Hybridoma cell strain secreting tetramethyl rhodamine monoclonal antibody and application of hybridoma cell strain

Abstract

The invention provides a hybridoma cell strain capable of secreting a tetramethyl rhodamine monoclonal antibody and application of the hybridoma cell strain, and relates to the technical field of immunochemistry. According to the invention, tetramethyl rhodamine and protein are connected to prepare a complete antigen, and a hybridoma cell strain secreting a monoclonal antibody is obtained through mouse immunization and cell screening, and the preservation number is CGMCC NO.46520. The monoclonal antibody secreted by the cell strain has good detection sensitivity and specificity to tetramethyl rhodamine, the IC50 value to tetramethyl rhodamine is 1.79 ng / mL, and the cross reaction rate to various tetramethyl rhodamine structural analogues is lt; therefore, a foundation is established for immunodetection of tetramethyl rhodamine, and the tetramethyl rhodamine immunodetection kit has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of immunochemical technology, and in particular to a hybridoma cell line that secretes a tetramethylrhodamine monoclonal antibody and its application. Background Technology

[0002] Tetramethylrhodamine is a fluorescent dye widely used in scientific research and diagnostics, commonly employed in experiments such as cell labeling, molecular detection, and substance tracking. However, with its increasing applications in environmental monitoring, drug research, and diagnostics, the problem of tetramethylrhodamine residues has become a growing concern. Tetramethylrhodamine itself possesses strong fluorescence properties, emitting significant light signals under ultraviolet light. Although it is generally non-toxic, its residues in certain experiments or production processes may have potential impacts on the environment, food, and human health. Studies have shown that long-term accumulation of tetramethylrhodamine may have negative effects on certain biological systems, particularly in terms of cytotoxicity and potential allergic reactions. For example, in some experiments, tetramethylrhodamine may interfere with cell structure and function, thereby affecting the accuracy of experimental results. Therefore, timely and accurate detection of tetramethylrhodamine residues has become an important task in environmental monitoring and biosafety.

[0003] To mitigate the negative impacts of tetramethylrhodamine residues on human health and the environment, many countries and regions have implemented strict regulations on its residue limits. Despite this, current methods for detecting tetramethylrhodamine residues still primarily rely on traditional instrumental analytical techniques, such as high-performance liquid chromatography (HPLC), fluorescence spectroscopy, and LC-MS / MS. While these instrumental methods offer high sensitivity and specificity, they also have drawbacks, including the need for complex sample pretreatment, expensive equipment, cumbersome operation, and highly specialized technical personnel.

[0004] To address these issues, there is an urgent need to develop rapid, simple, low-cost methods suitable for on-site testing. Enzyme-linked immunosorbent assay (ELISA), as a commonly used immunoassay method, has been widely applied in fields such as food safety and environmental monitoring due to its ease of operation, rapid detection speed, and low cost.

[0005] The key to detecting tetramethylrhodamine residues using ELISA lies in obtaining highly specific and sensitive monoclonal antibodies. Monoclonal antibodies can specifically bind to tetramethylrhodamine molecules, thus achieving efficient and sensitive detection. The preparation of monoclonal antibodies against tetramethylrhodamine typically requires hybridoma technology. Although there have been some advancements in existing technologies, successfully preparing monoclonal antibodies with high specificity and sensitivity remains a challenge in this field. Key issues include how to effectively design and prepare tetramethylrhodamine antigens, how to induce a strong immune response in mice through immunogenicity, and how to ensure that the final antibody exhibits good specificity and high sensitivity during detection. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention prepares a complete antigen by linking tetramethylrhodamine to a protein, and obtains a hybridoma cell line that secretes a monoclonal antibody of tetramethylrhodamine through mouse immunization and cell screening, with the accession number CGMCC NO.46520.

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes a tetramethylrhodamine monoclonal antibody, the hybridoma cell line having the accession number CGMCC NO.46520.

[0008] Furthermore, the method for preparing the hybridoma cell line includes the following steps:

[0009] Step 1: Prepare tetramethylrhodamine complete antigen. Emulsify the obtained tetramethylrhodamine complete antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen.

[0010] Step 2: The obtained immunogen was injected subcutaneously into BALB / c mice via the back for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0011] Step 3: Collect blood from mice that have undergone the above immunization process, and detect the serum immune titer and immunosuppressive ability of mice by indirect ELISA to screen out mice with high sensitivity of tetramethylrhodamine antibody in serum.

[0012] Step 4: The selected mice were immunized by intraperitoneal injection using tetramethylrhodamine complete antigen without Freund's adjuvant.

[0013] Step 5: Fuse spleen cells and myeloma cells from BALB / c mice after sprint immunization. The fused cells are then screened and cultured in HAT medium. Positive cell pores are detected using indirect ELISA, and the inhibitory effect of positive cell pores is further determined using indirect competitive ELISA. The positive cell pores with the best inhibition are subcloned using limiting dilution, and finally, hybridoma cell lines that secrete highly sensitive tetramethylrhodamine monoclonal antibodies are screened to obtain hybridoma cell lines.

[0014] Furthermore, in step 2, there is a one-month interval between the initial immunization and the booster immunization, and a 21-day interval between booster immunizations.

[0015] Furthermore, in step 4, there is an interval of 18-21 days between the booster immunization and the sprint immunization.

[0016] Furthermore, the initial immunization dose in step 2 is 100 μg / animal, and the booster immunization dose is 50 μg / animal.

[0017] Furthermore, in step 4, the sprint immunization dose is 25 μg / animal.

[0018] Furthermore, the immunization process in steps 2 and 4 includes one initial immunization, four booster immunizations, and one sprint immunization.

[0019] Furthermore, the blood collection in step 3 is performed on the 7th day after the completion of the 3rd immunization process.

[0020] Furthermore, the cell fusion in step 5 was performed 3 days after the sprint immunization ended.

[0021] Furthermore, the cell fusion in step 5 is performed using the polyethylene glycol (PEG4000) method.

[0022] Furthermore, the culture medium in step 5 is RPMI-1640 medium.

[0023] Furthermore, the number of subcloning operations in step 5 is 4.

[0024] A second objective of this invention is to provide a tetramethylrhodamine complete antigen, the structural formula of which is shown below:

[0025] .

[0026] A third objective of this invention is to provide the use of the above-mentioned hybridoma cell line or the above-mentioned tetramethylrhodamine complete antigen in the preparation of tetramethylrhodamine monoclonal antibodies.

[0027] A fourth objective of this invention is to provide a tetramethylrhodamine monoclonal antibody, which is obtained from the above-mentioned hybridoma cell line.

[0028] Furthermore, the preparation method of the tetramethylrhodamine monoclonal antibody includes the following steps:

[0029] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, each mouse was injected intraperitoneally with 1×10 6 Ascites was collected from tetramethylrhodamine monoclonal antibody hybridoma cells starting from day 7. The ascites was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method. The purified monoclonal antibody was stored at -20°C.

[0030] A fifth object of the present invention is to provide a composition comprising the above-described hybridoma cell line or the above-described tetramethylrhodamine monoclonal antibody.

[0031] The sixth object of the present invention is to provide the application of the above-mentioned hybridoma cell line or the above-mentioned tetramethylrhodamine monoclonal antibody in the preparation of tetramethylrhodamine detection products.

[0032] Furthermore, the tetramethylrhodamine detection products include test strips, reagent kits, or biochips.

[0033] A seventh object of the present invention is to provide a tetramethylrhodamine detection kit comprising the above-mentioned tetramethylrhodamine monoclonal antibody.

[0034] Furthermore, the tetramethylrhodamine detection kit also includes an ELISA plate, blocking solution, diluent, chromogenic agent, and stop solution.

[0035] Furthermore, the tetramethylrhodamine detection kit also includes a tetramethylrhodamine-coated antigen, the structural formula of which is shown below:

[0036] .

[0037] An eighth object of the present invention is to provide the use of the above-mentioned hybridoma cell line, the above-mentioned tetramethylrhodamine monoclonal antibody, the above-mentioned composition, or the above-mentioned tetramethylrhodamine detection kit in the detection of tetramethylrhodamine.

[0038] The beneficial effects of this invention are:

[0039] The monoclonal antibody secreted by the hybridoma cell line provided by this invention has good detection sensitivity and specificity for tetramethylrhodamine, and its IC50 value for tetramethylrhodamine is [not specified]. 50With a value of 1.79 ng / mL and a cross-reactivity rate of <0.1% for various tetramethylrhodamine structural analogs, it lays the foundation for the immunoassay of tetramethylrhodamine and can be used to prepare a series of tetramethylrhodamine detection products. The obtained ELISA detection kit has a recovery rate of 98.2-106% for tetramethylrhodamine in the test samples, showing good application prospects and important significance for multiple fields such as environmental monitoring, food safety, and drug research. Preservation of biological materials

[0040] A hybridoma cell line secreting tetramethylrhodamine monoclonal antibody has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line, deposited on April 17, 2025, with accession number CGMCC NO.46520. Attached Figure Description

[0041] 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:

[0042] Figure 1 The standard inhibition curve of tetramethylrhodamine monoclonal antibody against tetramethylrhodamine provided by this invention;

[0043] Figure 2 This study validates the sensitivity and specificity of the tetramethylrhodamine monoclonal antibody provided by this invention. Detailed Implementation

[0044] 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.

[0045] The culture media involved in the following examples are as follows:

[0046] 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.

[0047] The reagents involved in the following examples are as follows:

[0048] 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.

[0049] Phosphate buffered saline (PBS): 8.00 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.

[0050] PBST: PBS containing 0.05% Tween 20;

[0051] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0052] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, 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 again before use.

[0053] The detection methods involved in the following embodiments are as follows:

[0054] The method for detecting the inhibition rate of tetramethylrhodamine (TMR) was as follows: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the TMR standard was diluted to eight concentrations (0, 0.071, 0.21, 0.64, 1.94, 5.82, 17.46, and 52.38 ng / mL). Following the ic-ELISA procedure, the inhibition curves were plotted using OriginPro 8.5, and the IC50 was calculated. 50 .

[0055] Example 1: Synthesis of Tetramethylrhodamine Complete Antigen

[0056] Since tetramethylrhodamine is a small molecule that is not immunogenic and cannot stimulate an immune response in mice to produce antibodies, it is necessary to use protein conjugation technology to couple tetramethylrhodamine to proteins to give it immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. Given that the tetramethylrhodamine molecule contains the active carboxyl group, a design was made to directly link tetramethylrhodamine to proteins to prepare a complete antigen.

[0057] Weigh 2.41 mg of tetramethylrhodamine and 2.07 mg of N-hydroxysuccinimide (NHS), dissolve them in 200 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 3.44 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the tetramethylrhodamine solution. Stir at room temperature for 6-8 h to activate the antigen. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), and dissolve it completely. Slowly add the activated tetramethylrhodamine to the diluted KLH solution and stir overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by UV absorption scanning.

[0058] The structural formula of the obtained tetramethylrhodamine complete antigen is shown below:

[0059] .

[0060] Example 2: Synthesis of Tetramethylrhodamine-coated antigen

[0061] 3.19 mg of tetramethylrhodamine and 1.1 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with the solution at room temperature for 10 min. 4.55 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with the solution at room temperature for 6–8 h to obtain the activated tetramethylrhodamine solution. 6 mg of bovine serum albumin (BSA) was dissolved in carbonate buffer (CBS). The activated tetramethylrhodamine solution was slowly added to the protein dilution buffer and stirred overnight at room temperature. The reaction solution was then dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.

[0062] The structural formula of the obtained tetramethylrhodamine-coated precursor is shown below:

[0063] .

[0064] Example 3: Preparation of hybridoma cell lines secreting tetramethylrhodamine monoclonal antibody

[0065] 1. Acquisition of immunity in animals

[0066] Tetramethylrhodamine complete antigen was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; subsequent booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse); sprint immunizations did not use adjuvant, but were directly diluted with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between the sprint immunization and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), which detected the serum titer and inhibition.

[0067] 2. Cell fusion

[0068] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0069] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0070] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0071] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate at 37°C in a 5% CO2 incubator.

[0072] 3. Cell selection and cell line establishment

[0073] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0074] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, tetramethylrhodamine is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0075] Cell wells that showed good inhibition of tetramethylrhodamine standard were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0076] At least three subcloning operations were performed using the method described above to finally obtain a tetramethylrhodamine monoclonal antibody cell line.

[0077] Example 4: Preparation and Identification of Tetramethylrhodamine Monoclonal Antibody

[0078] 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 1×10 6 Tetramethylrhodamine hybridoma cells were used to collect ascites fluid starting from day 7. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method.

[0079] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibody is dissolved in 0.01M PBS solution (pH=7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0080] Using an indirect competitive ELISA, the tetramethylrhodamine monoclonal antibody was found to have good detection sensitivity and specificity (IC50) for tetramethylrhodamine. 50 With a value of 1.79 ng / mL, it can be used for the immunoassay of tetramethylrhodamine.

[0081] Cross-reactivity experiments were performed on tetramethylrhodamine structural analogs (tetramethylrhodamine, rhodamine 110, rhodamine R6G, rhodamine B, and sulfonylrhodamine B). The results showed that the absorbance values ​​in each well of the ELISA plate corresponding to rhodamine 110, rhodamine R6G, rhodamine B, and sulfonylrhodamine B did not differ significantly with the dilution concentration of the standard. Specific results are as follows: Figure 2 As shown.

[0082] These results indicate that the monoclonal antibody has a low cross-reactivity with other structural analogs and good specificity for tetramethylrhodamine.

[0083] Cross-reactivity rate (%) = (Tetramethylrhodamine IC50) 50 ) / (Similar IC 50 )×100%

[0084] Example 5: Application of Tetramethylrhodamine Monoclonal Antibody

[0085] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was applied to a tetramethylrhodamine ELISA addition and recovery assay. The specific steps are as follows:

[0086] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0087] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0088] (3) Prepare tetramethylrhodamine standard solutions of 0, 0.071, 0.21, 0.64, 1.94, 5.82, 17.46 and 52.38 ng / mL using phosphate buffer (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of tetramethylrhodamine monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0089] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37℃ for 30 min, then wash the plate and pat dry.

[0090] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0091] (6) Addition of recovery and sample pretreatment:

[0092] Wastewater was selected as the test sample.

[0093] After centrifugation, the test samples were passed through a 20-mesh standard sieve. Three 20 mL aliquots were taken and added to each sample at concentrations of 5 ppb, 10 ppb, and 20 ppb of tetramethylrhodamine standard (based on the antibody linear range and IC50). 50 Set the concentration to be added), then vortex to mix.

[0094] The recovery rate of tetramethylrhodamine was 98.2-106% when spiking was performed using an indirect competitive ELISA.

[0095] 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 tetramethylrhodamine monoclonal antibody, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46520.

2. A tetramethylrhodamine complete antigen, characterized in that, The structural formula of the tetramethylrhodamine complete antigen is shown below: 。 3. The use of the hybridoma cell line of claim 1 or the tetramethylrhodamine complete antigen of claim 2 in the preparation of tetramethylrhodamine monoclonal antibody.

4. A tetramethylrhodamine monoclonal antibody, characterized in that: The tetramethylrhodamine monoclonal antibody is obtained from the hybridoma cell line described in claim 1.

5. A composition, characterized in that: The composition comprises the hybridoma cell line of claim 1 or the tetramethylrhodamine monoclonal antibody of claim 4.

6. The use of the hybridoma cell line of claim 1 or the tetramethylrhodamine monoclonal antibody of claim 4 in the preparation of tetramethylrhodamine detection products.

7. The application according to claim 6, characterized in that: The tetramethylrhodamine detection products include test strips, kits, or biochips.

8. A tetramethylrhodamine detection kit, characterized in that: The tetramethylrhodamine detection kit contains the tetramethylrhodamine monoclonal antibody as described in claim 4.

9. The tetramethylrhodamine detection kit according to claim 8, characterized in that: The tetramethylrhodamine detection kit also includes an ELISA plate, blocking solution, diluent, chromogenic agent, and stop solution.

10. The use of the hybridoma cell line of claim 1, the tetramethylrhodamine monoclonal antibody of claim 4, the composition of claim 5, or the tetramethylrhodamine detection kit of claim 8 in the detection of tetramethylrhodamine.

Images