CN111778215B

CN111778215B - Hybridoma cell strain secreting gamithromycin monoclonal antibody and application thereof

Info

Publication number: CN111778215B
Application number: CN202010650604.6A
Authority: CN
Inventors: 胥传来; 路倩倩; 匡华; 徐丽广; 刘丽强; 宋珊珊; 吴晓玲; 胥欣欣; 胡拥明; 郑乾坤
Original assignee: Delisi Group Co Ltd; Jiangnan University
Current assignee: Delisi Group Co Ltd; Jiangnan University
Priority date: 2020-07-08
Filing date: 2020-07-08
Publication date: 2021-11-12
Anticipated expiration: 2040-07-08
Also published as: CN111778215A

Abstract

A hybridoma cell strain secreting a gamithromycin monoclonal antibody and application thereof, belonging to the field of food safety immunodetection. The hybridoma cell strain ABC11 secreting the gamithromycin monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 11 and 28 days, and the preservation number is CGMCC No. 19167. The gamithromycin monoclonal antibody secreted by the strain is used for analyzing and detecting the gamithromycin residue in food safety detection. The gamithromycin monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has better detection sensitivity and specificity (IC) for gamithromycin₅₀Value of 0.82ng/mL, crossover to the gamithromycin analog of less than 10%, crossover rate = (IC of gamithromycin)₅₀IC of/analogue₅₀）×100%）。

Description

Hybridoma cell strain secreting gamithromycin monoclonal antibody and application thereof

Technical Field

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

Background

Gamithromycin (Gamithromycin) is a novel semi-synthetic macrolide veterinary antibiotic special for animals, has the functions of bacteriostasis and sterilization by inhibiting the synthesis of bacterial RNA-dependent protein, and has the advantages of wide antibacterial spectrum, strong antibacterial activity, low residue, safety, high efficiency and the like. In foreign countries, gamithromycin has been widely used in the prevention and treatment of respiratory diseases in non-lactating cows, pigs. In China, the gamithromycin injection is registered and sold in the market in 2017 by the import veterinary drug of the rural part of agriculture, and is used for treating and preventing bovine respiratory system diseases caused by main pathogenic bacteria such as mannheimia haemolytica, pasteurella multocida and histophilus somni. However, the gamithromycin may have problems of improper use and the like in the administration process, remains in animal bodies, enters human bodies through human eating, has adverse effects on the health of human beings, and even threatens the life of human beings. Based on the attention of human health, the Food and Drug Administration (FDA) stipulates that the Maximum Residual Limit (MRL) value of gamithromycin in beef and pork is 0.1 mg/kg.

For detecting the veterinary drug residues of gamithromycin, 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 gamithromycin. 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 gamithromycin by using the enzyme-linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to the gamithromycin is obtained, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to the gamithromycin.

Disclosure of Invention

The invention aims to overcome the defects and provides a hybridoma cell strain ABC11 secreting a gamithromycin monoclonal antibody and application thereof. The gamithromycin monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) on the gamithromycin₅₀Value of 0.82 ng/mL) can be used for establishing an immunological detection method of gamithromycin and detecting the residue of the gamithromycin in food.

The technical scheme of the invention is that a hybridoma cell strain ABC11 secreting a gamithromycin monoclonal antibody has been 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 of West Lu 1 of North Chen, south China, the south China area, the preservation date of which is 2019, the 11 month and 28 days, and the preservation number of which is CGMCC No. 19167.

Gamithromycin monoclonal antibody: it is secreted and produced by the hybridoma cell strain ABC11 with the preservation number of CGMCC number 19167.

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

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

(2) injecting the Freund's adjuvant into BALB/c mouse via back subcutaneous injection for multiple times of immunization, wherein complete Freund's adjuvant is adopted for the first immunization, and incomplete Freund's adjuvant is adopted for 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 milbemycin 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 gamithromycin complete antigen without Freund adjuvant;

(5) fusing splenocytes and myeloma cells of the BALB/c mice subjected to the sprint immunization, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competitive ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain capable of secreting the gamithromycin monoclonal antibody;

the molecular formula of the gamithromycin hapten in the step (1) is as follows:

；

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

。

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 invention also provides application of the hybridoma cell strain ABC11 secreting the gamithromycin monoclonal antibody or the preparation method of the hybridoma cell strain ABC11 secreting the gamithromycin monoclonal antibody in preparation of the gamithromycin monoclonal antibody.

The invention provides a gamithromycin monoclonal antibody which is secreted and generated by a hybridoma cell strain ABC11 with the preservation number of CGMCC number 19167.

The invention provides a preparation method of the gamithromycin monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain ABC11 with the preservation number of CGMCC number 19167 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and storing the obtained monoclonal antibody at low temperature.

In one embodiment of the invention, the method is to take 8-10 weeks of ageBALB/c mice, each mouse was injected with 1mL of paraffin oil intraperitoneally, and 7 days later, each mouse was injected with 1X 10 intraperitoneally⁶Collecting ascites from 7 days, purifying the ascites by octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 deg.C.

The invention provides an application of the gamithromycin monoclonal antibody or the preparation method of the gamithromycin monoclonal antibody in the aspect of recognizing the gamithromycin.

The invention has the beneficial effects that: the gamithromycin monoclonal antibody obtained by the cell strain can be used for immunoassay detection, and has better detection sensitivity and specificity (IC) for the gamithromycin₅₀Value of 0.82ng/mL, crossover to the gamithromycin analog of less than 10%, crossover rate = (IC of gamithromycin)₅₀IC of/analogue₅₀）×100%。

Biological material sample preservation: a hybridoma cell strain ABC11 secreting gamithromycin monoclonal antibody is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences microorganism institute 3, West Lu No.1 Hospital, Kyoho, Beijing, and has a collection date of 2019, 11 and 28 days, and a collection number of CGMCC No. 19167.

Drawings

FIG. 1 is a standard curve for the inhibition of gamithromycin by the gamithromycin monoclonal antibody of the invention.

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 gamithromycin 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 selection of the optimal working point, the gamithromycin standards were diluted to 8 concentrations (0, 0.042, 0.12, 0.37, 1.11, 3.33, 10 and 30 ng/mL), followed by ic-ELISA protocol and finally mapped with originPro 8.5 (results as inFIG. 1), a standard inhibition curve of gamithromycin was obtained, and IC was calculated₅₀。

Example 1: synthesis of gamithromycin hapten

The gamithromycin micromolecules have no immunogenicity, and can not stimulate mice to generate immune response so as to generate antibodies, so that the gamithromycin is coupled to proteins by a protein connection technology to obtain the immunogenicity; the active groups commonly used in the protein coupling technology are amino, carboxyl, hydroxyl, sulfydryl and the like, and because the gamithromycin does not contain amino and carboxyl in the molecular structure and contains a plurality of hydroxyl groups, the carboxyl is derived from the analogue.

The structure of the derivatized gamithromycin hapten is as follows:

。

example 2: synthesis of gamithromycin complete antigen

Weighing 4.0mg of amikacin hapten (GAM-COOH) and 3.1mg of N-hydroxysuccinimide (NHS), dissolving in 300 μ L N, N-Dimethylformamide (DMF), and stirring at room temperature for 10 min; then, 4.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved sufficiently in 100. mu.L of DMF, and then added to GAM-COOH solution, followed by reaction for 4 to 6 hours with stirring at room temperature (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 GAM-COOH-KLH, and identifying by ultraviolet absorption scanning method.

Example 3: synthesis of Gamithromycin coating antigen

Dissolving 4.2mg of amikacin hapten (GAM-COOH) and 2.3mg of N-hydroxysuccinimide (NHS) in 300 μ L of anhydrous N, N-Dimethylformamide (DMF), and stirring at room temperature for 10min to obtain an amikacin hapten (GAM-COOH) solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 μ L of anhydrous DMF, adding into GAM-COOH solution, and reacting at room temperature for 4-6h under stirring 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; dialyzing the reaction solution with PBS solution to remove unreacted small molecule hapten to obtain coating antigen (GAM-COOH-OVA).

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

1. Obtaining animal immunity; 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, killing the mouse by a cervical dislocation method, immediately placing the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding 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 in logarithmic growth phase before fusion, collecting tumor cells during fusion, suspending in RPMI-1640 basic culture mediumIn the nutrient solution, counting 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; 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, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C 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 for screening.

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

subcloning for three times according to the method to finally obtain the gamithromycin monoclonal antibody cell strain ABC 11.

Example 5: preparation and identification of gamithromycin 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 10⁶The mitomycin hybridoma cells were added, 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 ℃.

IC determination of Gamithromycin monoclonal antibodies using indirect competitive ELISA₅₀The value is 0.82ng/mL, the crossover to the gamithromycin analogue is less than 10%, which shows that the gamithromycin has good sensitivity and can be used for immunoassay detection of the gamithromycin.

(crossover rate = (IC of gamithromycin)₅₀IC of/analogue₅₀）×100%）。

Example 6: application of gamithromycin monoclonal antibody

The monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites is applied to an ELISA (enzyme-linked immunosorbent assay) additive recovery test of gamithromycin, 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, 0.042, 0.12, 0.37, 1.11, 3.33, 10 and 30ng/mL amikacin standard solutions by using Phosphate Buffered Saline (PBS), respectively adding the standard solutions and sample extracting solutions to be detected into the closed enzyme label plate, wherein each well is 50 mu L, repeating 3 wells for each sample, adding 50 mu L of an anti-amikacin monoclonal antibody diluted by 1:32000 into each well, 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 well₂SO₄Stop solution, absorbance at 450 nm.

The standard curve for the inhibition of gamithromycin by gamithromycin monoclonal antibody is shown in FIG. 1, and gamithromycin is measured by ic-ELISAIC of monoclonal antibody₅₀The value is 0.82ng/mL, which indicates that the antibody has better sensitivity to the gamithromycin and can be used for immunoassay detection of the gamithromycin.

Claims

1. A hybridoma cell strain ABC11 secreting gamithromycin monoclonal antibody is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences microorganism institute 3, West Lu No.1 Hospital, Kyoho, Beijing, and has a collection date of 2019, 11 and 28 days, and a collection number of CGMCC No. 19167.

2. The gamithromycin monoclonal antibody is characterized in that: it is secreted and produced by hybridoma cell strain ABC11 with the preservation number of CGMCC number 19167 as claimed in claim 1.

3. A gamithromycin hapten, characterized by: the molecular formula is as follows:

。

4. a gamithromycin complete antigen characterized by: the molecular formula is as follows:

。

5. the process for preparing the gamithromycin complete antigen of claim 4, characterized by the steps of: dissolving the gamithromycin hapten GAM-COOH and N-hydroxysuccinimide NHS of claim 3 in anhydrous N, N-dimethylformamide DMF, and stirring to react to obtain a gamithromycin hapten GAM-COOH solution; dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in anhydrous DMF, adding into GAM-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; dialyzing the reaction solution with phosphate buffer PBS to obtain complete antigen GAM-COOH-KLH.

6. The use of a monoclonal antibody to gamithromycin as claimed in claim 2, wherein: the method is applied to the analysis and detection of the amikacin residue in food safety detection.