Anti-quinidine monoclonal antibodies, hybridoma cell lines and uses

Abstract

The application provides an anti-quinidine monoclonal antibody, a hybridoma cell strain and application, and belongs to the field of food safety immune detection. The preservation number of the hybridoma cell strain is CGMCC No. 45125. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity (IC 50 Value is 0.5 ng / mL) for immune analysis detection. The application can be used for preparing an immune detection kit and a colloidal gold test strip of quinidine, and provides a powerful detection method and means for the detection of quinidine residue.

Description

Technical Field

[0001] This invention belongs to the field of food safety immunoassay, specifically relating to hybridoma cell lines that secrete anti-quinidine monoclonal antibodies and their applications. Background Technology

[0002] Quinidine is derived from cinchona bark containing alkaloids and is an isomer of quinine. Its sulfate form is commonly used; it is a white, fine needle-like crystal, odorless, and extremely bitter. Quinidine is a membrane-depressant antiarrhythmic drug that acts directly on the myocardial cell membrane. It significantly prolongs the myocardial refractory period, reduces automaticity, conductivity, and myocardial contractility, and is particularly effective against non-sinus ectopic rhythms. It does not change or prolongs the action potential time of sinoatrial node cells, reduces conduction velocity, prolongs the effective refractory period, and reduces excitability. Its prolongation of the atrial refractory period is more pronounced than that of the ventricles, and it shortens the atrioventricular junctional refractory period.

[0003] Currently, quinidine analysis is primarily conducted using instrumental methods. While these methods offer high accuracy and sensitivity, their high cost and demanding requirements for testing environments and technicians limit their development. In recent years, the rapid advancements in immunoassay technology have made it possible to establish efficient and rapid immunoassay methods for quinidine, including enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatographic strips, and immunomagnetic beads. A crucial prerequisite for establishing these methods is the screening of highly specific monoclonal monomers against quinidine. While hybridoma cells can be used to prepare anti-quinidine monoclonal antibodies, further research is needed to ensure that the prepared hybridoma cell lines successfully secrete these antibodies, and to achieve high specificity and sensitivity in the secreted anti-quinidine monoclonal antibodies. Summary of the Invention

[0004] To address the problems existing in related technologies, this invention provides a hybridoma cell line that secretes anti-quinidine monoclonal antibodies and its applications. The monoclonal antibody prepared from this cell line has good affinity and detection sensitivity for quinidine, and can be used to establish an enzyme-linked immunosorbent assay (ELISA) method for quinidine, or to establish a rapid detection method using colloidal gold immunochromatographic strips. This lays the foundation for the research and development and promotion of indirect competitive ELISA kits and colloidal gold test strips.

[0005] On the one hand, the present invention provides a hybridoma cell line that secretes anti-quinidine monoclonal antibody, which was deposited on March 3, 2022 at the China General Microbiological Culture Collection Center, with the deposit name "Monoclonal Cell Line Diur", the accession number CGMCC No.45125, and the deposit address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides an anti-quinidine monoclonal antibody, which is secreted by a hybridoma cell line that secretes anti-quinidine monoclonal antibody and has accession number CGMCC No. 45125.

[0007] On the other hand, a method for preparing anti-quinidine monoclonal antibody is provided, comprising: taking BALB / c mice, injecting paraffin oil into the peritoneum, then injecting a hybridoma cell line with accession number CGMCC No.45125 into the peritoneum, collecting ascites fluid after injection, purifying the ascites fluid, and storing the obtained anti-quinidine monoclonal antibody at low temperature.

[0008] On the other hand, the application of the hybridoma cell line that secretes anti-quinidine monoclonal antibody is provided in the detection of quinidine, in the preparation of quinidine immunoassay kits, or in the preparation of quinidine detection colloidal gold test strips.

[0009] On the other hand, the application of the aforementioned anti-quinidine monoclonal antibody in quinidine detection, in the preparation of quinidine immunoassay kits, or in the preparation of quinidine detection colloidal gold test strips is provided.

[0010] Preferably, the application of the anti-quinidine monoclonal antibody in quinidine detection is applied to the detection of quinidine residues in food.

[0011] On the other hand, a kit is provided containing the aforementioned anti-quinidine monoclonal antibody.

[0012] On the other hand, the kit described is provided for the detection of quinidine residues in food.

[0013] On the other hand, a colloidal gold test strip is provided, containing the aforementioned anti-quinidine monoclonal antibody.

[0014] On the other hand, the colloidal gold test strip is provided for the detection of quinidine residues in food.

[0015] The basic steps for preparing the hybridoma cell line secreting anti-quinidine monoclonal antibody provided by this invention are as follows:

[0016] (1) Preparation and identification of immunogens: Quinidine and succinic anhydride were used to form an artificial hapten in anhydrous pyridine. The quinidine hapten was then conjugated with carrier proteins BSA and OVA by the carbodiimide method to prepare immunogens quinidine-BSA and coated proquinidine-OVA. After the reaction, the complete antigen and the unconjugated hapten were separated by dialysis. The complete antigen was identified by ultraviolet absorption scanning to obtain the immunogen.

[0017] (2) Immunization of mice: BALB / c mice aged 6-8 weeks were selected for immunization. Quinidine-BSA, the immunogen, was completely emulsified with Freund's adjuvant and then injected subcutaneously at multiple sites (except for sprint immunization). The first immunization used Freund's complete adjuvant, and subsequent booster immunizations used Freund's incomplete adjuvant at half the dose. Sprint immunizations did not use adjuvants; the adjuvant was diluted directly with physiological saline and injected intraperitoneally. The immunization dose for sprint immunization was half that of the booster immunizations. The interval between the first 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 last booster immunization was 18-21 days. Blood was collected from the tail, and the immunization effect in mice was tested using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0018] (3) Cell fusion and cell line establishment: Mouse spleen cells and mouse myeloma cells were fused using polyethylene glycol (PEG 4000) method and cultured in selective medium (HAT medium). One week after fusion, positive cell wells were detected by indirect ELISA, and the inhibitory effect of positive cell wells was further determined by indirect competitive ELISA. Positive cell wells with good inhibition were subcloned three times by limiting dilution method, and hybridoma cell lines that can secrete monoclonal antibodies against quinidine were finally screened to obtain hybridoma cell lines.

[0019] (4) Identification of hybridoma cell line characteristics: The characteristics were determined using an enzyme-labeled secondary antibody kit for identifying mouse monoclonal antibody Ig classes / subclasses; IC50... 50 Values, cross-reactivity, and affinity were determined by ELISA.

[0020] Compared with related technologies, the present invention has at least the following beneficial effects:

[0021] The monoclonal antibody secreted by the hybridoma cell line provided by this invention has good detection sensitivity and affinity for quinidine, IC50. 50 The value was 0.5 ng / mL. This provides a new method for establishing a rapid, simple, inexpensive, sensitive, and specific method for the detection of quinidine, and has practical application value.

[0022] Preservation of biological materials

[0023] A hybridoma cell line secreting anti-quinidine monoclonal antibody, classified and named as: Monoclonal cell line Diur, is deposited at: China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number: CGMCC No. 45125, and deposit date: March 3, 2022. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 This is the standard curve of the inhibition of quinidine by monoclonal antibodies. Detailed Implementation

[0026] The embodiments described below are merely illustrative of the invention and should not be construed as limiting the scope or content of the invention. The invention will be further illustrated below through these embodiments.

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

[0028] 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.

[0029] The reagents involved in the following examples are as follows:

[0030] 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.

[0031] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0032] PBST: PBS containing 0.05% Tween 20;

[0033] 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 fresh before use.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Quinidine inhibition rate assay: 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 0.01, 0.03, 0.1, and 0.3 μg / mL with antibody dilution buffer. After selecting the optimal operating point, quinidine standards were serially diluted to 0, 0.0041, 0.012, 0.037, 0.11, 0.33, 1, and 3 ng / mL, following the ic-ELISA procedure. Finally, graphs were plotted using OriginPro 8.5 (results are shown in the figure). Figure 1 (As shown), the standard inhibition curve was obtained, and IC was calculated. 50 .

[0036] This invention immunizes mice with quinidine complete antigen, fertilizes them with HAT selective medium, and screens the cell supernatant by ic-ELISA to obtain a hybridoma cell line that secretes highly specific antibodies against quinidine.

[0037] Example 1: Preparation and identification of artificial antigens:

[0038] 1.1 Preparation of quinidine hapten: 300 mg quinidine and 150 mg succinic anhydride were dissolved in 8 mL of anhydrous pyridine and refluxed at 60 °C for 12 h. After the reaction was complete, the reaction solution was evaporated to dryness by rotary evaporation, and the solid was dissolved in 6 mL of 50% methanol-water mixture. The mixture was extracted three times with ethyl acetate, and the organic phase was collected. 10 mL of 10% HCl (v / v) solution was added, and a white precipitate was formed. The precipitate was collected, washed with distilled water until neutral, and dried in an oven at 37 °C for later use.

[0039] The reaction route for the synthesis of a hapten is shown in the following diagram:

[0040]

[0041] 1.2 Preparation and identification of complete antigen quinidine-OVA: Weigh 4.5 mg of the prepared quinidine hapten, dissolve it in 300 μL DMF and stir slowly. Add 6.4 mg N-hydroxysuccinimide and 7.3 mg 1-ethylcarbodiimide hydrochloride sequentially, and react at room temperature for 6-8 h to obtain a mixture. Then weigh 10 mg of chicken ovalbumin OVA and dissolve it in 3 mL of carbonate buffer. Slowly add the obtained mixture to the chicken ovalbumin solution and react at room temperature for 14 h with stirring. Dialyze with 0.01 mol / L phosphate buffered PBS for 3 days to obtain the conjugate quinidine-OVA, which is stored at -20℃ for later use.

[0042] It should be noted that the complete antigen quinidine-BSA was obtained using the same preparation and identification methods as described above.

[0043] Example 2: Preparation of hybridoma cell lines secreting anti-quinidine monoclonal antibodies

[0044] 2.1 Acquisition of immunity in animals

[0045] Healthy 6-8 week old Balb / c mice were selected for immunization. The complete antigen quinidine-BSA was emulsified with an equal volume of Freund's adjuvant and administered to BALB / c mice via multiple subcutaneous injections in the neck and back (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; multiple 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), i.e., the titer and inhibition of mouse serum were measured.

[0046] 2.2 Cell Fusion and Screening

[0047] Three days after the initial immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0048] a. Blood was collected from the eyeballs of mice. After euthanizing the mice by cervical dislocation, the mice were immediately immersed in 75% alcohol for about 5 minutes for disinfection. The spleen of the mice was removed under aseptic conditions. The spleen cells were moderately ground with the rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a suspension of spleen cells. The suspension was collected and centrifuged (800 rpm, 6 minutes). The spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside for later use.

[0049] b. Collection of SP2 / 0 cells: 7-10 days before fusion, collect 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 × 10⁻⁶. 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.

[0050] c. Fusion process (7 min). At min 1, add 1 mL of PEG 1500 dropwise to the cells, 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. Then incubate at 37°C for 5 min. Centrifuge (800 rpm, 8 min), discard the supernatant, and 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.

[0051] 2.3 Cell screening and cell line establishment

[0052] 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. Screening consisted of two steps: first, quinidine-OVA was used as the coating antigen to screen positive cell wells using ic-ELISA; second, quinidine was used as a standard, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of the quinidine standard were selected for subcloning using limiting dilution, and the same method was used for detection seven days later. Subcloning was performed three times using the above method to finally obtain a cell line secreting anti-quinidine monoclonal antibody.

[0053] Example 3: Preparation of anti-quinidine monoclonal antibody

[0054] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Hybridoma cells were used to collect ascites fluid starting from day 7. The ascites fluid was purified using the caprylic acid-saturated ammonium sulfate method: under slightly acidic conditions, caprylic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulins. After centrifugation, the precipitate was discarded. Then, an equal volume of saturated ammonium sulfate solution was used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant was discarded, and the antibodies were dissolved in 0.01M PBS solution (pH 7.4). After dialysis to desalt the solution, the purified monoclonal antibodies were finally obtained and stored at -20°C.

[0055] Example 4: Identification of anti-quinidine monoclonal antibodies

[0056] 4.1 Coating: Dilute the coated proquinidine-OVA to the optimal working concentration of 0.1 μg / mL with 0.05 M pH 9.6 carbonate buffer and add 100 μL / well to the microplate wells. Incubate at 37℃ for 2 h.

[0057] 4.2 Washing: Discard the solution in the plate and wash 3 times with PBST washing solution, 200 μL per well each time, for 3 min each time, and then pat dry.

[0058] 4.3 Blocking: Block with CBS containing 0.2% gelatin, 200 μL per well, and react at 37°C 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.

[0059] 4.4 Sample Addition: Quinidine standard was serially diluted with phosphate-buffered saline (PBS) to 0, 0.0041, 0.012, 0.037, 0.11, 0.33, 1, and 3 ng / mL; each was added to a pre-blocked microplate well (50 μL / well). The antibody was then diluted to the optimal working concentration of 0.03 μg / mL and added to the microplate wells (50 μL / well). Each sample was repeated in triplicate. The plates were incubated at 37°C for 30 min, washed, and blotted dry. 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:3000) was added to each well, and the plates were incubated at 37°C for 30 min, washed, and blotted dry.

[0060] 4.5 Color development: Add 100 μL of TMB colorimetric solution to each well and react at 37°C in the dark for 15 min.

[0061] 4.6 Termination and Measurement: Add 50 μL of 2M H₂SO₄ stop solution to each well to terminate the reaction, then measure the OD₄50 value of each well using a microplate reader. Plot the standard inhibition curve using OriginPro 8.5 and calculate the IC₂. 50 .

[0062] The standard inhibition curve of anti-quinidine monoclonal antibody against quinidine is as follows: Figure 1 As shown.

[0063] Example 5 Specificity Experiment

[0064] The IC50 of the monoclonal antibody against quinidine was determined using an indirect competitive ELISA method. 50 The concentration was 0.5 ng / mL, and its IC50 against quinidine structural analogues was verified. 50 and cross-reactivity rate, cross-reactivity rate = (IC50 of quinidine) 50 IC of other compounds 50 )×100%, as shown in Table 1.

[0065] Table 1 IC50 of monoclonal antibody 2C7 against quinidine and crossreactants 50 and cross-reactivity

[0066]

[0067] Interfering substances: Quinine, Cinconidine, Cinconidine.

[0068] The monoclonal antibody exhibits a 100% cross-reactivity with quinidine, and cross-reactivity with similar compounds such as quinine, cinconidine, and cinconidine is less than 5%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity to quinidine, with an IC50 score of [missing value]. 50 The value was 0.5 ng / mL, indicating that the monoclonal antibody has high sensitivity and specificity. Example 6: Quinidine Immunoassay Kit

[0069] This embodiment provides a quinidine immunoassay kit, which includes quinidine monoclonal antibody prepared in Example 3, an ELISA plate, quinidine-coated antigen, quinidine standard solution, HRP-labeled goat anti-mouse IgG secondary antibody, and TMB chromogenic solution.

[0070] The principle behind the quinidine immunoassay kit is as follows: Quinidine content in the test sample is detected using an indirect competitive ELISA method. Quinidine-coated antigen is pre-coated in the wells of the ELISA plate. Quinidine standard solution or the test sample, quinidine monoclonal antibody, HRP-labeled goat anti-mouse IgG secondary antibody, and TMB chromogenic solution are added to construct a quinidine standard inhibition curve. The quinidine content in the test sample is determined based on the quinidine standard inhibition curve and the absorbance value of the test sample. Quinidine detection can be achieved using methods commonly used in this field.

[0071] Example 7: Quinidine detection colloidal gold test strip

[0072] This embodiment provides a colloidal gold test strip, comprising a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. A detection line and a control line are sequentially arranged on the nitrocellulose membrane. The colloidal gold conjugate pad is coated with quinidine monoclonal antibody prepared in Example 3. The detection line is printed from quinidine-coated antigen. The control line is printed from goat anti-mouse IgG secondary antibody. The assembly method of the colloidal gold test strip can be any method commonly used in the art.

[0073] The principle behind the quinidine detection using colloidal gold test strips is as follows: It utilizes an indirect competitive method to detect the presence of quinidine in the sample. If the sample does not contain quinidine, the test line will not develop color, while the control line will. If the sample contains quinidine, both the test line and the control line will develop color. Quinidine detection can be achieved using methods commonly used in this field.

[0074] As can be seen from the above embodiments, the synthetic steps of the artificial antigen for quinidine in this invention are simple and effective, and can be effectively used in immunoassays, providing a convenient approach for subsequent research and analysis. The monoclonal antibody secreted by the provided cell line has good specificity and detection sensitivity for quinidine, indicating that the antibody secreted by the cell line provided by this invention has good sensitivity to quinidine, IC50. 50 With a value of 0.5 ng / mL, and virtually no cross-reactivity with other reactants (<5%), it can be used for rapid immunoassay detection of quinidine content in food.

[0075] The above description only illustrates the technical solution of the present invention with reference to preferred embodiments. However, those skilled in the art should be able to make changes in specific implementation methods and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A hybridoma cell line secreting anti-quinidine monoclonal antibody was deposited on March 3, 2022, at the China General Microbiological Culture Collection Center (CGMCC) under the name "Diur Monoclonal Cell Line" and accession number CGMCC No. 45125. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. An anti-quinidine monoclonal antibody, characterized in that, It is secreted by the hybridoma cell line with accession number CGMCCNo.45125 as described in claim 1.

3. The application of the anti-quinidine monoclonal antibody as described in claim 2 in the preparation of a quinidine immunoassay kit or in the preparation of a quinidine detection colloidal gold test strip, wherein the application is for the detection of quinidine residues in food.

4. A reagent kit, characterized in that, It contains the antiquinidine monoclonal antibody as described in claim 2.

5. The kit according to claim 4 is used for the detection of quinidine drug residues in food.

6. A colloidal gold test strip, characterized in that, It contains the antiquinidine monoclonal antibody as described in claim 2.

7. The colloidal gold test strip according to claim 6 is used for the detection of quinidine residues in food.

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