CN112280744A

CN112280744A - Hybridoma cell strain secreting monoclonal antibody of hypnone and application thereof

Info

Publication number: CN112280744A
Application number: CN202011152452.3A
Authority: CN
Inventors: 胥传来; 路倩倩; 匡华; 徐丽广; 孙茂忠; 刘丽强; 宋珊珊; 吴晓玲; 郝昌龙; 胡拥明
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-01-29

Abstract

A hybridoma cell strain secreting a methaqualone monoclonal antibody and application thereof belong to the technical field of immunoassay. The hybridoma cell strain TZ1B1 secreting the hypnone monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 19173. The monoclonal antibody of the hypnone secreted by the strain is used for analyzing and detecting the residual hypnone in food safety detection. The medicine obtained by the inventionThe monoclonal antibody cell strain of the hypnone can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) on the hypnone₅₀Value of 0.96 ng/mL), a rate of cross-over to the hypnone analog of less than 10%, cross-over rate = (IC of hypnone)₅₀IC of/analogue₅₀）×100%。

Description

Hybridoma cell strain secreting monoclonal antibody of hypnone and application thereof

Technical Field

The invention relates to a hybridoma cell strain secreting a hypnone monoclonal antibody and application thereof, and belongs to the technical field of immunodetection.

Background

Hypnone (Methaqualone), also known as hyacifluorn, mequindox or mecloxine, belongs to the non-barbital class of central nerve tranquilizers, is clinically used for administration before neurasthenia, insomnia and anesthesia, and is often used as a sedative and hypnotic drug. Long-term use of hypnone can form dependence to cause toxicomania; the large dose of the traditional Chinese medicine composition can cause poisoning and is a national regulated psychotropic medicine. In 2007, the Ministry of agriculture clearly stipulates that the residue of hypnone in animal food is zero. However, the production process of the hypnone is simple, and the hypnone is still illegally used for livestock and poultry breeding along with the rapid development of the livestock breeding industry, so that the insecurity of animal food is caused.

For detecting the hypnone veterinary drug residue, a high performance liquid chromatography, a gas chromatography-mass spectrometry or a liquid chromatography-mass spectrometry combined method is usually adopted. However, these methods have the disadvantages of complicated sample pretreatment and long detection time, and are not suitable for rapid detection of a large number of samples, and in order to maintain the benefits of consumers, it is necessary to establish an efficient and rapid detection method for hypnone.

The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for the field rapid detection of a large number of samples, so the ELISA is widely applied to veterinary drug residue analysis. The precondition for detecting the hypnone by using the enzyme-linked immunosorbent assay is to obtain the monoclonal antibody with high specificity and high sensitivity to the hypnone, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to the hypnone.

Disclosure of Invention

The invention aims to overcome the defects and provide a hybridoma cell strain secreting the monoclonal antibody of hypnone and application thereof. The monoclonal antibody of hypnone secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) on the hypnone₅₀Value of 0.96 ng/mL) can be used for establishing an immunological detection method of the hypnone, and detecting the residual hypnone in the food.

The technical scheme of the invention is that a hybridoma cell strain TZ1B1 secreting monoclonal antibody of hypnone is preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, 3, of Xilu No.1, North Chen, the south China area, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2019, 11 months and 28 days, and the preservation number is CGMCC No. 19173.

The invention provides a preparation method of a hybridoma cell strain TZ1B1 secreting a monoclonal antibody of hypnone, which comprises the following steps:

(1) preparing a hypnone complete antigen by using a hypnone hapten, and preparing the obtained hypnone 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 subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high content of the hypnone antibody in the serum to obtain the immunized mice;

(4) carrying out the last boosting immunization on the screened mice by using an incomplete Freund adjuvant containing the antigen, and then carrying out the thrust immunization by intraperitoneal injection, wherein the thrust immunization is carried out by using an hypnone complete antigen without the Freund adjuvant;

(5) fusing splenocytes of BALB/c mice subjected to the sprint immunization with myeloma cells, culturing the fused cells through a 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, subcloning the positive cell pores with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain TZ1B1 capable of secreting the hypnone monoclonal antibody.

In one embodiment of the invention, the interval between the first immunization and the booster immunization in the steps (2) and (4) is one month, the interval between the booster immunization is 21 days, and the interval between the booster immunization and the sprint immunization is 18-21 days.

In one embodiment of the present invention, the first immunization dose of the steps (2) and (4) is 100. mu.g/mouse, the boosting immunization dose is 50. mu.g/mouse, and the sprint immunization dose is 25. mu.g/mouse.

In one embodiment of the present invention, the immunization process of steps (2) and (4) comprises 1 first immunization, 4 booster immunizations and 1 sprint immunizations.

In one embodiment of the present invention, the blood collection in step (3) is performed on day 7 after the 3 rd immunization course.

In one embodiment of the present invention, the cell fusion in step (5) is performed 3 days after the completion of the sprint immunization.

In one embodiment of the present invention, the cell fusion in step (5) is performed by a polyethylene glycol (PEG 4000) method.

In one embodiment of the present invention, the medium in the step (5) is RPMI-1640 medium.

In one embodiment of the present invention, the number of subclones in step (5) is 3.

The hypnone monoclonal antibody is secreted and produced by the hybridoma cell strain TZ1B1 with the preservation number of CGMCC number 19173.

The invention provides a preparation method of the hypnone monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain TZ1B1 with the preservation number of CGMCC number 19173 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained monoclonal antibody at low temperature.

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 an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

A hypnone hapten, which is MTQ-COOH for short, has a molecular formula as follows:

。

a hypnone complete antigen, called MTQ-COOH-KLH, of the formula:

。

the invention also provides application of the hypnone monoclonal antibody, and the application of the hypnone monoclonal antibody in analysis and detection of hypnone residues in food safety detection is provided.

The invention has the beneficial effects that: the monoclonal antibody of the hypnone obtained by the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) on the hypnone₅₀Value of 0.96 ng/mL), a rate of cross-over to the hypnone analog of less than 10%, cross-over rate = (IC of hypnone)₅₀IC of/analogue₅₀）×100%。

Biological material sample preservation: a hybridoma cell strain TZ1B1 secreting monoclonal antibody of hypnone is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, 3, Xilu 1, Beijing, the morning area, and the Kyoho area, and is classified and named as monoclonal cell strain, the preservation date is 2019, 11 months and 28 days, and the preservation number is CGMCC No. 19173.

Drawings

FIG. 1 shows a standard curve of inhibition of the monoclonal antibody of hypnone against hypnone.

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: PBS was added with 0.1% gelatin.

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 inhibition rate of the hypnone 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 standard hypnone was diluted to equal concentrations of 0, 0.027, 0.082, 0.25, 0.74, 2.22, 6.67, 20 and 60ng/mL, subjected to the procedure of IC-ELISA, and finally plotted against originPro 8.5 (results are shown in FIG. 1), to obtain the standard inhibition curve of hypnone, and IC was calculated₅₀。

Example 1: synthesis of hypnone hapten

The hypnone micromolecules have no immunogenicity, and can not stimulate mice to generate immune response so as to generate antibodies, so that the hypnone is coupled to proteins through a protein connection technology to obtain the immunogenicity; the active groups commonly used in protein coupling technology include amino, carboxyl, hydroxyl, sulfydryl and the like, and the structural derivation of carboxyl is needed because the molecular structure of the hypnone does not contain amino, carboxyl and hydroxyl.

The structure of the derivative hypnone hapten is as follows:

;

the derivatization process comprises the following four reactions:

1. charging 1b (1.2 eq.), Pd (OAc) into the reaction vessel₂(0.04 eq.)、P(O-TOl)₃(0.06 eq.) and 1a, NEt was added₃(0.3M), replacing gas by a freeze-pump circulation method for 3 times, stirring at 60 ℃ for 2h under Ar atmosphere, heating to 80 ℃ and stirring for 24h to obtain an intermediate product 2 a.

2. Adding 2a into a reaction container, adding 5-10mol of Pd/C, adding a proper amount of ethanol for dissolving, replacing gas with hydrogen for 3 times, stirring at room temperature for 4 hours under a hydrogen atmosphere, filtering to remove Pd/C, collecting filtrate, spin-drying, and separating by a column to obtain an intermediate product 3 a.

3. Adding 3a into a reaction container, adding 3 eq. NaOH aqueous solution and a proper amount of ethanol, reacting for 3h at 50 ℃, cooling, acidifying with hydrochloric acid to pH =2-4, extracting with ethyl acetate, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain an intermediate product 4 a.

4. Adding 4a, 4b (1.2 eq.), adding acetic acid (0.3M), refluxing for 3 days, cooling, extracting with ethyl acetate, drying with anhydrous sodium sulfate, spin-drying, and separating with column to obtain hypnone hapten.

Example 2: synthesis of hypnone complete antigen

Weighing 9.45mg hypnone hapten (MTQ-COOH) and 6.21mg N-hydroxysuccinimide (NHS), dissolving in 300 μ L N, N-Dimethylformamide (DMF), and stirring at room temperature for 10 min; then, 10.35mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved sufficiently in 100. mu.L of DMF, and then added to the MTQ-COOH solution, followed by stirring at room temperature for 4 to 6 hours (referred to as solution A). Taking 6mg KLH, diluting to 3mg/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 MTQ-COOH-KLH, and identifying by ultraviolet absorption scanning method.

Example 3: synthesis of hypnone coating antigen

Dissolving 4.2mg of hypnone hapten (MTQ-COOH) and 2.8mg of N-hydroxysuccinimide (NHS) in 300 μ L of anhydrous N, N-Dimethylformamide (DMF), and stirring at room temperature for 10min to obtain hypnone hapten (MTQ-COOH) solution; dissolving 4.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into MTQ-COOH, and stirring at room temperature to react for 4-6h to obtain solution A; diluting 6mg of egg albumin (OVA) with 1mL of Carbonate Buffer Solution (CBS) with the concentration of 0.01mmol/L to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small molecule hapten, and thus a coating antigen (MTQ-COOH-OVA) was obtained.

Example 4: preparation of hybridoma cell strain secreting monoclonal antibody of hypnone

1. Obtaining animal immunity: mixing and emulsifying the hypnone complete antigen and equivalent Freund's adjuvant, and performing neck and back subcutaneous multipoint injection immunization (except for sprint 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% CO₂Culturing in an incubator, wherein the number of SP2/0 tumor cells 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 PEG 4000 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; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; 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, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf 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: performing RPMI-1640 screening culture medium half-exchange on fused cells on the 3 rd day of cell fusion, performing total-exchange with RPMI-1640 transitional culture medium containing 20% fetal calf serum and 1% 100 XHT on the 5 th day, and taking cell supernatant on the 7 th day for screening;

the screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting hypnone 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 hypnone standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days;

and carrying out subcloning for three times according to the method to finally obtain the hybridoma cell strain TZ1B1 secreting the monoclonal antibody of the hypnone.

Example 5: preparation and identification of hypnone 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 10⁶Collecting ascites from the seventh day, and purifying the ascites by an 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 ℃.

IC determination of monoclonal antibodies to hypnone Using an Indirect competitive ELISA₅₀The value is 0.96ng/mL, the cross rate to the hypnone analogue is less than 10%, which shows that the reagent has good sensitivity to the hypnone and can be used for immunoassay detection of the hypnone. (Cross over Rate = (IC of hypnone)₅₀IC of/analogue₅₀）×100%）。

Example 6: application of hypnone monoclonal antibody

The monoclonal antibody prepared from hybridoma cell strain TZ1B1 through in vivo ascites is applied to an ELISA addition recovery test of hypnone, 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, 0.027, 0.082, 0.25, 0.74, 2.22, 6.67, 20 and 60ng/mL of a hypnone standard solution by using Phosphate Buffer Solution (PBS), respectively adding the standard solution and a sample extracting solution to be detected into a closed enzyme label plate, wherein each well is 50 mu L, each sample is repeatedly provided with 3 wells, 50 mu L of an anti-hypnone monoclonal antibody diluted by 1:32000 is added into each well, reacting at 37 ℃ for 0.5h, washing and drying the plate;

(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;

a standard curve for inhibition of hypnone by the monoclonal antibody of hypnone is shown in FIG. 1, and IC of the monoclonal antibody of hypnone is determined by IC-ELISA₅₀The value is 0.96ng/mL, which indicates that the antibody has better sensitivity to the hypnone and can be used for immunoassay detection of the hypnone.

Claims

1. A hybridoma cell strain TZ1B1 secreting monoclonal antibody of hypnone is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, 3, Xilu 1, Beijing, the morning area, and the Kyoho area, and is classified and named as monoclonal cell strain, the preservation date is 2019, 11 months and 28 days, and the preservation number is CGMCC No. 19173.

2. The monoclonal antibody of hypnone is characterized in that: it is secreted and produced by hybridoma cell strain TZ1B1 with the preservation number of CGMCC number 19173 as claimed in claim 1.

3. A hypnone hapten, comprising: it is abbreviated as MTQ-COOH, and the molecular formula is as follows:

。

4. a hypnone complete antigen, characterized by: the cartridge is named MTQ-COOH-KLH and has the following molecular formula:

。

5. a process for the preparation of the hypnone complete antigen as claimed in claim 4, characterized by the following steps: dissolving the hypnone hapten MTQ-COOH and N-hydroxysuccinimide NHS as defined in claim 3 in anhydrous N, N-dimethylformamide DMF, and reacting under stirring to obtain a hypnone hapten MTQ-COOH solution; dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in anhydrous DMF, adding into MTQ-COOH solution, and stirring for reaction to obtain solution A; diluting keyhole limpet hemocyanin KLH with carbonate buffer solution CBS to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; and dialyzing the reaction solution by phosphate buffer solution PBS to obtain the complete antigen MTQ-COOH-KLH.

6. The use of the monoclonal antibody to hypnone of claim 2, characterized by: the method is applied to the analysis and detection of the remaining hypnone in food safety detection.