and the pH of the antibody dilution was 7.2. The preparation of antibody dilutions was as described in example 3.

Example 5

This example provides an antibody dilution consisting of 50mM phosphate-citrate buffer, 50% (v/v) sounding stabilizer, 2% (w/w) trehalose, 2% (w/w) BSA,0.15M sodium chloride, 5% (v/v) glycerol, 1. Mu.g/mL aprotinin, 1. Mu.g/mL leupeptin, 1000. Mu.g/M LABESF,20% (v/v) Stabilzyme@select stabilizer,0.1% (v/v) PC300, 30. Mu.g/mL gentamicin, 0.5% (v/v) Tween-20,0.5% (v/v) Triton X-100, and the pH of the antibody dilution was 7.4. The preparation of antibody dilutions was as described in example 3.

Example 6

This example provides an antibody dilution consisting of 50mM phosphate-citrate buffer, 20% (v/v) sounding stabilizer, 2% (w/w) trehalose, 2% (w/w) BSA,0.15M sodium chloride, 5% (v/v) glycerol, 10. Mu.g/mL aprotinin, 10. Mu.g/mL leupeptin, 400. Mu.g/mL ABESF,10% (v/v) Stabilzyme@select stabilizer,0.1% (v/v) PC300, 30. Mu.g/mL gentamicin, 0.1% (v/v) Tween-20,0.1% (v/v) Triton X-100, and the pH of the antibody dilution was 7.4. The preparation of antibody dilutions was as described in example 3.

Table 2 antibody dilutions provided in examples 3 to 6

Example 7

This example provides an antibody dilution consisting of 10mM phosphate-citrate buffer, 5% (v/v) sounding stabilizer, 1% (w/w) trehalose, 0.5% (w/w) BSA,0.05M sodium chloride, 1% (v/v) glycerol, 5. Mu.g/mL aprotinin, 5. Mu.g/mL leupeptin, 100. Mu.g/mL ABESF,5% (v/v) Stabilzyme@select stabilizer,0.05% (v/v) PC300, 1. Mu.g/mL gentamicin, 0.05% (v/v) Tween-20,0.05% (v/v) Triton X-100, and antibody dilution pH 7.4. The preparation of antibody dilutions was as described in example 3.

Example 8

This example provides an antibody dilution consisting of 200mM phosphate-citrate buffer, 5% (v/v) sounding stabilizer, 5% (w/w) trehalose, 5% (w/w) BSA,0.35M sodium chloride, 10% (v/v) glycerol, 5. Mu.g/mL aprotinin, 5. Mu.g/mL leupeptin, 100. Mu.g/mL ABESF,5% (v/v) Stabilzyme@select stabilizer,0.5% (v/v) PC300, 300. Mu.g/mL gentamicin, 0.05% (v/v) Tween-20,0.05% (v/v) Triton X-100, and the pH of the antibody dilution was 7.4. The preparation of antibody dilutions was as described in example 3.

TABLE 3 Components of antibody dilutions provided in examples 7-8

Example 9

The content of the antibody dilution was substantially the same as that of example 3, except that: the antibody diluent does not contain the component of the sounding stabilizer, and the volume percentage of the stabilzyme@select stabilizer in the antibody diluent is adjusted to 10%, and other components and concentrations are kept unchanged.

Example 10

The content of the antibody dilution was substantially the same as that of example 3, except that: the antibody diluent does not contain the component of stabilzyme@select stabilizer, the volume percentage of the sounding stabilizer in the antibody diluent is adjusted to 10%, and other components and concentrations are kept unchanged.

Example 11

The content of the antibody dilution was substantially the same as that of example 3, except that: the antibody diluent does not contain three components of aprotinin, leupeptin and ABESF, and other components and concentrations are kept unchanged.

Example 12

The content of the antibody dilution was substantially the same as that of example 3, except that: the antibody diluent does not contain Tween20, and the volume percentage of Triton X-100 in the antibody diluent is adjusted to 0.1%. Other components and concentrations remained unchanged.

Example 13

The content of the antibody dilution was substantially the same as that of example 3, except that: the antibody diluent does not contain Triton X-100, and the volume percentage of Tween20 in the antibody diluent is adjusted to be 0.1%. Other components and concentrations remained unchanged.

Example 14

The content of the antibody dilution was substantially the same as that of example 3, except that: 2% dextran was used instead of the sounding stabilizer. Other components and concentrations remained unchanged.

Example 15

The content of the antibody dilution was substantially the same as that of example 3, except that: PMSF (phenylmethylsulfonyl fluoride) is used for replacing leupeptin, and other components and concentrations are kept unchanged.

Example 16

The content of the antibody dilution was substantially the same as that of example 3, except that: SDS (sodium dodecyl sulfate) was used instead of Triton X-100, and the other components and concentrations remained unchanged.

Example 17

The composition of the antibody dilution provided in this example was: 100mM phosphate, 2% sucrose, 2% trehalose, 2% BSA,0.15M sodium chloride, 0.5% casein, 0.1% PC300,0.05% SDS, pH 7.2.

Test example 1 sensitivity and Linear Range experiment

1. Sample to be measured: human urine samples (containing Cyfra21-1 at 1.988ng/mL and FDP at 35.12 μg/mL, measured by clinical examination) were subjected to gradient dilution by 0, 2, 4, 8, 16, 32, 64, 128, 256 and 512 times, respectively, to be tested.

2. The samples to be tested in the above step 1 were tested by using the Cyfra21-1 and FDP combined detection electrochemiluminescence immunoassay kit, the Cyfra21-1 electrochemiluminescence immunoassay kit B and the FDP electrochemiluminescence immunoassay kit B in example 3. The sensitivity test results are shown in Table 4 and Table 5.

The detection result shows that the sensitivity of the combined detection electrochemiluminescence immunoassay kit for the Cyfra21-1 and the FDP for detecting the Cyfra21-1 is 0.007ng/mL (see table 4), and the sensitivity for detecting the FDP is 0.083 mug/mL (see table 4); cyfra21-1 electrochemiluminescence immunoassay kit B the sensitivity of detecting Cyfra21-1 is 0.021ng/mL (see Table 5); FDP electrochemiluminescence immunoassay kit B the sensitivity of FDP detection was 0.251. Mu.g/mL (see Table 5).

TABLE 4 detection sensitivity statistics for the Combined detection kit of example 3

TABLE 5 detection sensitivity statistics for Cyfra21-1 electrochemiluminescence immunoassay kit B and FDP electrochemiluminescence immunoassay kit B of example 3

As can be seen from tables 4-5, the sensitivity of the combined detection electrochemiluminescence immunoassay kit for Cyfra21-1 and FDP prepared by using the antibody of the application is higher. The application greatly improves the sensitivity of the reagent through the special antibody.

3. Antibody dilutions were performed using the antibody dilutions of examples 4-17, respectively, as follows:

the biotin-labeled Cyfra21-1 monoclonal antibody (secreted by the hybridoma cell line having accession number GDMCC No. 63630) and the biotin-labeled FDP monoclonal antibody (secreted by the hybridoma cell line having accession number GDMCC No. 63631) were each diluted to 1.0. Mu.g/mL using the antibody dilutions of examples 4-17, respectively, to prepare a biotin-labeled solution. The ruthenium complex-labeled Cyfra21-1 monoclonal antibody (secreted by hybridoma cell line deposited under accession number GDMCC No. 63629) and the ruthenium complex-labeled FDP monoclonal antibody (secreted by hybridoma cell line deposited under accession number GDMCC No. 63628) were each diluted to 1.0. Mu.g/mL using the antibody dilutions of examples 4-17, respectively, to prepare ruthenium-labeled solutions.

Further, the electrochemical luminescence immunoassay kit for combined detection of Cyfra21-1 and FDP corresponding to examples 4 to 17 is prepared and assembled according to the method of example 3, and each sample to be tested in the above step 1 is detected, and the sensitivity of each example kit is obtained. The statistics of the detection results are shown in Table 6.

TABLE 6 detection sensitivity statistics for the Combined detection kits of examples 4-17

As can be seen from Table 6, the antibody diluted by the antibody dilutions in examples 3 to 8 had higher sensitivity in detecting Cyfra21-1 and FDP than examples 9 to 17. The application greatly improves the sensitivity of the reagent through the combination of the special antibody protecting agent, aprotinin and surfactant.

Example 3 is compared with examples 9-10 and example 14 to demonstrate that the synergistic effect of the stabilizer and the stabizyme@select stabilizer.

Example 3 is compared with examples 12-13 and example 16 to demonstrate that Triton X-100 and Tween20 combinations have synergistic effects.

Test example 2 correlation detection

The combined detection electrochemiluminescence immunoassay kit of Cyfra21-1 and FDP in comparative example 3 is consistent with the detection of clinical samples of the same commercial products.

The detection results are shown in Table 7, and compared with the detection results of the Roche electrochemiluminescence detection kit, the detection results of the kit provided by the application have the advantages that the correlation coefficient (r) of the primary linear regression equation is larger than 0.99, the kappa value is larger than 0.75, the detection results of the kit provided by the application and the Roche electrochemiluminescence detection kit are better in consistency, and further the detection of the kit prepared by the Cyfra21-1 monoclonal antibody and the FDP monoclonal antibody provided by the application is higher in accuracy.

TABLE 7 correlation detection result statistics

Test example 3 thermal stability experiment

The Cyfra21-1 and FDP combined detection electrochemiluminescence immunoassay kit of the example 3 is placed in an oven at 37 ℃ for thermal acceleration, and after 0,4,8 and 12 days of acceleration, the basic performances such as sensitivity, accuracy, repeatability and linearity are evaluated.

The evaluation results are shown in Table 8, and demonstrate that the kit of the present application is thermally stable, and has a stable performance at 37℃for 12 days or more without change.

TABLE 8 thermal stability test results

Test example 4 accuracy and precision detection

1. Accuracy:

an accuracy high value sample and an accuracy low value sample are prepared. The high-accuracy samples and the low-accuracy samples were tested 3 times per sample using the FDP, cyfra21-1 joint detection electrochemiluminescence immunoassay kit of example 3, and the relative deviation from the target value was calculated (relative deviation= (sample test value-target value)/target value x 100%). Wherein, the concentration of Cyfra21-1 in the high-accuracy sample and the low-accuracy sample for detecting Cyfra21-1 is 37.07ng/mL and 3.047ng/mL respectively. FDP concentrations in the high-and low-accuracy samples for FDP detection were 53.01. Mu.g/mL and 7.015. Mu.g/mL, respectively.

Through detection, in Cyfra21-1 detection, the relative deviation between the detection result of the high-value sample and the target value is 1.33%, and the relative deviation between the detection result of the low-value sample and the target value is 2.23%. In FDP detection, the relative deviation between the detection result of the high-value sample and the target value is 0.21%, and the relative deviation between the detection result of the low-value sample and the target value is-0.27%. Therefore, the kit has higher accuracy.

2. And (3) precision detection:

the same batch of reagents (i.e., FDP, cyfra21-1 combined detection electrochemiluminescence immunoassay kit of example 3) was used to test the precision high value sample and the precision low value sample, each sample was tested 10 times in succession, and the Coefficient of Variation (CV) was calculated. Wherein, the concentration of Cyfra21-1 in the high-precision sample and the low-precision sample for detecting Cyfra21-1 is 45.05ng/mL and 4.021ng/mL respectively. FDP concentration in the high-precision sample and the low-precision sample for FDP detection was 49.02. Mu.g/mL and 7.032. Mu.g/mL, respectively.

According to the detection, in the Cyfra21-1 detection, the variation Coefficient (CV) of the high-value sample is 1.47%, and the variation Coefficient (CV) of the low-value sample is 1.28%. In the FDP test, the Coefficient of Variation (CV) of the high value sample was 2.14%, and the Coefficient of Variation (CV) of the low value sample was 0.50%. Therefore, the kit has higher precision.

Test example 5 sensitivity and specificity of different detection kits for bladder cancer diagnosis

382 clinical samples were collected, of which 70 for bladder cancer and 312 for healthy people. The detection results are shown in Table 9, and the detection is performed by using the Cyfra21-1 and FDP combined detection electrochemiluminescence immunoassay kit of example 3, the Cyfra21-1 electrochemiluminescence immunoassay kit (A) and the FDP electrochemiluminescence immunoassay kit (A) respectively.

TABLE 9 statistics of sensitivity and specificity results of the respective kits of example 3 for diagnosis of bladder cancer

Detection type reagent box	ROC-AUC	Sensitivity (%)	Specificity (%)	PPV(％)	NPV(％)
						Cyfra21-1	0.929	91.43	79.40	56.31	96.70
FDP	0.857	74.29	92.95	46.08	95.17
						FDP+Cyfra21-1	0.935	93.57	96.41	58.37	97.75

The result shows that when the urine Cyfra21-1 and FDP of the bladder cancer patient are detected in a combined way, the kit has better diagnosis efficiency, and is an ideal kit for the early screening auxiliary diagnosis of bladder cancer and the dynamic monitoring of postoperative recurrence.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. The scope of the application is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.

Claims (10)

1. A hybridoma cell having a deposit number of GDMCCNo:63628 or said hybridoma cells are deposited with GDMCC No:63631.

2. a monoclonal antibody secreted by the hybridoma of claim 1.

3. The method for producing a hybridoma cell according to claim 1, comprising the steps of:

immunizing an animal with an immunogen to obtain spleen cells of the immunized animal;

after fusing the spleen cells and myeloma cells, screening to prepare positive fusion cells; and

Cloning the positive fusion cells to obtain the hybridoma cells.

4. Use of the hybridoma cell of claim 1 or the monoclonal antibody of claim 2 for the preparation of a product for diagnosis of bladder cancer or for aiding diagnosis or early screening or recurrence inspection.

5. A bladder cancer detection kit comprising the monoclonal antibody of claim 2.

6. The bladder cancer detection kit of claim 5, comprising a capture antibody and a detection antibody that specifically bind to different epitopes of FDP protein, respectively, one of the capture antibody and the detection antibody being the monoclonal antibody of claim 2.

7. The bladder cancer detection kit of claim 6, wherein the detection antibody is selected from the group consisting of a polypeptide having accession No. GDMCC No:63628 monoclonal antibody secreted by hybridoma cells and deposited under accession number GDMCC No: one of the monoclonal antibodies secreted by the hybridoma cells of 63631; the capture antibody is selected from the group consisting of the antibodies having accession number GDMCC No:63628 monoclonal antibody secreted by hybridoma cells and GDMCC No:63631, and another of the monoclonal antibodies secreted by the hybridoma cells.

8. The bladder cancer detection kit of claim 7, wherein the detection antibody is a peptide consisting of a peptide deposited under accession No. GDMCC No:63628, the capture antibody is a monoclonal antibody secreted by the hybridoma cell deposited under accession number GDMCC No:63631 hybridoma cells.

9. The bladder cancer detection kit of any one of claims 6-8, wherein the capture antibody is capable of being attached to a solid support comprising magnetic particles;

the detection antibody is connected with a marker; the label comprises one of ruthenium complex, acridinium ester, horseradish peroxidase, pyruvate kinase, alkaline phosphatase, glucose oxidase, fluorescein and colloidal gold.

10. The bladder cancer detection kit of any one of claims 5-8, further comprising an antibody diluent comprising 10-200 mM phosphate-citrate buffer, 1-50% (v/v) sounding stabilizer, 1-5% (w/w) trehalose, 0.5-5% (w/w) BSA, 0.05-0.35M sodium chloride, 1-10% (v/v) glycerol, 1-20 μg/mL aprotinin, 1-20 μg/mL leupeptin, 50-1000 μg/mL ABESF, 1-20% (v/v) stabizyme selector, 0.05-0.5% (v/v) PC, 1-300 μg/v, 0.01-0.01% (v/v) gentamicin.