Hybridoma cell strain secreting monoclonal antibody to 5-carboxy-x-rhodamine and analogues

Abstract

The application provides a hybridoma cell strain secreting 5-carboxy-X-rhodamine and analog monoclone antibody, and relates to the technical field of immune detection. The application takes 5-carboxy-X-rhodamine analog as a hapten to prepare a complete antigen, combines animal immunization and cell fusion screening to obtain a hybridoma cell strain, and the monoclone antibody secreted by the cell strain can be applied to the immune detection of 5-carboxy-X-rhodamine and analog, and the IC 50 of 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine and rhodamine 101 is 0.1 ng / mL, the IC 50 of sulforhodamine 101 is 10 ng / mL, and the immune detection product of 5-carboxy-X-rhodamine and analog is prepared based on the hybridoma cell strain, so that an efficient detection method and means are provided for 5-carboxy-X-rhodamine and analog.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes monoclonal antibodies against 5-carboxy-X-rhodamine and its analogues. Background Technology

[0002] Fluorescent labeling technology is one of the important analytical tools in modern biological and medical research, widely used in protein localization, nucleic acid detection, immunoassay, flow cytometry, and microscopic imaging. By binding fluorescent dyes to specific biomolecules, the visual analysis and quantitative detection of biomolecules can be achieved. Commonly used fluorescent dyes include fluorescein and its derivatives, rhodamine compounds, and cysteine, among which rhodamine dyes have become an important class of red fluorescent probes due to their high quantum yield, excellent photostability, suitable excitation / emission wavelengths, and good cell compatibility.

[0003] 5-Carboxy-X-Rhodamine (5-ROX) and 6-Carboxy-X-Rhodamine (6-ROX) are two structurally similar derivatives with different spatial configurations. Both exhibit strong red fluorescence and high photostability, and are therefore widely used in fluorescent probe labeling, real-time quantitative PCR (qPCR), nucleic acid hybridization analysis, oligonucleotide labeling, and protein modification. However, due to the different positions of their carboxyl groups, 5-ROX and 6-ROX may differ significantly in coupling efficiency, spatial orientation, and interaction with target molecules. Current techniques for distinguishing them mainly rely on chromatographic separation or mass spectrometry detection, lacking immunological tools that can directly identify their specific structural differences.

[0004] Sulforodamine 101 (SR101) is a red fluorescent dye containing a sulfonic acid group. Its water solubility is significantly higher than that of carboxyrodamine derivatives, and it is commonly used for cell staining, tissue imaging, and selective labeling of neurons (e.g., astrocyte staining). SR101 exhibits good photostability and red-region emission characteristics in biological samples, making it suitable for use in combination with other green or blue fluorescent probes. However, its sulfonic acid group limits conjugation methods, and direct linking to biomolecules often requires complex derivatization steps. Furthermore, antibody resources for detecting SR101 or specifically recognizing its characteristic groups are currently very limited, hindering quantitative detection, purity analysis, and conjugation efficiency assessment.

[0005] Rhodamine 101 chloride, a salt of rhodamine 101, possesses very high molar absorptivity and fluorescence quantum yield, and is widely used in dye preparation, fluorescence imaging, cell tracking, and spectral calibration. However, rhodamine 101 chloride still has limitations in water solubility, and the quaternary ammonium nitrogen atoms in its structure easily lead to non-specific interactions with biomolecules. Existing detection methods mainly rely on chromatography, mass spectrometry, or spectroscopic analysis, lacking immunological recognition methods for its structure, thus limiting its specific detection and quantitative analysis in complex systems.

[0006] In general, existing methods for detecting rhodamine dyes (including 5-ROX, 6-ROX, SR101, and rhodamine 101 chloride) mostly rely on spectral characteristics. These methods are not only easily affected by background fluorescence or sample matrix interference, but also lack the ability to distinguish between different structural isomers or substituent differences. Especially in high-throughput detection, rapid on-site detection, and trace quantitative analysis in biological systems, existing technologies struggle to meet the dual requirements of accurate qualitative analysis and high sensitivity.

[0007] Monoclonal antibodies, as highly specific and high-affinity biorecognition tools, have played a crucial role in protein detection, immunodiagnostics, and targeted therapy. However, research on antibodies targeting small-molecule fluorescent dyes, particularly rhodamine-based dyes, remains limited. Existing anti-rhodamine antibodies generally suffer from poor specificity, high cross-reactivity, and insufficient ability to distinguish different rhodamine derivatives, making it difficult to meet the requirements for accurate recognition of 5-ROX, 6-ROX, SR101, and rhodamine 101 chloride. Therefore, developing a monoclonal antibody or related immunoassay method capable of highly specifically recognizing these rhodamine-based dyes is of great significance for improving the analytical accuracy of labeled products, establishing stable and reproducible detection systems, and expanding their applications. Summary of the Invention

[0008] To solve the above-mentioned technical problems, the present invention uses 5-carboxy-X-rhodamine analogue as a hapten to prepare a complete antigen. Combined with animal immunization and cell fusion screening, a hybridoma cell line is obtained, with the preservation number CGMCC NO.46730. The monoclonal antibody secreted by this cell line can be used for the immunodetection of 5-carboxy-X-rhodamine and its analogues.

[0009] The first objective of this invention is to provide a hybridoma cell line that secretes monoclonal antibodies against 5-carboxy-X-rhodamine and its analogues, the hybridoma cell line having the accession number CGMCC NO.46730.

[0010] Further, the analogue is 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

[0011] Furthermore, the preparation of the hybridoma cell line includes the following steps:

[0012] Step S1: Prepare a complete antigen from the 5-carboxy-X-rhodamine hapten, and use the complete antigen to immunize animals.

[0013] Step S2: Collect blood from immunized animals and screen the serum immune titers and immunosuppressive abilities of the immunized animals;

[0014] Step S3: The spleen cells and myeloma cells of the selected immunized animals are fused and cultured to obtain the hybridoma cell line that secretes monoclonal antibodies of 5-carboxy-X-rhodamine and its analogues.

[0015] Further, in step S1, the animal immunization process includes primary immunization, booster immunization and sprint immunization. Primary immunization uses complete antigen and complete Freund's adjuvant, booster immunization uses complete antigen and incomplete Freund's adjuvant, and sprint immunization uses complete antigen.

[0016] Furthermore, the immunized animal is a mouse.

[0017] Furthermore, the complete antigen is obtained by conjugating a 5-carboxy-X-rhodamine hapten to a carrier protein.

[0018] Furthermore, the carrier protein is bovine serum albumin (BSA) or ovalbumin (OVA).

[0019] Furthermore, the structure of the 5-carboxy-X-rhodamine hapten is shown below:

[0020] .

[0021] Furthermore, the preparation of the complete antigen includes the following steps:

[0022] (1) The above 5-carboxy-X-rhodamine hapten was activated to obtain an activated solution;

[0023] (2) Add the activation solution obtained in step (1) above into the carrier protein solution and react to obtain the 5-carboxy-X-rhodamine complete antigen.

[0024] Further, in step (1), the activation involves dissolving the 5-carboxy-X-rhodamine hapten and adding ethylene glycol and sodium borohydride to react.

[0025] Furthermore, the 5-carboxy-X-rhodamine hapten was dissolved using N,N-dimethylformamide.

[0026] Further, in step (2), the solution after the reaction is dialyzed and separated to obtain the 5-carboxy-X-rhodamine complete antigen.

[0027] Further, in step (3), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0028] Furthermore, the carbonate buffer solution has a concentration of 0.01-0.5 mol / L and a pH of 8.0-10.0.

[0029] Preferably, the carbonate buffer solution is 0.5 mol / L.

[0030] Preferably, the pH is 9.6.

[0031] A second objective of this invention is to provide the use of the above-mentioned hybridoma antibody in the preparation of 5-carboxy-X-rhodamine and analogue monoclonal antibodies.

[0032] A third objective of this invention is to provide a monoclonal antibody against 5-carboxy-X-rhodamine and its analogues, said monoclonal antibody being prepared from the aforementioned hybridoma cell line.

[0033] Further, the analogue is 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

[0034] A fourth objective of this invention is to provide the application of the above-mentioned hybridoma cell line or the above-mentioned monoclonal antibody in the preparation of detection products for 5-carboxy-X-rhodamine and its analogues.

[0035] A fifth objective of this invention is to provide a detection product for 5-carboxy-X-rhodamine and similar substances, wherein the detection product contains the above-mentioned hybridoma cell line or the above-mentioned monoclonal antibody.

[0036] Furthermore, the detection products include test strips, reagent kits, or biochips.

[0037] Furthermore, the test strip includes a sample pad, a conjugate pad, an absorbent pad, and a cellulose acetate membrane. The conjugate pad is coated with a monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres. The kit includes an ELISA plate, a coated antigen, a standard solution of 5-carboxy-X-rhodamine and its analogues, an enzyme-labeled secondary antibody, and a colorimetric solution.

[0038] A sixth object of the present invention is to provide the use of the above-mentioned hybridoma cell line, the above-mentioned monoclonal antibody or the above-mentioned detection product in the detection of 5-carboxy-X-rhodamine and analogues.

[0039] The beneficial effects of this invention are:

[0040] This invention provides a hybridoma cell line capable of secreting monoclonal antibodies with high affinity, good specificity, and high detection sensitivity for 5-carboxy-X-rhodamine and its analogues. The IC50 of the 5-carboxy-X-rhodamine monoclonal antibody against 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, and rhodamine chloride 101 is [not specified in the original text]. 50 Both were 0.1 ng / mL, and the IC50 for sulforhodamine 101 was... 50 With a concentration of 10 ng / mL, it can be used to establish an enzyme-linked immunosorbent assay (ELISA) method for 5-carboxy-X-rhodamine and its analogues. This lays the foundation for the research and development and promotion of indirect competitive ELISA kits and colloidal gold test strips, and helps to realize the detection of 5-carboxy-X-rhodamine and its analogues. It also provides an immunoassay method and raw materials for the immunoassay of 5-carboxy-X-rhodamine and its analogues in food, and has practical application value.

[0041] Preservation of biological materials

[0042] A hybridoma cell line secreting monoclonal antibodies against 5-carboxy-X-rhodamine and its analogues has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCCNO.46730. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0044] Figure 1 The standard inhibition curve of the monoclonal antibody against 5-carboxy-X-rhodamine provided by this invention;

[0045] Figure 2 The standard inhibition curve of the monoclonal antibody against 6-carboxy-X-rhodamine provided by this invention;

[0046] Figure 3 The standard inhibition curve of the monoclonal antibody against rhodamine 101 provided by this invention;

[0047] Figure 4 The standard inhibition curve of the monoclonal antibody provided in this invention against sulforhodamine 101 is shown. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0049] This invention uses a 5-carboxy-X-rhodamine analogue as a hapten, conjugating the haptenue to a carrier protein using the carbodiimide method, and identifying successful conjugation using a UV spectrophotometer. For the initial immunization, BALB / c mice are immunized with a mixture of 100 μg / mouse of complete 5-carboxy-X-rhodamine antigen and an equal volume of complete Freund's adjuvant via subcutaneous injection at multiple sites on the neck and back. Multiple booster immunizations (50 μg / mouse) are administered with incomplete Freund's adjuvant. The final immunization is performed via intraperitoneal injection of 25 μg / mouse of complete 5-carboxy-X-rhodamine antigen (diluted with physiological saline, adjuvant-free). High titer and low IC50 are selected. 50 Mouse spleen cells were fused with SP2 / 0 myeloma cells using the PEG 4000 method. Hybrid cells of the three cell types were selected by selective culture medium. Cells were then screened by indirect competitive enzyme-linked immunosorbent assay and subcloned four times to finally obtain a monoclonal antibody hybridoma cell line.

[0050] The solution preparation methods involved in the examples are as follows:

[0051] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0052] Phosphate buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0053] PBST: PBS containing 0.05% Tween 20;

[0054] TMB colorimetric solution: Solution A: Na2HPO4 . 12H₂O 18.43 g, citric acid 9.33 g, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix fresh before use.

[0055] Example 1: Preparation of complete antigen

[0056] The 5-carboxy-X-rhodamine hapten (a 5-carboxy-X-rhodamine analog) is conjugated to a carrier protein using the carbodiimide method to obtain the 5-carboxy-X-rhodamine artificial antigen. The 5-carboxy-X-rhodamine hapten is then conjugated with bovine serum albumin (BSA) or ovalbumin (OVA) to obtain the complete antigen 5-ROX-EDC-BSA / OVA. 5-ROX-EDC-BSA is used as an immunogen to immunize mice, while 5-ROX-EDC-OVA is used as a coating agent for assays in mouse serum and fusion subclones.

[0057] The structure of the 5-carboxy-X-rhodamine hapten is shown below:

[0058] .

[0059] The preparation method of the complete antigen 5-ROX-EDC-BSA is as follows:

[0060] 1. Weigh 3.59 mg of the hapten 5-ROX, 2.32 mg of N-hydroxysuccinimide, and 3.86 mg of 1-ethylcarbodiimide hydrochloride, and dissolve them in 400 μL of N,N-dimethylformamide (the reaction solution is called solution A). Stir the reaction at room temperature for 4-6 h. Weigh 5.0 mg of BSA and add it to 2 mL of carbonate buffer solution (the dissolved BSA protein is called solution B). At room temperature, add solution A dropwise to solution B. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution and react overnight at room temperature to obtain the conjugate 5-ROX-EDC-BSA. The conjugation method for 5-ROX-EDC-OVA is similar to the above method.

[0061] 2. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 minutes and cool it, then store it in deionized water at 4℃ for later use; put the 5-ROX-EDC-BSA conjugate into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours, and dialyze for 3 days to obtain the complete antigen 5-ROX-EDC-BSA, which should be taken out and stored at -20℃.

[0062] The preparation process and structure of the complete antigen 5-ROX-EDC-BSA / OVA are shown below:

[0063] Example 2: Immunization in mice

[0064] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg / mouse of the complete 5-carboxy-X-rhodamine antigen (5-ROX-EDC-BSA) and an equal volume of complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites on the back of the neck. Four weeks later, a booster immunization was performed with half the dose of the complete antigen (50 μg / mouse), emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were administered at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and injected intraperitoneally. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused.

[0065] Example 3: Cell Fusion and Screening

[0066] (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 4000 (polyethylene glycol) method. The specific steps are as follows:

[0067] a. Collection of SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion should reach 1-4 × 10⁻⁴ cells / year. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0068] b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use.

[0069] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 dropwise to the cells, gradually increasing the speed. At min 2, allow the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 r / min for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to the medium. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0070] (2) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. The screening was carried out in two steps: first, positive cell wells were screened by ic-ELISA; second, the inhibitory effect of 5-carboxy-X-rhodamine standard was measured by ic-ELISA on the positive cells. Cell wells that showed good inhibition of 5-carboxy-X-rhodamine standard were selected and subcloned using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the 5-carboxy-X-rhodamine monoclonal antibody cell line FPF.

[0071] Example 4: Preparation and Identification of Monoclonal Antibodies

[0072] I. Sensitivity Experiment

[0073] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 2 × 10⁻⁶ g of paraffin oil. 6 Ascites fluid was collected from 5-carboxy-X-rhodamine hybridoma cells starting on day 7. The ascites fluid was then purified for antibody using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded after centrifugation. Next, an equal volume of saturated ammonium sulfate solution was used to precipitate IgG-type monoclonal antibodies. After centrifugation and discarding the supernatant, the antibodies were dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0074] (1) Coating: The original 5-ROX-EDC-OVA (prepared in the same way as 5-ROX-EDC-BSA, see Example 1) was diluted 3 times from 1 µg / mL with 0.05 M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0075] (2) Washing: Pour out the solution in the plate and wash with washing solution 3 times, 3 min each time.

[0076] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0077] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0078] (5) Color development: Take out the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0079] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0080] The IC50 of the monoclonal antibody 5-carboxy-X-rhodamine was determined by ic-ELISA. 50 The concentration was 0.1 ng / mL, indicating good sensitivity to 5-carboxy-X-rhodamine, making it suitable for immunoassay detection of 5-carboxy-X-rhodamine.

[0081] II. Specificity Tests

[0082] The IC50 of the monoclonal antibody against 5-carboxy-X-rhodamine was determined using an indirect competitive ELISA method. 50 The concentration was 0.1 ng / mL, and its IC50 for compounds such as sulforhodamine 101, 6-carboxy-X-rhodamine, and rhodamine 101 chloride was verified. 50 The cross-reactivity rate and cross-reactivity value are calculated as follows:

[0083] (IC50 of 5-carboxy-X-rhodamine) 50 IC of other compounds 50 () × 100%, as shown in Table 1.

[0084] Table 1. IC50 of monoclonal antibodies against 5-carboxy-X-rhodamine and analogues50 and cross-reactivity

[0085] Compound Name <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity 5-Carboxy-X-Rhodamine 0.1 100% 6-Carboxy-X-Rhodamine 0.1 100% Rhodamine chloride 101 0.1 100% Sulforodamine 101 10 1%

[0086] The monoclonal antibody prepared in this study exhibited 100% reactivity towards 5-carboxy-X-rhodamine, demonstrating high affinity and sensitivity to the target antigen. Cross-reactivity analysis showed that the antibody also possessed some recognition ability for structurally similar compounds to 5-carboxy-X-rhodamine, with a 100% cross-reactivity rate towards 6-carboxy-X-rhodamine and rhodamine chloride 101, and a 1% cross-reactivity rate towards sulforhodamine 101. These results suggest that the antibody can effectively recognize the main structure of rhodamine molecules and still exhibits some cross-binding activity towards derivatives with different substituents, indicating good structural selectivity and application specificity.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line that secretes monoclonal antibodies against 5-carboxy-X-rhodamine and its analogues, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46730, and the analogue is 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

2. The use of the hybridoma cell line of claim 1 in the preparation of monoclonal antibodies against 5-carboxy-X-rhodamine and its analogues.

3. A monoclonal antibody against 5-carboxy-X-rhodamine and its analogues, characterized in that: The monoclonal antibody is prepared from the hybridoma cell line described in claim 1.

4. The monoclonal antibody according to claim 3, characterized in that: The analogues are 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

5. The use of the hybridoma cell line of claim 1 or the monoclonal antibody of any one of claims 3-4 in the preparation of detection products for 5-carboxy-X-rhodamine and its analogues, characterized in that, The analogues are 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

6. A detection product for 5-carboxy-X-rhodamine and similar substances, characterized in that: The test product contains any of the monoclonal antibodies described in claims 3-4, and the analogue is 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

7. The testing product according to claim 6, characterized in that: The testing products include test strips, reagent kits, or biochips.

8. The testing product according to claim 7, characterized in that: The test strip includes a sample pad, a conjugate pad, an absorbent pad, and a cellulose acetate membrane. The conjugate pad is coated with a monoclonal antibody, which is labeled with colloidal gold or fluorescent microspheres. The kit includes an ELISA plate, a coated antigen, a 5-carboxy-X-rhodamine and analogue standard solution, an enzyme-labeled secondary antibody, and a colorimetric solution.

9. The use of the hybridoma cell line of claim 1, the monoclonal antibody of any one of claims 3-4, or the detection product of any one of claims 6-8 in the detection of 5-carboxy-X-rhodamine and its analogues, characterized in that, The analogues are 6-carboxy-X-rhodamine, rhodamine chloride 101, or sulforhodamine 101.

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