Preparation and application of a hybridoma cell strain and its secreted uniform monoclonal antibody recognizing tylosin / tilmicosin

Abstract

This invention belongs to the field of immunology and discloses a hybridoma cell line and the preparation and application of its secreted monoclonal antibody that uniformly recognizes tylosin / tilmicosin. The monoclonal antibody hybridoma cell line is named DES-AOAA-14D5, with accession number CGMCC NO: 45303. This invention utilizes decarboxymethyl tylosin and oxycarboxymethyl hydroxylamine to synthesize a hapten, conjugates a carrier protein to prepare a complete antigen, immunizes mice, and, through cell fusion and screening techniques, prepares a hybridoma cell line that can secrete a monoclonal antibody that uniformly recognizes tylosin / tilmicosin. IC50 50 The effective concentrations were 1.59 and 1.72 ng / mL, respectively, with a cross-reactivity rate as high as 92.44%. An immunoassay method for the simultaneous and accurate detection of tylosin / tilmicosin in milk was established using this antibody. The cut-off value for both tylosin and tilmicosin was 16 ng / mL, and no cross-reactivity was observed with other structural analogs. The detection method of this invention shows promising application prospects.

Description

Preparation and application of a hybridoma cell line and its secreted monoclonal antibody that uniformly recognizes tylosin / tilmicosin. Technical Field

[0001] This invention relates to the field of immunology, specifically to a hybridoma cell line and the monoclonal antibody it secretes that uniformly recognizes tylosin / tilmicosin, and its applications. Background Technology

[0002] Tylosin (TYL) is a macrolide antibiotic extracted from the culture medium of Streptomyces. Tilmicosin (TM) is a semi-synthetic derivative of tylosin, with a similar antibacterial spectrum. Both are widely used to treat various bacterial infections in animals. Furthermore, as feed additives, they can promote animal growth and improve feed utilization. However, after administration of TYL and TM, drug residues are widely distributed in the body fluids and tissues of livestock. Long-term consumption of these contaminated foods may cause allergic reactions. Therefore, my country has set the maximum residue limits (MRL) for TYL and TM in milk at 50 μg / kg. Establishing a rapid analytical method for the simultaneous detection of TYL / TM in milk has good application value.

[0003] Lateral flow immunoassay (LFA) is the most commonly used rapid analytical method. High-performance antibodies are crucial for establishing immunoassay methods, especially when detecting different compounds; the antibody's recognition ability determines the accuracy of the results. Literature reports indicate that antibodies derived from haptens using the molecular structures of TYL or TM often exhibit significantly different recognition abilities against TYL / TM. For example, antibodies prepared using TYL-AOAA show a cross-reactivity (CR) of only 27.57% against TM, while antibodies prepared using the hapten DES-AOAA show a CR of 51% against TM. If an immunoassay method for simultaneously detecting TYL / TM is established based on these antibodies, using TYL as the standard may result in weak antibody recognition of TM, leading to false negatives; conversely, using TM as the standard may result in strong antibody recognition of TYL, leading to false positives. Therefore, preparing monoclonal antibodies that uniformly recognize TYL / TM and establishing an LFA method for the simultaneous and accurate detection of TYL / TM in milk is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a hybridoma cell line, its secreted monoclonal antibody that uniformly recognizes TYL and TM, and its application. The monoclonal antibody secreted by the hybridoma cell line can simultaneously recognize TYL and TM with a cross-reactivity rate of 92.44%, and can be used to establish an accurate immunological analysis method for detecting TYL and TM.

[0005] In a first aspect, the present invention provides a DES-AOAA hapten and a complete antigen, the structural formula of which is shown below:

[0006]

[0007] Secondly, this invention provides a hybridoma cell line, DES-AOAA-14D5, prepared using the aforementioned hapten and complete antigen. Complete preservation information is as follows:

[0008] The accession number is CGMCC No.45303; the classification name is hybridoma cell line DES-AOAA-14D5; the depositary institution is China General Microbiological Culture Collection Center (CGMCC); the deposit address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing; the deposit date is August 26, 2022.

[0009] Monoclonal antibodies were further prepared using the hybridoma cell line DES-AOAA-14D5. This antibody targets the IC50 of TYL / TM. 50 The concentrations were 1.59 ng / mL and 1.72 ng / mL, respectively, with a CR of 92.44% based on TYL.

[0010] Thirdly, the present invention further provides an immunochromatographic test strip containing the monoclonal antibody.

[0011] Fourthly, the present invention further provides the application of the monoclonal antibody or the immunochromatographic test strip in the simultaneous and accurate detection of TYL and TM in milk.

[0012] Advantages of this invention: It provides a hybridoma cell line that secretes monoclonal antibodies that uniformly recognize TYL and TM. These monoclonal antibodies can simultaneously recognize TYL and TM with a cross-reactivity rate of 92.44%. This can be used to establish an accurate immunological assay for detecting TYL and TM. The test strips prepared with this monoclonal antibody exhibit high sensitivity and accuracy (IC50). 50 The concentrations were 2.91 and 2.78 ng / mL, respectively, which have good practical applicability for monitoring TYL / TM in milk. Attached Figure Description

[0013] Figure 1 shows the ultraviolet scan spectra of the coating antigen and immunogen provided in Examples 2 and 3 of the present invention;

[0014] Figure 2 is a mass spectrum of the hapten DES-AOAA ion scanning provided in Example 2 of the present invention;

[0015] Figure 3 shows the ELISA standard curve of the monoclonal antibody DES-AOAA-14D5 provided in Example 5 of the present invention;

[0016] Figure 4 is a color comparison diagram of colloidal gold with different K2CO3 addition amounts provided in Example 6 of the present invention;

[0017] Figure 5 is a bar graph showing the change in color intensity of the colloidal gold test strip coated with different original streaking concentrations of the detection line provided in Embodiment 6 of the present invention.

[0018] Figure 6 is a bar chart showing the change in color intensity of the detection line of the colloidal gold test strip with different antibody addition amounts provided in Example 6 of the present invention.

[0019] Figure 7 is a bar chart showing the change in color intensity of the detection line of the colloidal gold test strip with different probe addition amounts provided in Example 6 of the present invention.

[0020] Figure 8 shows the standard curve of colloidal gold test strip against TYL / TM provided in Example 6 of the present invention;

[0021] Figure 9 shows the specificity test results of the colloidal gold test strip provided in Example 6 of the present invention. Detailed Implementation

[0022] The present invention will be further illustrated by the following examples, but these examples do not limit the invention. Unless otherwise specified, all percentages are mass percentages.

[0023]

[0024] Example 1: Preparation of DES

[0025] Weigh 5g of tylosin tartrate and dissolve it in 300mL of pH 2.0 sulfuric acid solution. Incubate at 85℃ for 3 hours. After the reaction is complete, cool to 20℃ and transfer to a separatory funnel. Extract three times with 100mL of dichloromethane, discarding the dichloromethane layer. Adjust the pH of the aqueous phase to 8.8 with saturated sodium carbonate solution, extract three times with 100mL of dichloromethane, combine the dichloromethane layers, add 10g of anhydrous sodium sulfate, shake, and let stand overnight. Filter and evaporate to dryness under reduced pressure at 40℃. Add 5mL of dichloromethane to the residue, place in a -20℃ refrigerator, and let stand overnight. The precipitate is DES.

[0026]

[0027] Example 2: Preparation of the hapten DES-AOAA

[0028] Dissolve 72 mg of DES and 11 mg of O-carboxymethoxyamine in 4 mL of methanol, stir at room temperature for 2.5 h, and evaporate to dryness to obtain the hapten DES-AOAA. Mass spectrometry identification results are shown in Figure 2. The molecular ion peak [M+H]+ of the synthesized product is m / z 846.4583, indicating successful hapten synthesis.

[0029] Its structural formula is shown below:

[0030]

[0031] Example 3: Preparation of complete antigen

[0032] Dissolve 85 mg of DES-AOAA, 42 mg of N,N-dicyclohexylcarbodiimide and 22 mg of N,N-hydroxysuccinimide in 4 mL of N,N-dimethylformamide, react at room temperature for 12 h, and centrifuge at 1000 rpm to remove the precipitate, which is the activated hapten.

[0033] Dissolve 10 mg of hemocyanin (KLH) in 10 mL of 0.1 mmol sodium phosphate buffer. Add 0.1 mL of the activated hapten. Stir magnetically for 12 h in an ice bath. Dialyze the conjugate in phosphate buffer for 3 days to obtain the immunogen DES-AOAA-KLH, aliquot and store at -20°C for later use.

[0034] In the above steps, KLH was replaced with 30 mg bovine serum albumin (BSA) to obtain the conjugate DES-AOAA-BSA, which was used as the coating agent. Its structural formula is shown below:

[0035]

[0036] The hapten, immunogen, coating antigen, KLH, and BSA were scanned using a UV spectrophotometer. As shown in Figure 1, the hapten exhibited a maximum absorption peak at 289 nm, while KLH and BSA showed maximum absorption peaks at 279.5 nm and 278.5 nm, respectively. The maximum absorption peaks of the immunogen DES-AOAA-KLH and the coating antigen DES-AOAA-BSA were at 284 nm and 285 nm, respectively, showing a slight shift in UV absorption peaks, indicating successful synthesis of the immunogen and coating antigen.

[0037] Example 4: Preparation of Monoclonal Antibodies

[0038] 4.1 Animal Immunization

[0039] Using DES-AOAA-KLH prepared in Example 3 as the immunogen, female Balb / C mice were immunized. The initial immunization was emulsified with 100 μL of physiological saline and 100 μL of Freund's adjuvant and subcutaneously injected into multiple sites on the back. Every 3 weeks, booster immunizations were performed using 100 μL of physiological saline and 100 μL of Freund's incomplete adjuvant. Eight days after the third immunization, serum was collected from the tail vein of the mice and detected using indirect ELISA and indirect competitive ELISA methods. The original DES-AOAA-BSA coating was diluted 20,000 times, and the mouse serum was diluted 7,000 times for measurement. The results are shown in Table 1. All mice achieved good immunity and showed good recognition of TYL. Mice number 9 could simultaneously recognize TM, with inhibition rates of 57.9% and 45.73%, respectively. Mice number 9 were selected for fusion.

[0040] Table 1. Results of the 4th immunization with DES-AOAA-KLH immunogen

[0041]

[0042] 4.2 Cell Fusion and Screening

[0043] Three days prior to fusion, mice (model 9) were intraperitoneally injected with 300 μg of DES-AOAA-KLH for shock immunization. Myeloma cells sp2 / 0 and spleen cells were fused using PEG. Positive wells were indirectly selected by competitive ELISA using TYL / TM as a standard. Subcloning was performed using limiting dilution to establish a hybridoma cell line that stably secretes and recognizes TYL / TM, named DES-AOAA-14D5. Antibodies were prepared using the ascites induction method, purified using the caprylic acid-ammonium sulfate method, and identified as IgG2 using an antibody subtype identification kit.

[0044] The complete preservation information for hybridoma cell lines is as follows:

[0045] The accession number is CGMCC No.45303; the classification name is hybridoma cell line DES-AOAA-14D5; the depositary institution is China General Microbiological Culture Collection Center (CGMCC); the deposit address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing; the deposit date is August 26, 2022.

[0046] Example 5: Monoclonal Antibody Performance Testing

[0047] 5.1 Reagent Preparation

[0048] Carbonate buffer (CB, 0.05 mol / L, pH 9.6): Accurately weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in ultrapure water, and bring the volume up to 1000 mL.

[0049] Phosphate buffer (PBS, 10 mmol / L, pH 7.4): Accurately weigh 8 g NaCl, 0.2 g KCl, 2.9 g Na2HPO4·12H2O, and 0.2 g KH2PO4, dissolve them in ultrapure water, and bring the volume to 1000 mL.

[0050] Washing buffer (0.5% PBST): Measure 1000 mL of PBS, add 0.5 mL of Tween-20, and vortex to mix.

[0051] Blocking solution: Accurately weigh 3.0g of skim milk powder, add 100mL of PBS, and stir thoroughly until completely dissolved.

[0052] Substrate solution: The substrate solution is a tetramethylbenzidine (TMB) solution. Take 0.5 mL of substrate solution A and 0.5 mL of substrate solution B, mix them well, and use immediately.

[0053] Termination solution: Slowly add 100 mL of concentrated sulfuric acid dropwise to 800 mL of ultrapure water while stirring continuously.

[0054] 5.2 Indirect Competition ELISA Steps

[0055] Dilute the original DES-AOAA-BSA coating to 0.25 ng / L with CB solution, add 100 μL to each well, and refrigerate overnight at 4°C. Wash once with PBST solution, agitate dry, add 150 μL of blocking buffer to each well, and block at 37°C for 2 h. After agitation, add 50 μL of PBS solution or TYL solution (0.1, 0.3, 0.9, 2.7, 8.1, 24.3 ng / mL) to each well, then add 50 μL of 0.05 ng / L DES-AOAA-14D5 solution, and incubate at 37°C for 30 min. Wash once with PBST solution, agitate dry, add 100 μL of enzyme-labeled secondary antibody to each well, and incubate at 37°C for 30 min. Wash three times with PBST solution, agitate dry, add 100 μL of substrate mixture to each well, and incubate at 37°C for 10 min. Add 50 μL of stop solution to each well. Measure OD using a microplate reader. 450 Values. A standard curve is calculated based on the data read from the ELISA reader. The cross-reactivity rate and IC50 are used as the basis for the curve. 50 The antibody performance was comprehensively evaluated, and the results are shown in Table 2 and Figure 3. The results indicate that, using DES-AOAA-BSA as the coating antigen, the IC5 value of the DES-AOAA-14D5 antibody against TYL / TM is [missing information]. 50 The values ​​were 1.59 and 1.72 ng / mL, respectively, and the cross-reactivity rate was 92.44%, indicating that the antibody can uniformly recognize TYL / TM.

[0056] Table 2. Monoclonal antibody performance assay

[0057]

[0058] Example 6: Construction of a colloidal gold lateral flow chromatography immunoassay method

[0059] 6.1 Solution Preparation

[0060] Carbonate buffer (CB, 0.05 mol / L, pH 9.6): Accurately weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in ultrapure water, and bring the volume up to 1000 mL.

[0061] Phosphate buffer (PBS, 10 mmol / L, pH 7.4): Accurately weigh 8 g NaCl, 0.2 g KCl, 2.9 g Na2HPO4·12H2O, and 0.2 g KH2PO4, dissolve them in ultrapure water, and bring the volume to 1000 mL.

[0062] Sample dilution buffer (0.5% PBST): Weigh 1000 mL of PBS, add 0.5 mL of Tween-20, and vortex to mix.

[0063] Sample pad treatment solution: Weigh 2g bovine serum albumin, 10g sucrose, 200μL Tween-20, and 40μL proclin 300, dissolve in PBS and bring the volume to 200mL;

[0064] Secondary antibody dilution solution: Weigh 1g of bovine serum albumin and 20μL of Proclin 300, dissolve in PBS and bring the volume to 100mL.

[0065] Preparation of 1% chloroauric acid by mass: Weigh 0.6g of chloroauric acid and add it to 60mL of ultrapure water, mix well, and store at 4℃ for later use.

[0066] Preparation of 1% (by mass) trisodium citrate: Dissolve 0.684g of trisodium citrate dihydrate in 60mL of ultrapure water and seal for later use.

[0067] K2CO3 solution (0.1mol / L): Accurately weigh 1.38g of K2CO3 and dilute to 100mL with ultrapure water.

[0068] Gold-labeled antibody resuspension: Accurately weigh 1g of bovine serum albumin, 20μL of Proclin 300, 1g of dextran 40000, and 0.05g of PEG 20000. Dissolve in PBS and bring the volume to 100mL.

[0069] 6.2 Preparation of Colloidal Gold

[0070] Take a 100mL Erlenmeyer flask, add 100mL of ultrapure water, stir and heat to boiling, then quickly add 1mL of 1% (w / w) chloroauric acid solution. After the chloroauric acid aqueous solution boils, quickly add 1mL of 1% (w / w) trisodium citrate solution. The solution changes from golden yellow to purple-red within 2 minutes, and becomes wine-red after 8 minutes. Cool to room temperature, make up to 100mL, and store at 4℃.

[0071] 6.3 Preparation of colloidal gold-labeled antibodies

[0072] Rinse a series of 1.5 mL EP tubes with ultrapure water. Add 1 mL of colloidal gold to each tube and adjust the pH by adding 0, 0.5, 1, 1.5, 2, and 2.5 μL of 0.1 M K2CO3 solution, respectively. Mix well. Add 6 μg of DES-AOAA-14D5, mix well, and let stand for 25 min. Add 30 μL of 10% BSA solution, mix well, and let stand for 20 min. Centrifuge at 8500 rpm for 10 min, discard the supernatant, resuspend the precipitate in gold-labeled antibody resuspension buffer to 0.2 mL, mix well, and store at 4 °C for later use.

[0073] When the amount of K2CO3 added is too small, the pH value of the colloidal gold solution is lower than the isoelectric point of the antibody. The positively charged antibody will react with the negatively charged colloidal gold to form large particles, disrupting the stability of the colloidal gold, causing the colloidal gold particles to aggregate and changing its color. When the amount of K2CO3 added is too large, the pH value of the colloidal gold solution is too high, which is not conducive to the experiment. As shown in Figure 4, it can be clearly seen that when the amount of K2CO3 added is 2 and 2.5 μL, the solution color is red in both cases. Since the amount of K2CO3 added is not conducive to the experiment, the optimal pH condition is selected as 2 μL of 0.1M K2CO3.

[0074] 6.4 Construction of test strips

[0075] The test strip consists of a sample pad, an NC membrane, absorbent paper, and a PVC base. The sample pad is cut into 5cm wide pieces, thoroughly soaked in the sample pad treatment solution, and then placed in a drying oven at 37°C for 12 hours. The coating agent is diluted to 0.35mg / mL with CB and streaked to form the detection line (T line). Goat anti-mouse IgG is diluted to 0.5mg / mL with secondary antibody dilution buffer and streaked to form the control line (C line). The strips are then dried overnight at 37°C. The sample pad, NC membrane, and absorbent paper are sequentially attached to the PVC base, cut into 3.2mm wide test strips, and sealed for storage.

[0076] 6.5 Optimization of test strip streaking concentration

[0077] Accurately transfer 7 μL of gold-labeled antibody into each well, add 200 μL of skim milk containing 0, 2, or 8 ng / mL TYL, and insert DES-AOAA-BSA-coated test strips with original scratch concentrations of 0.7, 0.35, and 0.175 mg / mL into each well. Incubate for 8 min. Use a gold-labeled immunochromatographic analyzer to read the corresponding values ​​and determine the optimal scratch concentration based on the color development and competition results.

[0078] The results are shown in Figure 5. When the etching concentration was 0.175 mg / mL, the T-line reading was 602, indicating a light color development that was difficult to interpret visually. When the etching concentration was 0.7 mg / mL, the T-line reading was 894, showing good color development, but the inhibition rates at TYL concentrations of 2 ng / mL and 8 ng / mL were 41.72% and 62.75%, respectively, indicating poor competition and low sensitivity. When the etching concentration was 0.35 mg / mL, the T-line reading was 784, and the inhibition rates at TYL concentrations of 2 ng / mL and 8 ng / mL were 44.64% and 74.11%, respectively. Therefore, 0.35 mg / mL was selected as the optimal etching concentration for further optimization.

[0079] 6.6 Optimization of Antibody Addition Amount

[0080] Add 6, 10, and 14 μg of DES-AOAA-14D5 to 200 μL of skim milk containing 0, 2, and 8 ng / mL TYL, respectively, insert test strips, and react for 8 min.

[0081] The results are shown in Figure 6. When the antibody dosage was 6 μg, the T-line reading was 612, indicating a light color development that was difficult to interpret visually. When the antibody dosage was 14 μg, the T-line reading was 912, showing good color development, but the inhibition rates at TYL levels of 2 ng / mL and 8 ng / mL were 38.15% and 81.46%, respectively, indicating low sensitivity. When the antibody dosage was 10 μg, the T-line reading was 784, showing good color development, and the inhibition rates at TYL levels of 2 ng / mL and 8 ng / mL were 44.64% and 88.39%, respectively. Therefore, a 10 μg antibody dosage was selected for subsequent experiments.

[0082] 6.7 Optimization of the amount of gold-labeled antibody added

[0083] Add 5, 7, and 9 μL of gold-labeled antibody to 200 μL of skim milk containing 0, 2, and 8 ng / mL TYL, respectively, insert the test strip, and react for 8 min.

[0084] The results are shown in Figure 7. When the amount of gold-labeled antibody added was 5 μL, the T-line reading was 402, indicating a light color development that was difficult to interpret visually. When the amount of gold-labeled antibody added was 9 μL, the T-line reading was 912, showing good color development, but the inhibition rates at TYL of 2 ng / mL and 8 ng / mL were 12.28% and 57.34%, respectively, indicating low sensitivity. When the amount of gold-labeled antibody added was 7 μL, the T-line reading was 784, showing good color development, and the inhibition rates at TYL of 2 ng / mL and 8 ng / mL were 47.45% and 88.39%, respectively. Therefore, a gold-labeled antibody addition of 7 μL was selected for establishing the standard curve.

[0085] 6.8 Establishment of the Standard Curve

[0086] Based on the above optimized conditions, 7 μL of gold-labeled antibody was added, and TYL / TM was diluted with skim milk to concentrations of 0, 1, 2, 4, 8, and 16 ng / mL. 200 μL was added to each well, and the test strip was inserted, allowing the reaction to proceed for 8 min. A standard curve was plotted with the logarithm of the TYL / TM standard concentration on the x-axis and the colorimetric values ​​detected by the colorimeter on the y-axis. The cut-off value and IC50 value were then plotted. 50 The values ​​are determined, and a standard curve is established.

[0087] The results are shown in Figure 8. Visual observation revealed a cut-off value of 16 ng / mL for all values. Quantitative analysis yielded the IC50 value of TYL / TM. 50 The values ​​were 2.91 and 2.78 ng / mL, respectively, with linear ranges of 1.27–6.61 ng / mL and 1.05–7.34 ng / mL, respectively, and a cross-reactivity rate of 104.68%.

[0088] 6.9 Specificity test

[0089] The LFA was established using 20 ng / mL TYL, 20 ng / mL TM, and 1 mg / L standards of tiamulin, avermectin, streptomycin, erythromycin, and spiramycin to investigate its specificity. The results are shown in Figure 9. The LFA established in this invention showed almost no cross-reactivity with the above antibiotics (CR < 0.01%), and can accurately detect TYL / TM simultaneously.

[0090] 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 hybridoma cell line DES-AOAA-14D5, characterized in that, It is deposited by the China General Microbiological Culture Collection Center, with accession number CGMCC No. 45303, and classified as a hybridoma cell line.

2. A monoclonal antibody, characterized in that, The monoclonal antibody is produced by the hybridoma cell line DES-AOAA-14D5 as described in claim 1.

3. A colloidal gold immunochromatographic test strip, characterized in that, The antigen used was selected from the decarboxymethyl tylosin-oxycarboxymethyl hydroxylamine complete antigen, and its molecular structure is as follows: The antibody used is the monoclonal antibody as described in claim 2.

4. The application of the monoclonal antibody as described in claim 2 for the simultaneous detection of tylosin and tilmicosin in milk using colloidal gold immunochromatographic test strips.

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