Hybridoma cell strain secreting lasalocid monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a lasalocid monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention has high sensitivity and specificity to lasalocid, and the IC50 value of lasalocid is 200ng / mL, so that trace lasalocid in a sample can be detected. In addition, the monoclonal antibody disclosed by the invention has no cross reaction on analogues of lasalocid, such as salinomycin, salinomycin, monensin and Hainanmycin, so that the monoclonal antibody has good specificity. The invention provides an immunological method for detecting the content of lasalocid in a sample, and has practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to a hybridoma cell line secreting lasalocidin monoclonal antibody and application thereof. Background Art

[0002] Coccidiosis is an intestinal protozoan infection that particularly affects poultry. These infections lead to economic losses due to decreased growth rate, reduced egg production and increased mortality. To control the disease, a range of different coccidiostats are used as feed additives. Currently, the European Union (EU) has approved 11 different anticoccidiostats for the prevention of coccidiosis in chickens, turkeys and rabbits (Regulation (EC) No 1831 / 2003). The drugs currently used to treat coccidiosis in chickens mainly include sulfonamides and polyether ionophore antibiotics. The disease is characterized by high incidence, and long-term intervention is often required during the breeding process. Long-term medication will bring certain toxic side effects. If the dosage of the drug cannot be scientifically controlled, it is easy to develop drug resistance. In addition to developing drug resistance, these drugs may also develop cross-resistance, so the effectiveness of the drugs will be reduced. We must continue to increase research and development efforts on the existing basis to find drugs with better therapeutic effects. However, the development process of new drugs is very long. When using drugs, we must ensure that the effects of the drugs can be fully exerted, use the drugs rationally and scientifically, and continuously improve the prevention and control of poultry coccidiosis.

[0003] Lasalocid (LAS), also known as lasalocid, is an antibiotic produced by fermentation of Streptomyces lasalori. Lasalocid usually exists in the form of sodium salt in production applications, also known as lasalocid sodium. Lasalocid has the function of regulating rumen fermentation, which can improve and improve the feed utilization rate of ruminants. Lasalocid can also act on coccidia sporozoites and first-generation merozoites, so it can be used to prevent coccidia infection in livestock and poultry.

[0004] At present, in order to prevent consumers from being harmed by drug residues, many countries have stipulated the maximum limit standards of lasalocid in different tissues. In China, the maximum amount of lasalocid in beef and milk products is 700μg / kg; the maximum amount of lasalocid in chicken products is 1200μg / kg; the maximum amount of lasalocid in egg products is 400μg / kg. The content of lasalocid in food should be strictly controlled. Therefore, the research on the detection method of lasalocid is of great significance.

[0005] In recent years, the detection of polyether antibiotics is mainly instrumental analysis. Chen Tong et al. used liquid chromatography-tandem mass spectrometry to detect the residues of four polyether antibiotics in feed. The concentrations of the four antibiotics were linearly correlated in the range of 20.0-800 ng / mL, and the detection limit was less than 3.58 μg / kg; Wang Yingyu et al. established a method for detecting anticoccidial drug residues in chicken skin and adipose tissue by ultra-high performance liquid chromatography-tandem mass spectrometry. The method was linearly correlated in the concentration range of 15-200 μg / kg, among which the detection limit of lasalocid was 5.0 μg / kg and the quantitative limit was 15.0 μg / kg. These methods are complicated to operate and the sample processing is cumbersome, which is not conducive to the detection of large quantities of samples in actual production. It is urgent to establish a detection method with simple operation, high sensitivity and strong specificity. Enzyme-linked immunosorbent assay (ELISA) is an efficient, sensitive and rapid detection method, and is also increasingly used in food safety detection. However, the monoclonal antibodies used in the enzyme-linked immunosorbent assay in the prior art have poor sensitivity and specificity. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a monoclonal antibody with high specificity and high sensitivity to lasalocid.

[0007] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting lasalocid monoclonal antibody and its application. The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to lasalocid, wherein the IC 50 The value is 200ng / mL, and there is no cross reaction to the structural analogs of lasalocid such as salinomycin, methyl narasin, monensin and hainanmycin, so it can accurately detect low concentrations of lasalocid.

[0008] The first object of the present invention is to provide a hybridoma cell strain, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46226.

[0009] Furthermore, the hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is as shown in Formula I:

[0010]

[0011] Furthermore, the complete antigen is obtained by coupling the hapten to a carrier protein.

[0012] Furthermore, the carrier protein includes bovine serum albumin.

[0013] The second object of the present invention is to provide a use of the hybridoma cell line in detecting lasalocid.

[0014] The third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line.

[0015] The fourth object of the present invention is to provide a use of the above monoclonal antibody in detecting lasalocid.

[0016] A fifth object of the present invention is to provide a detection product for lasalocid, which comprises the above-mentioned monoclonal antibody.

[0017] Furthermore, the detection product also includes a coating source.

[0018] Furthermore, the coating agent is prepared by coupling a hapten to a carrier protein, wherein the carrier protein comprises chicken ovalbumin.

[0019] Beneficial effects of the present invention:

[0020] The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity and specificity to lasalocid, wherein the IC 50 The value is 200ng / mL, and there is no cross reaction to the structural analogs of lasalocid such as salinomycin, methyl narasin, monensin and hainanmycin, so it can accurately detect low concentrations of lasalocid.

[0021] Biomaterial Deposit

[0022] The monoclonal cell line EZH was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No. 46226, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0024] Figure 1 is the inhibition standard curve of lasalocid monoclonal antibody;

[0025] Figure 2 The invention relates to a method for preparing lasalocid hapten. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0027] The culture medium involved in the following examples is as follows:

[0028] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine 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, p-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, niacinamide 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 in a small amount of double distilled water respectively and mix them, add double distilled water to about 800 mL and mix well, adjust the pH to 9.6, add double distilled water to 1000 mL, and store at 4°C for later use.

[0031] Phosphate buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and made up to 1000 mL;

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

[0033] TMB colorimetric solution: Solution A: Na2HPO4 . 12H2O 18.43g, citric acid 9.33g, pure water to 1000mL; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain TMB colorimetric solution, which should be mixed before use.

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

[0035] Lasalocid inhibition rate detection method: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.3, 0.1, 0.03 and 0.01 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the best working point, the lasalocid standard was diluted to 8 concentrations (0, 7.5, 22.2, 66.7, 200.0, 600.0, 1800.0 and 5400.0 ng / mL), and the ic-ELISA operation steps were followed. Finally, OriginPro 8.5 was used to make a graph (the results are shown in Figure 2). Figure 1 As shown), obtain the standard inhibition curve of lasalocid and calculate IC 50 .

[0036] Example 1: Synthesis of Lasalocid Hapten

[0037] like Figure 2 As shown, the product having a carboxyl group, namely the hapten LAS, is obtained by reacting the lasalocid original drug with ethyl 4-aminobutyrate. The specific steps are:

[0038] Reaction A solution: Weigh lasalocid (50 mg, 0.81 mmol), NHS (150 mg, 1.013 mmol) and EDC (260 mg, 1.013 mmol) and dissolve in DMF, activate for 30 min, and slowly add 4-aminobutyric acid ethyl ester (0.675 mmol) to the activated lasalocid to generate ester. The mixture is then purified and evaporated to dryness. Next, 1M sodium hydroxide and anhydrous ethanol are mixed in a volume ratio of 1:2. 1M sodium hydroxide and anhydrous ethanol are added in a volume ratio of 1:2, and the reaction is carried out at a temperature of 60°C for 4h, and then extracted with ethyl acetate. After stratification, the upper solution is collected and blown dry under nitrogen to obtain a crude sample.

[0039] The crude sample was purified by recrystallization and dissolved in methanol. After being completely dissolved, the crude sample was allowed to stand overnight at room temperature to precipitate a brown precipitate, which was filtered with filter paper, washed with petroleum ether 2-3 times, and dried at 50°C to obtain a brown precipitate (37.5 mg), which was the lasalocid hapten shown in Formula I. The yield was 75.0%.

[0040]

[0041] Example 2: Preparation of Lasalocid Complete Antigen and Coating

[0042] The hapten was coupled with the carrier protein by the carbodiimide method to obtain the artificial lasalocidin antigen. The hapten prepared in Example 1 was coupled with bovine serum albumin BSA to obtain the complete antigen LAS-EDC-BSA; or coupled with chicken ovalbumin OVA to obtain the coating original LAS-EDC-OVA.

[0043] The preparation method of complete antigen LAS-EDC-BSA is as follows:

[0044] Weigh 0.756 mg of the lasalocid hapten prepared in Example 1, 0.618 mg of N-hydroxysuccinimide, and 1.0 mg of 1-ethylcarbodiimide hydrochloride, dissolve them in 300 μL of N,N-dimethylformamide (called solution A), and react at room temperature for 4-6 hours with stirring. Weigh 6.0 mg of BSA (LAS to BSA molar ratio is 20:1), add 2 mL of carbonate buffer solution (called solution B after BSA protein is dissolved), add solution A dropwise into solution B at room temperature, adjust the pH of the mixture to 8-9 with 1 M NaOH solution, and react at room temperature overnight to obtain the conjugate LAS-EDC-BSA.

[0045] Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, store it in deionized water at 4°C for later use; put the conjugate LAS-EDC-BSA conjugate solution into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours. Dialyze for 3 days to obtain the complete antigen LAS-EDC-BSA, take it out and store it at -20°C.

[0046] The preparation method of LAS-EDC-OVA coating is as follows:

[0047] Weigh 0.756 mg of the lasalocidin hapten prepared in Example 1, 0.618 mg of N-hydroxysuccinimide, and 1.0 mg of 1-ethylcarbodiimide hydrochloride, dissolve them in 300 μL of N,N-dimethylformamide (called solution A), and react at room temperature for 4-6 hours with stirring. Weigh 6.0 mg of chicken egg white albumin (OVA) (LAS to BSA molar ratio is 20:1), add 2 mL of carbonate buffer solution (OVA protein is dissolved and called solution B), add solution A dropwise into solution B at room temperature, adjust the pH of the mixture to 8-9 with 1M NaOH solution, and react at room temperature overnight to obtain the conjugate LAS-EDC-OVA.

[0048] Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, store it in deionized water at 4°C for later use; put the conjugate LAS-EDC-OVA conjugate solution into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours. Dialyze for 3 days to obtain the complete antigen LAS-EDC-OVA, take it out and store it at -20°C.

[0049] Example 3: Preparation of hybridoma cell lines secreting lasalocidin monoclonal antibodies

[0050] 1. Immunization of mice

[0051] For the first immunization, the complete antigen of lasalocid was mixed with an equal amount of complete Freund's adjuvant and emulsified at a dose of 100 μg / mouse for injection, and BALB / c mice were immunized by multiple subcutaneous injections on the back of the neck; after a lapse of 4 weeks, a booster immunization was performed, and the dose of the complete antigen was halved (50 μg / mouse) and mixed with incomplete Freund's adjuvant for emulsification, and the intervals between subsequent booster immunizations were 3 weeks; for the sprint immunization, the dose was halved again (25 μg / mouse), and the complete antigen was diluted with saline and injected intraperitoneally into the mice. After the third immunization, the mice were tail-cut and blood was collected for testing, and the titer and IC of the mouse serum were tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Low mice were fused;

[0052] 2. Cell fusion and screening

[0053] 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:

[0054] a. Collect 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. Before fusion, the number of SP2 / 0 tumor cells should reach 1-4×10 7 , to ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0055] b. After killing the mouse by dislocating the neck, immediately sterilize it in 75% alcohol, soak it for about 5 minutes, remove the mouse spleen by aseptic operation, grind it moderately with a syringe rubber tip and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect it in a 50mL sterile centrifuge tube and centrifuge it at 1200r / min for 8 minutes. Wash the spleen cells with RPMI-1640 medium, pick out the larger tissue impurities, and repeat the operation three times. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside;

[0056] c. Fusion process 7min: In the first minute, 1mL of PEG 4000 was added to the cells from slow to fast speed; in the second minute, the tube was left to stand and held tightly with both hands. In the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 1mL of RPMI-1640 medium was added every 30s; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s. Then, the cells were incubated at 37°C for 5min. Centrifuged at 800r / min for 10min, the supernatant was discarded, the cells in the centrifuge tube were gently knocked apart, and RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT was added thereto, and added to the 96-well cell plate at 200μL / well, and cultured in a 37°C, 5% CO2 incubator.

[0057] 3. Cell screening and cell line establishment

[0058] On the third day after cell fusion, the fused cells were half-changed with HAT medium; on the fifth day, the medium was completely changed with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on the seventh day, the cell supernatant was taken for screening. The screening was divided into two steps: the first step was to screen the positive cell wells by ic-ELISA method, and the second step was to select the lasalocid standard and measure the inhibitory effect of the positive cells by ic-ELISA method. The cell wells with better inhibition of the lasalocid standard were selected, subcloned by limiting dilution method, and tested by the same method after seven days. Four subclonings were performed according to the above method, and finally a hybridoma cell line secreting lasalocid monoclonal antibody was obtained.

[0059] Example 4: Preparation and identification of lasalocidin monoclonal antibodies

[0060] 8-10 week old BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, 2 × 10 6 Lasalocidin hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-saturated ammonium sulfate method. Under acidic conditions, caprylic acid can precipitate other miscellaneous proteins except IgG immunoglobulin in the ascites, and then centrifuge and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibodies, centrifuge, discard the supernatant, dissolve with 0.01M PBS solution (pH 7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20℃.

[0061] (1) Coating: The original LAS-EDC-OVA was diluted 3-fold from 1 μg / mL with 0.05 M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37°C for 2 h.

[0062] (2) Washing: Pour off the solution in the plate and wash with washing solution three times, each time for 3 minutes.

[0063] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 h. Wash and dry for later use.

[0064] (4) Sample addition: dilute the antiserum (blood is collected from mice by tail amputation, and then diluted with antibody diluent to obtain the antiserum) from 1:1000 onwards and add to the coated wells of each dilution at 100 μL / well and react at 37°C for 30 min. After thorough washing, add 1:3000 diluted HRP-sheep anti-mouse IgG at 100 μL / well and react at 37°C for 30 min.

[0065] (5) Color development: Take out the ELISA plate, 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.

[0066] (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 an ELISA reader. 450 value.

[0067] Determination of IC of monoclonal antibody lasalocidin by ic-ELISA 50 The value is: 200ng / mL, indicating that it has good sensitivity to lasalocid and can be used for lasalocid immunoassay detection.

[0068] Specificity experiments

[0069] Determination of IC of monoclonal antibodies against lasalocid using indirect competitive ELISA 50 The IC values ​​of the samples were 200 ng / mL, and the IC values ​​of the samples were verified for salinomycin, methyl salinomycin, monensin, hainanmycin and other compounds. 50 And the cross-reaction rate, the cross-reaction value is calculated as follows:

[0070] Cross-reaction rate (%) = (IC 50 / IC of other compounds 50 )×100%, as shown in Table 1.

[0071] Table 1 IC of lasalocid monoclonal antibodies against lasalocid and cross-linkers 50 and cross-reaction rate

[0072] <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate Lasalocid 200 100％ Salinomycin ＞10000 ＜1％ Narasin ＞10000 ＜1％ Monensin ＞10000 ＜1％ Hainanmycin ＞10000 ＜1％

[0073] The crossover of the monoclonal antibody to lasalocid is 100%, and the crossover to salinomycin, methyl narasin, monensin, and hainanmycin is less than 1%. This indicates that the monoclonal antibody obtained by the present invention has a high sensitivity (IC 50 value is 200ng / mL) and has high specificity (cross-reaction rate with analogues is less than 1%).

[0074] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line, characterized in that The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46226.

2. The hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by 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 coupling the hapten to a carrier protein.

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

5. Use of the hybridoma cell line according to any one of claims 1 to 4 in detecting lasalocid.

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

7. Use of the monoclonal antibody according to claim 6 in detecting lasalocid.

8. A lasalocid detection product, characterized in that: The detection product comprises the monoclonal antibody according to claim 6.

9. The detection product according to claim 8, characterized in that: The detection product also includes a coating source.

10. The detection product according to claim 9, characterized in that: The coating is prepared by coupling hapten with carrier protein, wherein the carrier protein comprises chicken ovalbumin.

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