Hybridoma cell strain secreting a linezolid monoclonal antibody and application thereof

Abstract

This invention relates to a hybridoma cell line that secretes linezolid monoclonal antibodies and its applications, belonging to the field of immunoassay technology. Through multiple screening processes, this invention has obtained a hybridoma cell line that secretes linezolid monoclonal antibodies. The linezolid monoclonal antibody secreted by this hybridoma cell line exhibits excellent affinity and sensitivity to linezolid, with a high IC50 value for linezolid. 50 The concentration reaches 50 ng / mL, and there is no cross-reactivity with linezolid structural analogs, such as terdizolid and contezolid. Therefore, the monoclonal antibody of this invention can be used to prepare immunoassay products for linezolid, providing an efficient detection method and means for the detection of linezolid residues in food.

Description

Technical Field

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

[0002] Linezolid (LNZ) is an oxazolidinone antibiotic with excellent antibacterial activity against multidrug-resistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Since its introduction, it has become one of the key drugs in clinical treatment of complicated skin and soft tissue infections, pneumonia, and serious diseases caused by vancomycin-resistant enterococci. Linezolid can cause serious adverse reactions, such as thrombocytopenia and lactic acidosis. However, due to its indispensable role in human medicine, the potential or inappropriate use of linezolid in animal husbandry, and the resulting residues and resistance problems, constitute a serious global public health issue. Surveillance reports from the European Centre for Disease Prevention and Control (ECDC) clearly indicate that linezolid-resistant enterococci carrying mobile resistance genes (such as optrA and poxtA) are not only increasing in the proportion of hospital-acquired infections, but are also frequently detected in community environments and food animals. These drug-resistant bacteria and their genes can be directly transmitted to humans through the food chain, or promote the expansion of the drug-resistant gene pool through environmental emissions, seriously threatening the long-term efficacy of such core antibacterial drugs.

[0003] Currently, there are few analytical methods for determining linezolid content, mainly including instrumental methods such as gas chromatography-tandem mass spectrometry (GC-MS / MS), high-performance liquid chromatography-UV, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). These methods are complex to operate and require cumbersome sample preparation, which is not conducive to the detection of large batches of samples in actual production. Therefore, there is an urgent need to establish a detection method that is simple to operate, highly sensitive, and highly specific. Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive, and rapid detection method, and it is increasingly being used in food safety testing. However, the prerequisite for using ELISA to detect linezolid is to obtain a monoclonal antibody with high specificity and high sensitivity to linezolid. Existing monoclonal antibodies have low IC50 values ​​for linezolid. 50 The lowest concentration was 0.28 μg / mL (280 ng / mL), but its sensitivity was still poor, so a new monoclonal antibody was needed. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a hybridoma cell line for detecting low concentrations of linezolid in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes linezolid monoclonal antibodies and its applications. Through multiple screening processes, this invention has obtained a hybridoma cell line capable of secreting linezolid monoclonal antibodies. The linezolid monoclonal antibody secreted by this hybridoma cell line exhibits excellent affinity and sensitivity to linezolid, with a high IC50 value for linezolid. 50 The concentration reaches 50 ng / mL, and there is no cross-reactivity with linezolid structural analogs, such as terdizolid and contezolid. Therefore, the monoclonal antibody of this invention can be used to prepare immunoassay products for linezolid, providing an efficient detection method and means for the detection of linezolid residues in food.

[0006] The first objective of this invention is to provide a hybridoma cell line, which is proposed to be taxonomically named a monoclonal cell line and has the accession number CGMCC No. 46739.

[0007] The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0008] .

[0009] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0010] Furthermore, the carrier protein includes chicken oocyte albumin.

[0011] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of linezolid.

[0012] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0013] A fourth objective of this invention is to provide an application of the above-described monoclonal antibody in the detection of linezolid.

[0014] A fifth objective of this invention is to provide an application of the above-described monoclonal antibody in the detection of linezolid.

[0015] A sixth object of the present invention is to provide a detection product for linezolid, the detection product comprising the above-mentioned monoclonal antibody.

[0016] Furthermore, the test product also includes a coating agent.

[0017] Furthermore, the coating is prepared from a hapten-coupled carrier protein; the carrier protein includes bovine serum albumin.

[0018] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0019] The monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits good specificity and detection sensitivity against linezolid (IC50 for linezolid). 50 With a concentration of 50 ng / mL and no cross-reactivity with structural analogs of linezolid, such as terdizolid and contezolid, it can be used to detect linezolid residues, providing an immunoassay method and raw materials for the immunoassay of linezolid residues in food, and has practical application value.

[0020] Preservation of biological materials

[0021] The monoclonal cell line EJSG was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46739, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0022] 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.

[0023] Figure 1 The standard inhibition curve of the linezolid monoclonal antibody prepared for the example is shown. Detailed Implementation

[0024] 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.

[0025] The culture media involved in the following examples are as follows:

[0026] 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.

[0027] The reagents involved in the following examples are as follows:

[0028] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000mL. Store at 4℃ for later use.

[0029] Phosphate buffered saline (PBS): Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12 H2O in 800mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and bring the volume to 1000mL.

[0030] PBST: PBS containing 0.05% Tween 20;

[0031] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0032] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix again before use.

[0033] The detection methods involved in the following embodiments are as follows:

[0034] Linezolid inhibition rate assay: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, linezolid standard was diluted to eight concentrations (0, 6.25, 12.5, 25, 50, 150, 450, 1250 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), obtain the standard inhibition curve of linezolid and calculate IC50. 50 .

[0035] Example 1: Preparation of linezolid hapten

[0036] Purchase the synthetic intermediate for linezolid technical: 3-fluoro-4-(4-morpholino)-aniline, CSA number 93246-53-8.

[0037] Example 2: Preparation of complete antigen

[0038] The hapten LNZ was conjugated to a carrier protein using the diazotization method to obtain the linezolid artificial antigen. Specifically, the hapten LNZ purchased in Example 1 was conjugated with bovine serum albumin (BSA) to obtain the complete antigen LNZ-EDC-BSA.

[0039] The method for preparing the complete antigen LNZ-EDC-BSA is as follows:

[0040] a. Weigh 7.6 mg of the hapten LNZ purchased in Example 1 into a reaction flask, dissolve it in 300 μL of N,N-dimethylformamide, add 20 μL of 1M HCl solution dropwise to the reaction flask to adjust the pH to between 2 and 3, react in an ice bath at 4°C for 30 min, then add 200 μL of freshly prepared sodium nitrite solution dropwise to the reaction flask, react in an ice bath at 4°C for 1 h (this is called solution A). Weigh 6.0 mg of BSA (the molar ratio of LNZ to BSA is 20:1), add 2 mL of carbonate buffer solution (the dissolved BSA protein is called solution B), add solution A dropwise to solution B, adjust the pH of the mixture to 8-9 with 1M NaOH solution, react in an ice bath at 4°C for 4-6 h, and the conjugate LNZ-EDC-BSA is obtained.

[0041] 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 LNZ-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 LNZ-EDC-BSA, which should be taken out and stored at -20℃.

[0042] Example 3: Preparation of the coating agent

[0043] In Example 1, the purchased hapten LNZ was conjugated with chicken ovalbumin OVA to obtain coated progenitor LNZ-EDC-OVA. The preparation method of coated progenitor LNZ-EDC-OVA is as follows:

[0044] a. Weigh 4.3 mg of the hapten LNZ purchased in Example 1 into a reaction flask, dissolve it in 200 μL of N,N-dimethylformamide, add 20 μL of 1M HCl solution dropwise to the reaction flask to adjust the pH to between 2 and 3, react in an ice bath at 4°C for 30 min, then add 200 μL of freshly prepared sodium nitrite solution dropwise to the reaction flask, react in an ice bath at 4°C for 1 h (this is called solution A). Weigh 4.0 mg of OVA (the molar ratio of LNZ to OVA is 20:1), add 2 mL of carbonate buffer solution (the dissolved OVA protein is called solution B), add solution A dropwise to solution B, adjust the pH of the mixture to 8-9 with 1M NaOH solution, react in an ice bath at 4°C for 4-6 h, and the conjugate LNZ-EDC-OVA is obtained.

[0045] 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 LNZ-EDC-OVA 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 LNZ-EDC-OVA, which should be taken out and stored at -20℃.

[0046] Example 4: Preparation of hybridoma cell lines

[0047] 1. Mouse immunization

[0048] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg linezolid 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;

[0049] 2. Cell fusion and screening

[0050] (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:

[0051] 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 be 1-4 x 10⁻⁴ cells. 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.

[0052] b. After euthanizing the 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.

[0053] 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.

[0054] (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, linezolid standard was selected, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of linezolid 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 linezolid monoclonal antibody cell line.

[0055] Example 4: Preparation and Identification of Monoclonal Antibodies

[0056] 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 Linezolid 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.

[0057] (1) Coating: The original LNZ-EDC-OVA was diluted 3 times from 1 µg / mL with 0.05M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0058] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0059] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0060] (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.

[0061] (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.

[0062] (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.

[0063] The IC50 of the monoclonal antibody linezolid was determined by ic-ELISA. 50 The result was 50 ng / mL, indicating good sensitivity to linezolid and its suitability for linezolid immunoassay.

[0064] The IC50 of the monoclonal antibody against linezolid was determined using an indirect competitive ELISA method. 50 The concentration was 50 ng / mL, and its IC50 for compounds such as terdizodil and continazolamide was verified. 50 And the cross-reactivity rate, the formula for calculating the cross-reactivity rate is: Cross-reactivity rate = IC50 of linezolid 50 IC of other compounds 50 ×100%, as shown in Table 1.

[0065] Table 1. IC50 of linezolid monoclonal antibody against linezolid and its cross-links 50 and cross-reactivity

[0066]

[0067] 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, characterized in that, The proposed taxonomic name for the hybridoma cell line is monoclonal cell line, and its accession number is CGMCC No. 46739.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes chicken oocyte albumin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of linezolid.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of linezolid.

8. A detection product for linezolid, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared from a hapten coupled with a carrier protein; the carrier protein includes bovine serum albumin.

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