A hybridoma cell strain and a 4-methylaminoantipyrine residual marker monoclonal antibody secreted by the hybridoma cell strain and application

Abstract

This invention relates to the field of immunology, and more particularly to a hybridoma cell line and its secreted monoclonal antibody against the metamizole residue marker 4-methylaminoantipyrine (MAA), and its applications. This invention uses 4-aminoantipyrine to react with methyl 4-bromomethylbenzoate, followed by alkaline hydrolysis to obtain a hapten (Formula I). ​​An immunogen is then synthesized and used to immunize mice. After cell fusion and screening, a hybridoma cell line capable of secreting a monoclonal antibody recognizing MAA is prepared, named Metamizole-6A4. This hybridoma cell line has a high efficiency in secreting monoclonal antibodies. The secreted monoclonal antibody can accurately detect MAA, IC50, and other markers. 50 The concentration is 4.06 ng / mL, with high specificity. Cross-reactivity with aminopyrine and its metabolites 4-aminoantipyrine, 4-formamidoantipyrine, 4-acetamidoantipyrine, sulfadiazine, norfloxacin, lincomycin, tylosin, penicillin G, streptomycin, and gentamicin is less than 5%. It can accurately detect MAA residues in food. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically, it relates to a hybridoma cell line and its secreted monoclonal antibody against 4-methylaminoantipyrine (MAA), a residual marker of aminopyrine, and its application. Technical Background

[0002] Dipyrone (DIP) is a pyrazolone antipyretic and analgesic widely used in animal husbandry. In animals, it is rapidly metabolized into 4-methylaminoantipyrine (MAA), a potent pharmacodynamic agent, which serves as a residue marker for DIP. Furthermore, it can be further metabolized into 4-formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA), which can also be metabolized to 4-acetylaminoantipyrine (AAA). DIP and its metabolites entering the food chain can damage the urinary, hematopoietic, and circulatory systems in humans. The maximum residue limits (MRLs) for MAA in milk in China and the European Union are 100 μg / kg and 50 μg / kg, respectively. Therefore, rapid and accurate detection of MAA is crucial.

[0003] Immunoassay methods based on antigen-antibody specific reactions, such as colloidal gold test strip immunoassays, offer advantages such as high sensitivity and specificity, simple operation, and short reaction time, making them particularly advantageous for high-throughput rapid screening of samples in the field. The core reagent in immunoassay technology is the antibody. Currently, the reported half-inhibitory concentration (IC50) of monoclonal antibodies against MAA is... 50 The concentration was 8.08 ng / mL, but the IC50 of this antibody against the major metabolites of DIP, AAA and FAA, was [not specified]. 50 The values ​​reached 0.98 ng / mL and 0.82 ng / mL, respectively, with a cross-reactivity rate as high as approximately 1000%. However, using this antibody to establish an immunoassay method is prone to false positives. Therefore, it is essential to rationally design novel haptens and prepare highly specific MAA monoclonal antibodies. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art by designing and synthesizing a new hapten and preparing hybridoma cells with high specificity for MAA monoclonal antibodies. The monoclonal antibodies secreted by the hybridoma cell line can accurately detect the DIP residual marker MAA.

[0005] In a first aspect, the present invention provides a novel MAA immune hapten, the structural formula of which is shown in formula (I):

[0006]

[0007] The present invention further provides an immunogen obtained by reacting the immune hapten with the carrier protein keyhole hemocyanin (KLH), as shown in formula (II).

[0008]

[0009] Secondly, the present invention provides an MAA-coated hapten, as shown in Formula (III).

[0010]

[0011] The present invention further provides a coated antigen obtained by coating the hapten-coupled carrier protein bovine serum albumin, as shown in Formula (IV).

[0012]

[0013] Thirdly, the present invention provides a hybridoma cell line, Analgin-6A4, which is deposited by the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 45304, and is prepared by synthesizing an immunogen from an immune hapten and a coating antigen from a coating hapten.

[0014] The innovation of this invention lies in disclosing a new MAA hapten, immunogen, and its preparation method. Immunizing animals with the MAA immunogen can yield highly specific monoclonal antibodies against MAA. Based on this, immunoassay methods including, but not limited to, enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography can be established to accurately detect MAA residues in food.

[0015] Advantages of this invention: MAA antibodies can be prepared using the conjugate of the hapten and carrier protein provided by this invention, with IC50... 50 The value was 4.06 μg / mL. The cross-reactivity with aminopyrine and its metabolites, 4-aminoantipyrine, 4-formamidoantipyrine, and 4-acetamidoantipyrine was all less than 3%, indicating good specificity and promising application prospects.

[0016] The hybridoma cell line Analgin-6A4 was deposited by the China General Microbiological Culture Collection Center (CGMCC) on August 26, 2022, with accession number CGMCC No. 45304, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0017] Figure 1 This is a mass spectrometry image of the daughter ions of an immune hapten.

[0018] Figure 2 This is a UV absorption spectrum for identifying immunogens.

[0019] Figure 3 This is the UV absorption spectrum for identifying the coated antigen.

[0020] Figure 4 The image shows the MAA drug inhibition curve of monoclonal antibody 6A4. Detailed Implementation

[0021] The following embodiments of the present invention are merely illustrative of the invention and should not be construed as limiting the scope or content of the invention. The present invention will now be further illustrated through these embodiments.

[0022] Example 1. Synthesis of Formula I immune hapten

[0023] 0.50 g of 4-aminoantipyrine, 0.68 g of potassium carbonate, and 0.56 g of methyl 4-bromomethylbenzoate were added to 20 mL of acetonitrile solution and refluxed overnight at 80 °C. The liquid was evaporated to dryness, and the mixture was extracted three times with 30 mL of purified water and 30 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness. The residue was purified by silica gel column chromatography with dichloromethane:methanol = 100:1 as the eluent to give 0.226 g of a pale yellow solid, with a yield of 26.2%.

[0024] 0.4 g of the above pale yellow solid product was dissolved in 10 mL of a 10% NaOH ethanol / water (1:2) solution and stirred overnight. The mixture was extracted three times with 10 mL of ethyl acetate, and the aqueous phase was collected. The pH was adjusted to 3 with 0.1 M HCl, and the precipitated solid was filtered and dried in a 37°C oven to obtain 0.252 g of Formula I immunohapten, with a yield of 65.6%. Mass spectrometry identification results are shown in the appendix. Figure 1 The molecular ion peak [M+H]+ of the synthesized product is 338.15 m / z, indicating that the synthesis was successful.

[0025] Example 2. Synthesis of Immunogens

[0026] Weigh 20 mg of hapten I, 30 mg of N,N'-dicyclohexylcarbodiimide, and 20 mg of N-hydroxysuccinimide, dissolve them in 1.0 mL of N,N-dimethylformamide, and react at room temperature for 12 h. Centrifuge at 8000 rpm for 10 min, and take 0.2 mL of the supernatant. Add it to 5 mL of 0.01 M phosphate buffer (pH 7.4) containing 5 mg of keyhole hemocyanin. Stir at 4 °C for 12 h to obtain the protein conjugate. Dialyze against 0.01 M phosphate buffer (pH 7.4) for 3 days to obtain the immunogen. After UV identification, see attached figure. Figure 2The maximum absorption wavelength of the immunogen was 285 nm, which is slightly different from the maximum absorption wavelength of the immune hapten (290 nm) and the carrier protein keyhole hemocyanin (280 nm), indicating that the immunogen was successfully prepared.

[0027] Example 3. Synthesis of the coating agent

[0028] 20 mg of 4-aminoantipyrine (Formula III) was dissolved in 1.0 mL of N,N-dimethylformamide and added dropwise to a carbonate buffer solution containing 130 mg of bovine serum albumin at pH 9.6. Then, 40 μL of 50% glutaraldehyde aqueous solution was added dropwise to the protein solution, and the reaction was allowed to proceed at room temperature for 6 hours. The resulting protein conjugate was dialyzed against 0.01 M phosphate buffer (pH 7.4) for 3 days to obtain the coated antigen. UV identification was performed (see attached image). Figure 3 The maximum absorption wavelength of the coating antigen was 270 nm, which was different from the maximum absorption wavelength of the coated hapten (288 nm) and the carrier protein bovine serum albumin (280 nm), indicating that the coating antigen was successfully prepared.

[0029] Example 4. Preparation of monoclonal antibodies

[0030] 4.1 Animal Immunization

[0031] Nine female Balb / c mice, approximately 6 weeks old, were immunized with the immunogen. For the first immunization, each mouse was given 100 μL of immunogen (1 mg / mL) emulsified with an equal volume of Freund's complete adjuvant via multiple subcutaneous injections at the nape of the neck. A booster immunization was administered 21 days later, using Freund's incomplete adjuvant for emulsification. After the fourth immunization, blood was collected from the tail, and serum was diluted 3000-fold. The immunogen was then diluted 5000-fold and plated. Serum titers and inhibition rates against MAA were measured using indirect enzyme-linked immunosorbent assay (iELISA) and indirect competitive enzyme-linked immunosorbent assay (icELISA). The results are shown in Table 1. The results indicate that all mice achieved good immunization efficacy. The absorbance of MAA at concentrations of 5 ng / mL and 50 ng / mL showed a significant decreasing trend, indicating that the obtained antiserum specifically recognized MAA. Mice (number 3) were selected for a pulse immunization to induce cell fusion.

[0032] Table 1. Results of the 4th immunization

[0033]

[0034] 4.2 Cell Fusion and Screening

[0035] Three days prior to fusion, mice (model II) were intraperitoneally immunized with a pulse immunization containing 300 μg of immunogen. Spleen cells were prepared from the spleens of the immunized mice and fused with myeloma cells using PEG1450. Positive cell wells secreting anti-MAA were screened using indirect ELISA and indirect competitive ELISA. Cloning was performed three times consecutively using limiting dilution to establish a stable hybridoma cell line, aminopyrine-6A4, that secretes anti-MAA.

[0036] 4.3 Ascites preparation

[0037] Seven days prior to inoculation, mice were pretreated with 0.5 mL of Freund's incomplete adjuvant via intraperitoneal injection. The number of aminopyrine-6A4 cells was adjusted to approximately 10 per mouse. 6 Hybridoma cells were then inoculated into the peritoneal cavity of mice. Once the mice's abdomens became significantly distended, ascites fluid was collected, which yielded the anti-MAA monoclonal antibody.

[0038] Example 5. Testing the performance of monoclonal antibodies

[0039] 5.1 Reagent Preparation

[0040] Carbonate buffer (pH 9.6): Accurately weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, and bring the volume to 1000 mL with ultrapure water.

[0041] Phosphate buffer (pH 7.4): Accurately weigh 8.00 g NaCl, 0.20 g KH2PO4, 2.90 g Na2HPO4·12H2O, and 0.20 g KCl, and bring the volume to 1000 mL with ultrapure water. Stir thoroughly to dissolve.

[0042] Washing solution: Accurately weigh 8.00g NaCl, 0.20g KH2PO4, 2.90g Na2HPO4·12H2O, and 0.20g KCl. Make up to 1000mL with ultrapure water, add 0.5mL Tween-20, and stir thoroughly to dissolve.

[0043] Blocking solution: Accurately weigh 3.0g of skim milk powder, add 100mL of phosphate buffer, and stir thoroughly until completely dissolved.

[0044] Substrate colorimetric solution: The substrate colorimetric solution is a tetramethylbenzidine (TMB) solution. Mix equal volumes of substrate colorimetric solutions A and B, and prepare fresh before use.

[0045] Termination solution: Slowly add 100 mL of concentrated sulfuric acid dropwise to ultrapure water and finally bring the volume to 1 L, stirring constantly during the process.

[0046] 5.2 Procedures for Indirect ELISA and Indirect Competitive ELISA

[0047] Coating: Dilute the coating agent to 0.8 μg / mL with carbonate buffer and add 100 μL / well to the microplate. Coat overnight in a light-protected environment at 4°C. After coating, wash three times with washing buffer.

[0048] Blocking: Add 150 μL of blocking solution to each well and incubate at 37°C for 1 hour. After incubation, wash three times with washing solution.

[0049] Sample loading: Dilute the antibody or mouse serum into a series of gradients. First, add 50 μL of PBS to the microplate, then add 50 μL of serially diluted antibody to the microplate. Incubate at 37°C for 30 minutes. After incubation, wash three times with washing buffer.

[0050] Enzyme-labeled secondary antibody reaction: Add 100 μL / well of goat anti-mouse enzyme-labeled secondary antibody (5000-fold dilution) to the microplate and incubate at 37°C for 30 minutes. After incubation, wash the plate three times with washing buffer.

[0051] Colorimetric reaction and termination: Add 100 μL of the prepared substrate colorimetric solution to each well of the microplate and incubate at 37°C for 15 minutes. Then, add 50 μL of the stop solution to each well to terminate the colorimetric reaction.

[0052] Colorimetric assay: Place the ELISA plate on an ELISA reader and read the absorbance (OD) at 450 nm. 450 Numerical values ​​are used to calculate and analyze data.

[0053] The procedure for indirect competitive ELISA is basically the same as that for indirect ELISA, except that the PBS in the sample is replaced with a series of standard concentrations.

[0054] 5.3 Selection of Optimal Coating Concentration / Antibody Dilution Factor

[0055] The original coating agent was diluted to 1.20, 0.60, and 0.30 μg / mL for coating; the antibody was diluted to 80,000, 40,000, and 30,000; the target MAA concentrations were set to 0, 0.33, 1, 3, 9, and 81 ng / mL; each concentration was tested in triplicate using indirect competitive ELISA. A curve was fitted using Origin 8.5 with the logarithm of drug concentration as the x-axis and OD value as the y-axis, and the IC50 was calculated. 50 and linear range (IC) 20 -IC 80 (Table 2) shows that the optimal coating concentration is 0.60 ng / mL and the optimal antibody dilution factor is 40,000.

[0056] Table 2. Performance parameters of aminopyrine-6A4 at different coating concentrations

[0057]

[0058] 5.4 Establishment of the Standard Curve

[0059] Using the optimized coating concentration and antibody dilution factor described above, MAA concentrations were set to 0, 0.33, 1, 3, 9, and 81 ng / mL, with three replicates for each concentration, and the assays were repeated three times. The four-parameter equation was plotted using Origin software; see attached figure. Figure 4 IC 50 The effective concentration was 4.06 ng / mL, with a linear range of 0.66-24.91 ng / mL. The regression equation was y = 0.1674 + 1.496 / [1 + (x / 4.0605)]. 0.7642 ], R 2 It is 0.997.

[0060] 5.5 Cross-reactivity determination

[0061] Using the ELISA established above, the IC50 values ​​of aminopyrine and its metabolites 4-aminoantipyrine, 4-formamidoantipyrine, and 4-acetamidoantipyrine, as well as other common antibiotics such as sulfadiazine, norfloxacin, lincomycin, tylosin, penicillin G, streptomycin, and gentamicin were determined. 50 Following the above-described indirect competitive enzyme-linked immunosorbent assay (ELISA) procedure, calculate its IC50. 50 The cross-reactivity rate (CR) was measured. The results are shown in Table 3. The cross-reactivity rate of metamizole-6A4 with 4-aminoantipyrine was 2.09%, and there was no cross-reactivity with other metamizole and its metabolites or antibiotics. This indicates that the MAA antibody prepared based on the novel hapten of the present invention has good specificity and good application prospects.

[0062] Table 3. Cross-reactivity rate of Analgin-6A4

[0063]

[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A hapten, characterized in that, The hapten has a structure shown in Formula I:

2. An immunogen, characterized in that, obtained by reacting the hapten of claim 1 and keyhole limpet hemocyanin (KLH), having a structure shown in Formula II:

3. A hybridoma cell strain, Anisomycin-6A4, characterized in that, Preserved by Institute of Microbiology, Chinese Academy of Sciences, with a preservation number of GCMCC No. 45304.

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