CN110747173B

CN110747173B - Hybridoma cell strain HOT capable of secreting tricaine monoclonal antibody and application thereof

Info

Publication number: CN110747173B
Application number: CN201911272054.2A
Authority: CN
Inventors: 胥传来; 沈心怡; 匡华; 徐丽广; 刘丽强; 宋珊珊; 吴晓玲; 胡拥明; 曹玉朋
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2022-03-15
Anticipated expiration: 2039-12-12
Also published as: CN110747173A

Abstract

A hybridoma cell strain HOT secreting a tricaine monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain HOT for secreting the tricaine monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18514. The tricaine monoclonal antibody secreted and obtained by the strain is used for analyzing and detecting the residual tricaine in the aquatic product safety detection. The tricaine monoclonal antibody obtained by the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) to tricaine₅₀A value of 41.8ng/mL, a crossing rate of the tricaine analog of less than 10%, and a crossing rate = (IC of tricaine)₅₀IC of/analogue₅₀) X 100 percent), can realize the detection of the residual quantity of the tricaine in the aquatic products, and has practical application value.

Description

Hybridoma cell strain HOT capable of secreting tricaine monoclonal antibody and application thereof

Technical Field

The invention relates to a hybridoma cell strain HOT secreting a tricaine monoclonal antibody and application thereof, belonging to the field of food safety immunodetection.

Background

Tricaine (Tricaine, TMS), chemical name ethyl-3-aminobenzoate methanesulfonate (MS-222), which is a white crystal or powder, readily soluble in water; the anesthetic is a fishery anesthetic approved by the FDA in the United states and applicable to edible fishes, and has been widely applied to living transportation and marked releasing of the fishes due to the advantages of low use concentration, long acting time, quick recovery, no toxic or side effect and the like. It can be conducted to fish brain sensing center through gill and skin in water solution, and can inhibit fish reflex and activity, slow fish movement, slow respiration, and reduce metabolism, thereby entering into quasi-dormancy state. However, the amount of the tricaine used by aquatic product distributors in the market is not always considered, and after the aquatic products are transported to a destination, the aquatic products are not temporarily cultured in a drug-off period, so that high anesthetic residues still exist in live fish bodies of consumers. This brings with it the potential risks for the safety of consumption of the water products, which have an adverse effect on the health of humans, whose side effects are mainly manifested as allergic reactions, urinary and hematopoietic disorders, and possibly even carcinogenesis. In order to ensure the edible safety of aquatic products, strict international regulations are put on the use of tricaine in aquatic products. For example, the FDA in the united states stipulates that edible fish anesthetized with tricaine must be sold in the market after a drug holiday of 21 d, and the drug holiday stipulated in canada is 5 d. And China has no clear related use and limit standards for the anesthetic used on the edible aquatic products after entering the circulation link.

At present, methods for detecting the residual tricaine include colorimetry, gas chromatography, liquid chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. The methods have the advantages of high sensitivity, accurate qualitative determination and the like; meanwhile, the method has the defects of complicated sample pretreatment, expensive used equipment, difficulty in popularization and use in a larger range and the like, and is not suitable for rapid detection of a large number of samples. Therefore, it is very important to establish a high-efficiency, sensitive and rapid detection method for tricaine.

The enzyme-linked immunosorbent assay (ELISA) is an efficient and rapid detection method, has the advantages of simple sample pretreatment, low detection cost, simple and convenient operation and the like, is suitable for the field rapid detection of a large number of samples, and is widely applied to the analysis of residues of aquatic products and veterinary drugs. The precondition for detecting the tricaine by using the enzyme linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to the tricaine is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the tricaine is very critical.

Disclosure of Invention

The invention aims to overcome the defects and provide a hybridoma cell strain HOT secreting a tricaine monoclonal antibody and application thereof. The tricaine monoclonal antibody secreted by the hybridoma cell strain has better detection sensitivity and specificity (IC) to tricaine₅₀A value of 41.8ng/mL, less than 10% crossover to the tricaine analog, = (IC of tricaine)₅₀IC of/analogue₅₀) X 100 percent) can be used for establishing an immunological detection method of the tricaine and detecting the residual of the tricaine in the aquatic products.

According to the technical scheme, the hybridoma cell strain HOT secreting the tricaine monoclonal antibody is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms, and is classified and named as a monoclonal cell strain by the microbiological research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Kyoho, wherein the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18514.

The invention provides a preparation method of a hybridoma cell strain secreting a tricaine monoclonal antibody, which comprises the following steps:

(1) preparing a tricaine complete antigen by using a tricaine hapten, and preparing the obtained tricaine complete antigen into an antigen-containing Freund adjuvant and an antigen-containing incomplete Freund adjuvant;

(2) injecting the obtained Freund adjuvant containing the antigen into a BALB/c mouse body through back subcutaneous injection for multiple times of immunization, wherein the complete Freund adjuvant containing the antigen is adopted for the first immunization, and the incomplete Freund adjuvant containing the antigen is adopted for the boosting immunization;

(3) collecting blood from the mice after the 3 rd immunization process on the 7 th day, detecting the serum immune titer and the immune suppression capability of the mice through indirect ELISA, and screening the mice with high content of the tricaine antibody in the serum to obtain the immunized mice;

(4) performing last boosting immunization on the screened mice by using incomplete Freund adjuvant, and performing thrust immunization by intraperitoneal injection, wherein the thrust immunization is performed by using a tricaine complete antigen without Freund adjuvant;

(5) fusing splenocytes and myeloma cells of BALB/c mice by a polyethylene glycol (PEG 4000) method after 3 days of completion of the spurt immunization, culturing the fused cells by RPMI-1640 culture medium, detecting positive cell pores by using indirect ELISA, further determining the inhibition effect of the positive cell pores by using an indirect competitive ELISA method, carrying out subcloning on the positive cell pores with the best inhibition for 3 times by a limiting dilution method, and finally screening out a hybridoma cell strain HOT capable of secreting a tricaine monoclonal antibody.

The multiple immunization process in the steps (2) and (4) comprises 1 first immunization, 4 boosting immunizations and 1 sprint immunization; one month is left between the first immunization and the boosting immunization, 21 days are left between the boosting immunization, and 18-21 days are left between the boosting immunization and the sprint immunization; the first immunization dose is 100 mug/mouse, the boosting immunization dose is 50 mug/mouse, and the sprint immunization dose is 25 mug/mouse.

The molecular formula of the tricaine hapten TMS-1 in the step (1) is as follows:

；

structural analog mol. Wt: 137.14.

The molecular formula of the tricaine complete antigen in the step (1) is as follows:

。

the preparation method of the tricaine complete antigen in the step (1) comprises the following steps: weighing 2.4mg of tricaine hapten TMS-1, 8.0mg of N-hydroxysuccinimide NHS, dissolving in 300 mu L N of N-dimethylformamide DMF, and stirring at room temperature for reaction for 10 min; weighing 11.4mg dicyclohexylcarbodiimide DCC, fully dissolving with 100 microliter DMF, adding into TMS-1 solution, stirring at room temperature for 6-8h to obtain solution A; taking 7mg KLH, diluting to 5mg/mL by using 0.01M carbonate buffer CBS to obtain solution B, dropwise adding the solution A into the solution B slowly, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain the tricaine complete antigen TMS-1-KLH.

The invention provides an application of the hybridoma cell strain HOT secreting the tricaine monoclonal antibody or the preparation method of the hybridoma cell strain HOT secreting the tricaine monoclonal antibody in preparation of the tricaine monoclonal antibody.

The invention provides a tricaine monoclonal antibody which is secreted and generated by a hybridoma cell strain HOT with the preservation number of CGMCC No. 18514.

The invention provides a preparation method of the tricaine monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain HOT with the preservation number of CGMCC No.18514 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and storing the obtained low-temperature monoclonal antibody.

In one embodiment of the invention, the method is to take BALB/c mice 8-10 weeks old, inject 1mL paraffin oil into the abdominal cavity of each mouse, and inject 1X 10 paraffin oil into the abdominal cavity of each mouse 7 days later⁶Collecting ascites from 7 days, purifying the ascites by octanoic acid-ammonium sulfate method,the obtained monoclonal antibody is stored at-20 ℃.

The invention provides an application of the tricaine monoclonal antibody or the preparation method of the tricaine monoclonal antibody in analysis and detection of the residual tricaine in aquatic product safety detection.

The invention has the beneficial effects that: the tricaine monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has better detection sensitivity and specificity (IC) to tricaine₅₀A value of 41.8ng/mL, less than 10% crossover to the tricaine analog, = (IC of tricaine)₅₀IC of/analogue₅₀）×100%）。

Biological material sample preservation: a hybridoma cell strain HOT secreting a tricaine monoclonal antibody is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 10 months and 14 days and the preservation number of CGMCC No. 18514.

Drawings

FIG. 1 is a standard curve of the inhibition of tricaine by a tricaine 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.

The media involved in the following examples are as follows:

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, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.

The reagents involved in the following examples are as follows:

carbonate Buffer (CBS): weighing Na₂CO₃ 1.59 g，NaHCO₃2.93 g 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, 0.2g KCl, 0.2g KH₂PO₄，2.9g 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;

antibody dilution: adding 0.1% gelatin into PBS;

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. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.

The detection methods referred to in the following examples are as follows:

the method for detecting the inhibitory rate of the tricaine comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.03, 0.1, 0.3 and 1. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03, 0.1, 0.3 and 1. mu.g/mL with antibody diluent. After selecting the optimal working point, the tricaine standard was diluted to 8 concentrations (0, 2, 6, 18, 50, 150, 450, 1350 ng/mL), subjected to the IC-ELISA protocol, plotted against originPro 8.5 (results are shown in FIG. 1), and IC standard inhibition curves were calculated₅₀。

Example 1: synthesis of tricaine hapten

Because the small molecule of the tricaine has no immunogenicity, the small molecule of the tricaine can not stimulate a mouse to generate immune response so as to generate an antibody, the tricaine needs to be coupled to protein by a protein connection technology so as to obtain the immunogenicity; active groups commonly used in the protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, and a tricaine structural analogue (TMS-1) is used as a hapten in the experiment.

The structure of the tricaine hapten (TMS-1) is as follows:

structural analog mol. Wt: 137.14.

Example 2: synthesis of tricaine complete antigen

Weighing 2.4mg of tricaine hapten (TMS-1) and 8.0mg of N-hydroxysuccinimide (NHS), dissolving in 300 mu L N of N-Dimethylformamide (DMF), and stirring at room temperature for reaction for 10 min; then 11.4mg Dicyclohexylcarbodiimide (DCC) was weighed out and dissolved in 100. mu.L DMF, and added to TMS-1 solution, and the reaction was stirred at room temperature for 6 to 8 hours (referred to as solution A). Taking 7mg of KLH, diluting the KLH to 5mg/mL (called as solution B) by using 0.01M Carbonate Buffer Solution (CBS), then slowly adding the solution A into the solution B dropwise, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen TMS-1-KLH, and identifying by a UV absorption scanning method.

Example 3: synthesis of tricaine coatingen

Dissolving 4.0mg of tricaine hapten (TMS-1) in 400 mu L of anhydrous N, N-Dimethylformamide (DMF), and stirring in an ice bath to obtain a tricaine hapten (TMS-1) solution; adding 8.3 mu L of tri-n-butylamine into the TMS-1 solution, carrying out ice bath reaction for 15min, adding 4.5 mu L of isobutyl chloroformate, and carrying out ice bath reaction for 1h to obtain solution A; diluting 7mg of egg albumin (OVA) with 1.4mL of 0.01mmol/L Carbonate Buffer Solution (CBS) to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; and dialyzing the reaction solution by using a PBS solution to remove unreacted small molecule hapten to obtain the coating antigen (TMS-1-OVA).

Example 4: preparation of hybridoma cell strain HOT secreting tricaine monoclonal antibody

1. Obtaining animal immunity: mixing and emulsifying a tricaine complete antigen and an equivalent amount of Freund's adjuvant, and performing neck and back subcutaneous multipoint injection immunization (except puncture immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;

2. cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:

a. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200 rpm, 8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;

b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂In the incubator, SP2/0 tumor cell number is required to reach (1-4) x 10 before fusion⁷Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;

c. the fusion process is 7 min: 1min, 1mL of PEG4000 was added to the cells dropwise from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; at 5min and 6min, 2mL of RPMI-1640 was added dropwise over 1minA culture medium; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800 rpm, 10 min), discarding the supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, placing at 37 ℃ and 5% CO₂Culturing in an incubator;

3. 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: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting tricaine as a standard substance, and determining the inhibition effect of positive cells by using the ic-ELISA method;

selecting a cell hole with good inhibition on a tricaine standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days;

and subcloning for three times according to the method to finally obtain the tricaine monoclonal antibody cell strain HOT.

Example 5: preparation and identification of tricaine monoclonal antibody

Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Tricaine hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.

Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Determination of IC of tricaine monoclonal antibody Using Indirect competitive ELISA₅₀The value is 41.8ng/mL, the crossing rate to the tricaine analogue is less than 10 percent, which shows that the sensitivity to the tricaine is good, and the method can be used for the tricaineAnd (4) detecting by immunoassay.

(Cross Rate = (IC of tricaine)₅₀IC of/analogue₅₀）×100%）。

Example 6: application of tricaine monoclonal antibody

The monoclonal antibody prepared from hybridoma cell strain HOT through in-vivo ascites is applied to an ELISA (enzyme-linked immuno sorbent assay) addition recovery test of tricaine, and the method specifically comprises the following steps:

(1) coating a 96-well enzyme label plate with coating antigen with the concentration of 0.3 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor for three times, wherein 200 mu L of the washing liquor in each well is used for 3min, and drying by beating;

(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 3min, and drying;

(3) respectively preparing 0, 2, 6, 18, 50, 150, 450 and 1350ng/mL tricaine standard solutions by using Phosphate Buffered Saline (PBS), respectively adding the standard solutions and the extract of a sample to be detected into the closed enzyme label plate, respectively, adding 50 mu L of each sample into each hole, repeating 3 holes for each sample, adding 50 mu L of an anti-tricaine monoclonal antibody diluted by 1:32000 into each hole, reacting at 37 ℃ for 0.5h, washing the plate, and drying;

(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;

(5) adding 100 μ L of TMB 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;

the standard curve for the inhibition of the tricaine monoclonal antibody is shown in FIG. 1, and the IC of the tricaine monoclonal antibody is determined by IC-ELISA₅₀The value is 41.8ng/mL, which indicates that the antibody has better sensitivity to the tricaine and can be used for immunoassay detection of the tricaine.

Claims

1. A hybridoma cell strain HOT secreting tricaine monoclonal antibodies is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute No. 3, West Lu No.1, North Cheng, south China area, Beijing city, and is classified and named as a monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18514.

2. A tricaine monoclonal antibody characterized by: it is secreted and produced by hybridoma cell strain HOT with the preservation number of CGMCC No.18514 as claimed in claim 1.

3. The use of the tricaine monoclonal antibody of claim 2, wherein: the method is used for analyzing and detecting the residual tricaine in the safety detection of aquatic products.

4. The hybridoma cell strain HOT secreting a tricaine monoclonal antibody according to claim 1, wherein: the molecular formula of the tricaine complete antigen adopted for preparing the cell strain is as follows:

。

5. the hybridoma cell line HOT secreting a tricaine monoclonal antibody according to claim 4, wherein: the preparation method of the tricaine complete antigen comprises the following steps: weighing 2.4mg of tricaine hapten TMS-1, 8.0mg of N-hydroxysuccinimide NHS, dissolving in 300 mu L N of N-dimethylformamide DMF, and stirring at room temperature for reaction for 10 min; weighing 11.4mg dicyclohexylcarbodiimide DCC, fully dissolving with 100 microliter DMF, adding into TMS-1 solution, stirring at room temperature for 6-8h to obtain solution A; taking 7mg KLH, diluting to 5mg/mL by using 0.01M carbonate buffer CBS to obtain solution B, dropwise adding the solution A into the solution B slowly, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain the tricaine complete antigen TMS-1-KLH.

6. The hybridoma cell line HOT secreting a tricaine monoclonal antibody according to claim 4, wherein: the molecular formula of the tricaine hapten adopted for preparing the cell strain is as follows:

。