Hybridoma cell strain secreting cy3.5 monoclonal antibody and application thereof

Abstract

This invention relates to a hybridoma cell line that secretes a Cy3.5 monoclonal antibody and its application, belonging to the field of immunoassay. The hybridoma cell line of this invention was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46741. The Cy3.5 monoclonal antibody secreted by this cell line exhibits excellent affinity and sensitivity to Cy3.5, with an IC50 of [missing value]. 50 Reaching 10 ng / mL, the IC50 for Cy5.5 50 An immunoassay product based on this hybridoma cell line, with a concentration of up to 50 ng / mL, can be used for the detection of Cy3.5 and its analogues, providing an efficient detection method and means for the residual detection of Cy3.5.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more particularly to a hybridoma cell line that secretes Cy3.5 monoclonal antibody and its applications. Background Technology

[0002] Fluorescent labeling technology is a biotechnology that binds fluorescent substances to target molecules, cellular structures, or biological particles, enabling visual detection and analysis through fluorescence signals. Its core principle is that when a labeled substance is irradiated with excitation light of a specific wavelength, it undergoes a transition and releases fluorescence of a characteristic wavelength. This signal can be precisely captured using equipment such as fluorescence microscopes and flow cytometers. Commonly used labels include Cy series dyes, rhodamine series dyes, and fluorescent quantum dots. This technology combines high sensitivity, high specificity, and real-time visualization, and is widely used in cell imaging, molecular interaction analysis, immunoassay, and nucleic acid quantification, making it one of the core tools in biomedical and molecular biology research. Cy series dyes are commonly used near-infrared organic fluorescent dyes in fluorescent labeling technology. With their broad excitation spectrum, narrow emission spectrum, high fluorescence intensity, and superior resistance to photobleaching compared to traditional dyes such as FITC, they have become a core tool in the field of biological detection.

[0003] Cy3.5 is an important subtype of Cy-series organic fluorescent dyes, belonging to the orange-red fluorescence band. Its excitation wavelength is approximately 581 nm, and its emission wavelength is approximately 594 nm. Its spectral characteristics fall between Cy3 and Cy5, effectively avoiding interference from the autofluorescence of biological samples. It features high fluorescence quantum yield and superior photobleaching resistance compared to traditional rhodamine dyes. It is often covalently coupled to biomolecules such as antibodies, nucleic acid probes, and peptides through active groups (such as N-hydroxysuccinimide ester). Its core applications are concentrated in immunofluorescence detection, flow cytometry, and fluorescence in situ hybridization (FISH). It is particularly suitable for use in combination with Cy3 and Cy5 in multicolor fluorescent labeling experiments to achieve simultaneous imaging of multiple targets.

[0004] Cy5.5 is a Cy-series near-infrared fluorescent dye with an excitation wavelength of approximately 678 nm and an emission wavelength of approximately 694 nm. Located within the low-absorption near-infrared window of biological tissues, it exhibits strong light penetration and extremely low background fluorescence. It demonstrates excellent fluorescence stability, significantly superior resistance to photobleaching compared to Cy5, and strong chemical modifiability, enabling efficient conjugation with antibodies, aptamers, and nanocarriers.

[0005] Cy series dyes are cyanine fluorescent dyes that bind to nucleic acid probes or fragments through covalent labeling or non-covalent binding, generating specific fluorescent signals under excitation light. The detection of Cy dyes essentially represents source control of the entire nucleic acid detection process: firstly, quality control across the entire chain from dye synthesis to labeling application ensures the reliability of each step; secondly, it reduces experimental failures and misinterpretations due to dye quality issues, lowering research and clinical risks; and finally, it ensures the comparability of experimental results from different laboratories and batches, promoting the standardization of detection. Current detection methods for Cy series dyes (such as Cy3, Cy3.5, and Cy5.5) are mainly instrumental, such as UV-Vis spectrophotometry, high-performance liquid chromatography, and Raman spectroscopy. These methods are easily affected by background fluorescence or sample matrix interference, lack the ability to distinguish between different structural isomers or substituent differences, are expensive, not portable, require specialized personnel, and lack rapid detection capabilities.

[0006] Therefore, developing a monoclonal antibody or related immunoassay method that can highly specifically recognize the above-mentioned Cy dyes is of great significance for improving the analytical accuracy of labeled products, establishing a stable and reproducible detection system, and expanding its applications. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a Cy3.5 monoclonal antibody and its applications. This invention provides a Cy3.5 hapten and a hybridoma cell line that secretes a Cy3.5 monoclonal antibody. The prepared product is used for research on Cy3.5 immunoassay methods, providing an essential artificial antigen for future research. The antibody prepared from this cell line exhibits good specificity and detection sensitivity for Cy3.5 and can be used to establish immunological detection methods for Cy3.5 and its analogues.

[0008] This invention is achieved through the following technical solution:

[0009] The first objective of this invention is to provide a hybridoma cell line that secretes a Cy3.5 monoclonal antibody. This hybridoma cell line was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46741.

[0010] In one embodiment of the present invention, the hybridoma cell line is obtained by immunizing animals with an immunogen obtained by conjugating Cy3.5 hapten with a carrier protein.

[0011] In one embodiment of the present invention, the structural formula of the Cy3.5 hapten is as follows:

[0012] .

[0013] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin and / or chicken oocyte albumin;

[0014] And / or, the animals include mice.

[0015] A second objective of this invention is to provide a Cy3.5 monoclonal antibody secreted by the hybridoma cell line.

[0016] A third object of the present invention is to provide a composition comprising the hybridoma cell line and / or the Cy3.5 monoclonal antibody.

[0017] A fourth objective of this invention is to provide a kit comprising one or more of the hybridoma cell line, the Cy3.5 monoclonal antibody, and the composition described herein.

[0018] A fifth objective of this invention is to provide a test strip comprising one or more of the hybridoma cell line, the Cy3.5 monoclonal antibody, and the composition described herein.

[0019] A sixth objective of this invention is to provide a chip comprising one or more of the hybridoma cell line, the Cy3.5 monoclonal antibody, and the composition described herein.

[0020] A seventh objective of this invention is to provide the application of the hybridoma cell line, the Cy3.5 monoclonal antibody, the composition, the kit, the test strip, or the chip in the detection of Cy3.5 or Cy5.5; the application does not involve the diagnosis or treatment of diseases.

[0021] The eighth object of the present invention is to provide a method for preparing a hybridoma cell line that secretes monoclonal antibodies against Cy3.5 and its analogues, comprising the following steps:

[0022] S1. Prepare a complete antigen from the Cy3.5 hapten, and use the complete antigen to immunize animals;

[0023] S2. Blood samples are collected from immunized animals to screen for serum immunogenicity and immunosuppressive capacity.

[0024] S3. The spleen cells and myeloma cells of the selected immunized animals are fused and cultured to obtain the hybridoma cell line that secretes Cy3.5 monoclonal antibody.

[0025] In one embodiment of the present invention, 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.

[0026] In one embodiment of the present invention, the animal is a mouse.

[0027] In one embodiment of the present invention, in step S1, the structure of the Cy3.5 hapten is as follows:

[0028] .

[0029] In one embodiment of the present invention, in step S1, the Cy3.5 complete antigen is obtained by conjugating the above-mentioned Cy3.5 hapten with a carrier protein.

[0030] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin (KLH), ovalbumin (OVA), etc.

[0031] In one embodiment of the present invention, the Cy3.5 complete antigen is coupled to a carrier protein via a carbodiimide method. The specific preparation method includes the following steps:

[0032] (1) Activate the above Cy3.5 hapten to obtain an activation solution;

[0033] (2) Add the activation solution obtained in step (1) above into the carrier protein solution and react to obtain Cy3.5 complete antigen.

[0034] In one embodiment of the present invention, in step (1), the activation is to dissolve the Cy3.5 hapten, add 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to react.

[0035] In one embodiment of the present invention, N,N-dimethylformamide is used to dissolve the Cy3.5 hapten.

[0036] In one embodiment of the present invention, in step (2), the solution after the reaction is dialyzed and separated to obtain the Cy3.5 complete antigen.

[0037] In one embodiment of the present invention, in step (3), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0038] In one embodiment of the present invention, the carbonate buffer solution has a concentration of 0.01-0.5 mol / L (preferably 0.05 mol / L) and a pH of 8.0-10.0 (preferably 9.6).

[0039] In one embodiment of the present invention, in step S2, serum immunotiter and immunosuppressive capacity are detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0040] In one embodiment of the present invention, the above-mentioned Cy3.5 detection kit is used for residual analysis and detection of Cy3.5 in biological reagents (such as nucleic acid detection technology).

[0041] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0042] This invention provides a hybridoma cell line that secretes a Cy3.5 monoclonal antibody and its applications. The Cy3.5 hapten synthesis procedure provided by this invention is simple and efficient, and can be fully utilized in immunoassays, providing a convenient approach for future research. The complete antigen prepared based on this hapten, the hybridoma cell line obtained by immunizing mice with the complete antigen, and the monoclonal antibody secreted by it exhibit good specificity and detection sensitivity for Cy3.5 (IC5 of the Cy3.5 monoclonal antibody against Cy3.5 is [value missing]). 50 With a concentration of 10 ng / mL, this invention enables the detection of Cy3.5 residues, providing an immunoassay method and raw materials for the immunoassay of Cy3.5 residues in biological agents, and has practical application value. In addition, the monoclonal antibody of this invention exhibits an IC50 value for Cy5.5. 50 It also has good detection sensitivity, reaching 50 ng / mL.

[0043] Preservation of biological materials:

[0044] A hybridoma cell line, YLG, secreting a Cy3.5 monoclonal antibody, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCC No. 46741. Attached Figure Description

[0045] 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.

[0046] Figure 1 The standard inhibition curve of the Cy3.5 monoclonal antibody prepared for the embodiments of the present invention;

[0047] Figure 2 The standard inhibition curve of Cy3.5 monoclonal antibody prepared for the embodiments of the present invention against Cy5.5 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 Cy3.5-carboxylic acid, a structural analog of Cy3.5, as a hapten. The hapten is conjugated to a carrier protein using the carbodiimide method, and the success of the conjugation is determined using a UV spectrophotometer. For the initial immunization, 100 μg of complete Cy3.5 antigen is emulsified with an equal volume of complete Freund's adjuvant and administered via multiple subcutaneous injections at the nape of the neck to BALB / c mice. Multiple booster immunizations (50 μg / mouse) are administered using incomplete Freund's adjuvant. The final immunization is performed via intraperitoneal injection of 25 μg of complete Cy3.5 antigen (diluted with physiological saline and without adjuvant). 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 from the three cell lines were selected using selective culture medium. These cells were then further screened using an indirect competitive enzyme-linked immunosorbent assay (ELISA) and subjected to four subcloning processes to obtain a monoclonal antibody hybridoma cell line. The monoclonal antibody secreted by this cell line exhibited good specificity and detection sensitivity (IC50) for Cy3.5. 50 With a concentration of 10 ng / mL, this invention enables the detection of Cy3.5 residues in Cy-based dyes used as biological agents, providing a raw material for the immunoassay of Cy3.5 residues in nucleic acid detection reagents and possessing practical application value. This invention also successfully synthesized a Cy3.5 artificial antigen; the synthesis steps are simple and effective, and it can be fully utilized in immunoassays, providing an essential artificial antigen for future research.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0051] (1) The culture media involved in the following examples are as follows:

[0052] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0053] (2) The reagents involved in the following examples are as follows:

[0054] 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.

[0055] 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.

[0056] PBST: PBS containing 0.05% Tween 20;

[0057] 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.

[0058] (3) The detection methods involved in the following embodiments are as follows:

[0059] Cy3.5 inhibition rate detection method: The optimal antigen and antibody concentrations for icELISA were selected using a checkerboard assay. The antigen was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, Cy3.5 standards were diluted to concentrations of 100 ng / mL, 33.33 ng / mL, 11.11 ng / mL, 3.70 ng / mL, 1.23 ng / mL, 0.41 ng / mL, and 0.13 ng / mL, following the icELISA procedure. Finally, Origin 2024 was used to plot the Cy3.5 standard inhibition curve, and the IC50 was calculated. 50 .

[0060] Cy5.5 inhibition rate detection method: The optimal antigen and antibody concentrations for icELISA were selected using a checkerboard assay. The antigen was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, Cy5.5 standards were diluted to concentrations of 100 ng / mL, 33.33 ng / mL, 11.11 ng / mL, 3.70 ng / mL, 1.23 ng / mL, 0.41 ng / mL, and 0.13 ng / mL, following the icELISA procedure. Finally, Origin 2024 was used to plot the Cy5.5 standard inhibition curve, and the IC50 was calculated. 50 .

[0061] Example 1: Preparation of complete antigen

[0062] The Cy3.5 artificial antigen is obtained by conjugating the hapten Cy3.5-COOH (purchased from Duoying Biotechnology, catalog number D10128-25) to a carrier protein using the carbodiimide method. Specifically, the hapten Cy3.5-COOH can be conjugated with keyhole cyanin (KLH) to obtain the complete antigen Cy3.5-EDC-KLH; or conjugated with chicken ovalbumin (OVA) to obtain the complete antigen Cy3.5-EDC-OVA.

[0063] The preparation method of the complete antigen Cy3.5-EDC-KLH is as follows:

[0064] a. Weigh 1.78 mg of the hapten Cy3.5-COOH, 1.035 mg of N-hydroxysuccinimide, and 1.72 mg of 1-ethylcarbodiimide hydrochloride, and dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of KLH (Cy3.5-COOH to KLH molar ratio of 2000:1), and add 2 mL of carbonate buffer solution (the dissolved KLH protein is referred to as 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 Cy3.5-EDC-KLH. The conjugation method for Cy3.5-EDC-OVA is similar to the above method.

[0065] b. 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 conjugate Cy3.5-EDC-KLH / OVA 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 Cy3.5-EDC-KLH / OVA, which should be taken out and stored at -20℃.

[0066] Example 2 Immunization in mice

[0067] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg of Cy3.5 complete antigen 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 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.

[0068] Example 3 Cell Fusion and Screening

[0069] (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:

[0070] 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. 7To 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.

[0071] 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.

[0072] 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.

[0073] (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 using ic-ELISA; second, Cy3.5 standard was selected, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of Cy3.5 standard were selected, and subcloning was performed 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 Cy3.5 monoclonal antibody cell line.

[0074] Example 4: Preparation and Identification of Monoclonal Antibodies

[0075] 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. 6Cy3.5 hybridoma cells were used, and ascites fluid was collected starting from 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 by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. 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.

[0076] (1) Coating: The original Cy3.5-EDC-OVA was diluted 3-fold from 1 µg / mL with 0.05 M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0077] (2) Washing: Pour out the solution in the plate and wash with washing solution 3 times, 3 min each time.

[0078] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0079] (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.

[0080] (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.

[0081] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using an ELISA reader.

[0082] The IC50 of monoclonal antibody Cy3.5 was determined by ic-ELISA. 50 The value was 10 ng / mL, indicating good sensitivity to Cy3.5, and it can be used for Cy3.5 immunoassay detection.

[0083] Example 5 Specificity Experiment

[0084] The IC50 of the monoclonal antibody against Cy3.5 was determined using an indirect competitive ELISA method. 50 The concentration was 10 ng / mL (standard inhibition curve shown in figure). Figure 1 As shown), for Cy5.5 IC 50 The concentration was 50 ng / mL (standard inhibition curve shown in figure). Figure 2 (as shown), and verified its IC50 for compounds such as Cy3, Cy5, Cy7, Cy7.5, and X-rhodamine. 50 The cross-reactivity rate and cross-reactivity value are calculated as follows:

[0085] (Cy3.5 IC) 50 IC of other compounds 50 () × 100%, as shown in Table 1.

[0086] Table 1. IC50 of Cy3.5 monoclonal antibody against Cy3.5 and its cross-links 50 and cross-reactivity

[0087]

[0088] The monoclonal antibody showed 100% cross-linking to Cy3.5 and 20% cross-linking to Cy5.5, with cross-linking to Cy3, Cy5, Cy7, Cy7.5, and X-rhodamine all less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity (IC50) to Cy3.5. 50 It has a value of 10 ng / mL and also exhibits high specificity (cross-reactivity with analogs is less than 1%).

[0089] 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 a Cy3.5 monoclonal antibody, characterized in that: The hybridoma cell line was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46741.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with an immunogen derived from the conjugation of Cy3.5 hapten with a carrier protein.

3. The hybridoma cell line according to claim 2, characterized in that, The structural formula of the Cy3.5 hapten is shown below: 。 4. The hybridoma cell line according to claim 2, characterized in that, The carrier proteins include keyhole hemocyanin and / or chicken oocyte albumin; And / or, the animals include mice.

5. A Cy3.5 monoclonal antibody, characterized in that, Produced by the hybridoma cell line described in any one of claims 1-4.

6. A composition, characterized in that, The composition comprises the hybridoma cell line of any one of claims 1-4 and / or the Cy3.5 monoclonal antibody of claim 5.

7. A reagent kit, characterized in that, The kit comprises one or more of the hybridoma cell lines of any one of claims 1-4, the Cy3.5 monoclonal antibody of claim 5, and the composition of claim 6.

8. A test strip, characterized in that, The test strip comprises one or more of the hybridoma cell lines of any one of claims 1-4, the Cy3.5 monoclonal antibody of claim 5, and the composition of claim 6.

9. A chip, characterized in that, The chip comprises one or more of the hybridoma cell lines of any one of claims 1-4, the Cy3.5 monoclonal antibody of claim 5, and the composition of claim 6.

10. The use of the hybridoma cell line of any one of claims 1-4, the Cy3.5 monoclonal antibody of claim 5, the composition of claim 6, the kit of claim 7, the test strip of claim 8, or the chip of claim 9 in the detection of Cy3.5 or Cy5.5; the use does not relate to the diagnosis and treatment of disease.

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