CN110343669B - A hybridoma cell line DNC secreting anti-triclabendazole monoclonal antibody and its application - Google Patents

Abstract

A hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain DNC secreting the anti-triclabendazole monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17395. The monoclonal antibody secreted by the hybridoma cell strain DNC has good affinity and high sensitivity to triclabendazole and 50% inhibition concentration IC of triclabendazole₅₀Is 1.77ng/mL, can be used for preparing an immunoassay kit and a colloidal gold test strip of the triclabendazole, and provides a powerful detection method and means for the detection of the triclabendazole in animal-derived food.

Description

Hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof

Technical Field

The invention relates to a hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.

Background

Triclabendazole belongs to a novel benzimidazole type anthelmintic, has good expelling and killing effects on fasciola hepatica, fasciola gigantica and fasciola frontalis in various developmental stages of cattle and sheep, and has low toxicity. Therefore, it is widely used in veterinary clinical practice for the prevention and treatment of trematosis in ruminant livestock.

A few reports about a method for detecting residual triclabendazole drug in animal tissues exist at home and abroad, and the method mainly adopts a high performance liquid chromatography-fluorescence detector detection method and a liquid chromatography-tandem mass spectrometry method. The extraction methods are also different, and there are liquid-liquid extraction, liquid-solid extraction, etc. The instrumental detection method can carry out quantitative analysis and has lower detection limit, but generally needs expensive instruments and complex operation, has long pretreatment and detection time, and seriously restricts the popularization of the detection methods. The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirement on technical personnel and the like, so that the immunoassay method is suitable for rapid screening of a large number of samples. The invention aims to provide a preparation method of a monoclonal antibody hybridoma cell strain with higher affinity and detection sensitivity to triclabendazole. Lays a foundation for research and development of indirect competition ELISA kits and colloidal gold test strips.

Disclosure of Invention

The monoclonal antibody prepared by the cell strain has good affinity and sensitivity to triclabendazole, and can be used for establishing an enzyme-linked immunosorbent assay method for triclabendazole or establishing a rapid detection method for colloidal gold immunochromatographic test strips.

The technical scheme of the invention is that a hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen, west, of the Korean area, Beijing, and is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17395.

The anti-triclabendazole monoclonal antibody is produced by secreting the hybridoma cell strain DNC which has the preservation number of CGMCC No.17395 and secretes the anti-triclabendazole monoclonal antibody.

The application of the anti-triclabendazole monoclonal antibody is used for detecting triclabendazole residues in food.

The preparation steps of the hybridoma cell strain DNC secreting the anti-triclabendazole monoclonal antibody provided by the invention are as follows:

(1) synthesis of hapten: carboxyl is derived through the reaction of 3-chloroperbenzoic acid, methyl 4-hydroxybutyrate and triclabendazole so as to facilitate the connection of carrier protein;

(2) preparation and identification of immunogen: taking a triclabendazole derivative (TCD-H) as a raw material, connecting the triclabendazole derivative with an amino group of a protein carrier by an activated ester method, separating a complete antigen and an uncoupled small molecular hapten by dialysis after the reaction is finished, and identifying the complete antigen by an ultraviolet absorption scanning method;

(3) immunization of mice: and selecting BALB/c mice of 6-8 weeks old for immunization. Emulsifying immunogen and Freund's adjuvant completely, injecting the emulsified immunogen and Freund's adjuvant into mice subcutaneously at multiple points, adopting Freund's complete adjuvant for first immunization, using Freund's incomplete adjuvant for boosting immunization, mixing the immune dose of the stimulated immunization with normal saline uniformly, and directly injecting the mixture into abdominal cavity; the intervals between immunizations were three weeks. After the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition;

(4) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 2000) method, culturing by HAT culture medium, detecting positive cell holes by indirect ELISA, further determining the inhibition effect of the positive cell holes by indirect competition ELISA, carrying out three times of subcloning on the positive cell holes with the best inhibition by a limiting dilution method, and finally screening to obtain a hybridoma cell strain DNC;

(5) and (3) identification of the properties of hybridoma cell strains: determining by using an enzyme-labeled secondary antibody kit for identifying mouse monoclonal antibody Ig class/subclass; IC (integrated circuit)₅₀Values, cross-reactivity and affinity were determined by ELISA.

The preparation process of the complete antigen adopted by the hybridoma cell strain DNC comprises the following specific steps:

(1) synthesis of hapten:

5g of TCD was first dissolved in 100mL of methylene chloride/tetrahydrofuran (v/v, 1:1), 4g of 3-chloroperbenzoic acid was added, and the resulting mixed solution was stirred at room temperature for 3 hours. Then 100mL of 1M Na₂S₂O₃The solution was added to the above solution and the aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine and concentrated under reduced pressure to give compound C1, which precipitated as a yellow solid;

5g of Compound C1 were dissolved in 100mL of DMF, 4g of NaHS was added, and the mixture was stirred at 90 ℃ overnight. After the reaction was completed, 300mL of deionized water was added, and the aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine and concentrated under reduced pressure to give crude compound C2 as a white solid;

4.0g of Compound C2, 0.6 g of methyl 4-hydroxybutyrate, 6.4g of triphenylphosphine were dissolved in N, N-dimethylformamide 50mL and cooled to 0 ℃.2g of diisopropyl azodicarboxylate are added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and quenched by addition of deionized water. The aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine, MgSO₄Drying and vacuum concentrating to obtain compound C3 as brown liquid;

1.0g of Compound C3 was dissolved in 20mL of 50% tetrahydrofuran aqueous solution, and 0.5g of LiOH-H was added₂O, stirring for 1h at room temperature. After the reaction was completed, the solution was concentrated in vacuo, the pH of the mixed solution was adjusted to 3, 1M hydrochloric acid solution was added, and then the aqueous phase was extracted three times with 20mL of ethanol. The combined organic fractions were washed with 200mL brine in Na₂SO₄Drying, and concentrating under reduced pressure to obtain TCD-H hapten as white solid;

(2) synthesis of complete antigen: taking 4.5mg of the hapten, adding 5.0 mg of EDC, namely 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 3.7mg of N-hydroxysuccinimide NHS, dissolving by using N, N-dimethylformamide DMF, stirring at room temperature, and activating for 6 hours; dissolving 15mg of keyhole limpet hemocyanin KLH in 3mL of carbonate buffer solution CB with the pH value of 0.05M and 9.6; and dropwise adding the activating solution into a KLH solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 3 days, and subpackaging and storing at-20 ℃.

The invention has the beneficial effects that: the anti-triclabendazole monoclonal antibody obtained by the cell strain has good detection sensitivity and affinity for triclabendazole; the invention also provides a novel method for synthesizing the triclabendazole hapten and the immunogen, the synthesis steps are simplified and effective, and the idea and the method for synthesizing the immunogen are provided for the research of people in the future.

Biological material sample preservation: a hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute No. 3, West Lu No.1 institute of North Chen West road, Kyowa, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2019, 3 months and 7 days, and the preservation number is CGMCC No. 17395.

Drawings

FIG. 1 is a synthetic pathway for triclabendazole hapten.

FIG. 2 is a UV absorption spectrum characterization of the immunogen.

FIG. 3 is a standard inhibition curve for triclabendazole monoclonal antibody.

Detailed Description

The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.

According to the invention, a mouse is immunized by a triclabendazole complete antigen, cell fusion and HAT selective medium culture are carried out, and cell supernatants are screened by indirect ELISA and indirect competitive ELISA, so that the monoclonal antibody hybridoma cell strain with good affinity and sensitivity to triclabendazole is finally obtained.

Example 1: preparation of anti-triclabendazole monoclonal antibody hybridoma cell strain DNC

1. Synthesis of hapten: the synthetic path is shown in FIG. 1

5g of TCD was first dissolved in 100mL of methylene chloride/tetrahydrofuran (v/v, 1:1), 4g of 3-chloroperbenzoic acid was added, and the resulting mixed solution was stirred at room temperature for 3 hours. Then 100mL of 1M Na₂S₂O₃The solution was added to the above solution and the aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine and concentrated under reduced pressure to give compound C1, which precipitated as a yellow solid;

5g of Compound C1 were dissolved in 100mL of DMF, 4g of NaHS was added, and the mixture was stirred at 90 ℃ overnight. After the reaction was completed, 300mL of deionized water was added, and the aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine and concentrated under reduced pressure to give crude compound C2 as a white solid;

4.0g of Compound C2, 0.6 g of methyl 4-hydroxybutyrate, 6.4g of triphenylphosphine were dissolved in N, N-dimethylformamide 50mL and cooled to 0 ℃.2g of diisopropyl azodicarboxylate are added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and quenched by addition of deionized water. The aqueous phase was extracted three times with dichloromethane. The combined organic portions were washed with 200mL brine, MgSO₄Drying and vacuum concentrating to obtain compound C3 as brown liquid;

1.0g of Compound C3 was dissolved in 20mL of 50% tetrahydrofuran aqueous solution, and 0.5g of LiOH-H was added₂O, stirring for 1h at room temperature. After the reaction was completed, the solution was concentrated in vacuo, the pH of the mixed solution was adjusted to 3, 1M hydrochloric acid solution was added, and then the aqueous phase was extracted three times with 20mL of ethanol. The combined organic fractions were washed with 200mL brine in Na₂SO₄Drying, and concentrating under reduced pressure to obtain TCD-H hapten as white solid.

2. Synthesis of complete antigen: adding 5.0 mg EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7mg NHS (N-hydroxysuccinimide) into 4.5mg of the above hapten, dissolving with DMF (N, N-dimethylformamide), stirring at room temperature, and activating for 6 h; another 15mg of KLH (keyhole limpet hemocyanin) was dissolved in 3mL of a 0.05M CB (carbonate buffer solution) solution having a pH of 9.6; and dropwise adding the activating solution into a KLH solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 3 days, and subpackaging and storing at-20 ℃.

3. Animal immunization: healthy BALB/c mice 6-8 weeks old were selected for immunization. After a triclabendazole complete antigen (1 mg/mL) is emulsified uniformly with an equal amount of Freund's adjuvant, BALB/c mice are immunized by subcutaneous multi-point injection, and each 100 mu L of the antigen is injected. The Freund complete adjuvant is adopted for the first immunization, the Freund incomplete adjuvant is used for strengthening the immunization, the immune dose is half of that of the former immune dose during the spurting immunization, and the mixture is directly injected into the abdominal cavity after being uniformly mixed with the normal saline; the intervals between immunizations were three weeks. After the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition; the best-suppressed mice were selected, immunized by boosting 18 days after five immunizations, and prepared for fusion.

4. Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 2000) method, and the specific steps are as follows:

(1) taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells;

(2) collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells;

(3) mixing splenocytes and SP2/0 cells at a ratio of 2-10: 1, centrifuging, fusing with PEG for 1 min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 × HAT, adding into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in an incubator.

5. Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened. The screening is divided into two steps: in the first step, positive cell holes are screened by indirect ELISA, and in the second step, triclabendazole is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competition ELISA. And selecting a cell hole with good inhibition on triclabendazole, performing subcloning by using a limiting dilution method, and detecting by using the same method. This was repeated three times to obtain cell line DNC.

6. Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶And (3) collecting ascites from the 7 th day, purifying the ascites by an octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

And (3) carrying out immunoglobulin subtype identification on the monoclonal antibody obtained by ascites purification by using a mouse monoclonal antibody subtype identification kit, wherein the subtype is IgG2b type, and is specifically shown in Table 1.

TABLE 1 subtype identification of triclabendazole monoclonal antibodies

Determination of IC of monoclonal antibody for triclabendazole Using Indirect competitive ELISA₅₀1.77 mu g/L, and verified its IC for tadalafil and the like₅₀And the cross-reactivity ratio are shown in Table 2.

TABLE 2 Trichlorobendazole monoclonal antibody IC for Trichlorobendazole, aminobenzimidazole, clopyralid, amitraz₅₀And cross reaction rate

7. The application of the antibody comprises the following steps: the monoclonal antibody prepared from hybridoma cell strain DNC through in-vivo ascites is applied to an ELISA addition recovery test of triclabendazole, and the method specifically comprises the following steps:

(1) coating 0.1 mu g/mL triclabendazole diluted by Carbonate Buffer Solution (CBS) as a coating source to coat a 96-hole enzyme label plate, coating 100 mu L of triclabendazole in each hole at 37 ℃ for 2 h, washing the plate with PBST washing liquor three times, wherein each time is 200 mu L, each time is 3 min, and patting to dry;

(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3 min, and drying;

(3) phosphate Buffered Saline (PBS) is used for preparing 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 mu g/L of triclabendazole standard solution respectively. Respectively adding the standard solution and the extract of the sample to be detected into the closed enzyme label plate, wherein each hole is 50 mu L, each sample is repeatedly provided with 3 holes, and each hole is added with 50 mu L1: 16000 diluted anti-triclabendazole monoclonal antibody, reacting for half an hour at 37 ℃, washing and drying;

(4) add 100 μ L per well of PBS 1: reacting a goat anti-mouse IgG secondary antibody marked by HRP and diluted by 3000 at 37 ℃ for half an hour, washing the plate and drying the plate;

(5) adding 100 μ L of TMB color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well₂SO₄Stopping solution, measuring the light absorption value at 450 nm;

(6) adding and recovering and sample pretreatment: 5g of fresh or rewarming (refrigerated storage) mutton is taken, and three triclabendazole standard substances with different dosages are added, wherein the dosages are respectively 5 ng, 10ng and 20 ng. Placing the mixture into a 50mL centrifuge tube, slowly dropping 1mL of 50% potassium hydroxide solution, fully oscillating on a vortex mixer, slowly dropping 20mL of ethyl acetate, oscillating on the vortex mixer for 10min, and then placing the mixture into a centrifuge to centrifuge for 5min at 3000 r/min. 4mL of the supernatant was removed from the other centrifuge tube, blown dry with nitrogen, and 1mL of 10% methanol in PBS was added for reconstitution, and 50. mu.L was used for detection. The additive recovery tests were performed by indirect competitive ELISA with recovery rates of 91.2%, 102.1%, 99.4%, respectively.

Solution preparation:

carbonate Buffer (CBS): weighing Na₂CO₃ 1.59g，NaHCO₃2.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;

phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2gKCl，0.24g KH₂PO₄，3.62g Na₂HPO₄·12 H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

PBST: PBS containing 0.05% Tween 20;

TMB color development liquid: solution A: na (Na)₂HPO₄·12H₂18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The volume ratio of the solution B to the solution B is 1: 5, mixing to obtain the TMB color developing solution which is mixed at present.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. That is, all equivalent changes and modifications made within the scope of the present invention should be considered to be within the technical scope of the present invention.

Claims (4)

1. A hybridoma cell line DNC that secretes anti-triclabendazole monoclonal antibody has been deposited in CGMCC, General Microbiology Center of China Microorganism Culture Collection Management Committee, address No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, classified as monoclonal cell line, preservation date March 7, 2019, preservation number CGMCCNo.17395. 2 . An anti-triclabendazole monoclonal antibody, characterized in that: it is secreted and produced by the hybridoma cell line DNC that secretes the anti-triclabendazole monoclonal antibody with the deposit number of CGMCC No. 17395 described in claim 1 . 3. the application of the described anti-triclabendazole monoclonal antibody of claim 2, is characterized in that: be used for the detection of triclbendazole residue in food. 4. the preparation method of the complete antigen that hybridoma cell line DNC described in claim 1 adopts is characterized in that concrete steps are as follows: (1) Synthesis of hapten: First, 5 g of TCD was dissolved in 100 mL of dichloromethane/tetrahydrofuran (v/v, 1:1), 4 g of 3-chloroperbenzoic acid was added, and the resulting mixed solution was stirred at room temperature for 3 hours; then 100 mL of 1M Na ₂ S ₂ The O solution was added to the above solution, and the aqueous phase was extracted _three times with dichloromethane; the combined organic part was rinsed with 200 mL of brine, and concentrated under reduced pressure to obtain compound C1, which was precipitated as a yellow solid; Dissolve 5g of compound C1 in 100mL of DMF, add 4g of NaHS, and stir at 90°C overnight; after the reaction, add 300mL of deionized water, and extract the aqueous phase with dichloromethane three times; the combined organic part is washed with 200mL of brine, and concentrated under reduced pressure to obtain Crude compound C2 is a white solid; 4.0 g of compound C2, 0.6 g of methyl 4-hydroxybutyrate, and 6.4 g of triphenylphosphine were dissolved in 50 mL of N, N-dimethylformamide, cooled to 0°C; 2 g of diisoazodicarboxylate was added dropwise propyl ester; the reaction mixture was stirred at 0 °C for 2 hours, and quenched by adding deionized water; the aqueous phase was extracted three times with dichloromethane; the combined organic part was washed with 200 mL of brine, dried over MgSO ₄ , and concentrated in vacuo to give compound C3, as brown liquid; 1.0 g of compound C3 was dissolved in 20 mL of 50% tetrahydrofuran aqueous solution, 0.5 g of LiOH-H ₂ O was added, and stirred at room temperature for 1 h; after the reaction, the solution was concentrated in vacuo, the pH of the mixed solution was adjusted to 3, and 1 M hydrochloric acid solution was added , and then extracted the aqueous phase three times with 20 mL of ethanol; the combined organic part was rinsed with 200 mL of brine, dried over Na ₂ SO ₄ , and concentrated under reduced pressure to obtain TCD-H hapten as a white solid; (2) Synthesis of complete antigen: take 4.5mg of the above hapten, add 5.0mg EDC, namely 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 3.7mg N- Hydroxysuccinimide NHS, dissolved in N,N-dimethylformamide DMF, stirred at room temperature, and activated for 6h; another 15mg of keyhole limpet hemocyanin KLH was dissolved in 3mL, 0.05M, pH9.6 carbonate buffer solution CB; the above activation solution was added dropwise to the KLH solution, and after stirring at room temperature overnight, the immunogen was removed for PBS dialysis for 3 days, and stored in aliquots at -20°C.

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