CN112939832B - Tiamulin hapten TMLO, artificial antigen, antibody and preparation method and application thereof - Google Patents

Tiamulin hapten TMLO, artificial antigen, antibody and preparation method and application thereof Download PDF

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CN112939832B
CN112939832B CN202110170648.3A CN202110170648A CN112939832B CN 112939832 B CN112939832 B CN 112939832B CN 202110170648 A CN202110170648 A CN 202110170648A CN 112939832 B CN112939832 B CN 112939832B
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tiamulin
tmlo
hapten
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沈玉栋
张政
杨金易
徐振林
王弘
雷红涛
孙远明
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South China Agricultural University
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Abstract

The invention discloses tiamulin hapten TMLO, an artificial antigen, an antibody, a preparation method and application thereof. The invention firstly provides a tiamulin hapten TMLO, and further uses an artificial antigen obtained by coupling the tiamulin hapten and carrier protein as an immunogen to immunize a mouse to prepare the tiamulin monoclonal antibody. The antibody prepared by the invention has better titer, specificity and affinity, the minimum detection limit of the antibody on tiamulin is 0.09ng/mL, and IC (integrated Circuit) 50 0.53ng/mL, and the linear range is 0.21-1.35 ng/mL; the method has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity, and has good application prospect and wide development space in the rapid and effective detection of tiamulin.

Description

Tiamulin hapten TMLO, artificial antigen, antibody and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a tiamulin hapten TMLO, an artificial antigen, an antibody, and a preparation method and application thereof.
Background
Tiamulin (TML) is a commonly used veterinary drug additive, has an antibacterial spectrum similar to that of macrolides, is classified as a narrow spectrum antibiotic, and has good antibacterial activity against bacteria, mildews and the like. When the TML is used together with other medicines such as sulfonamide synergist, sulfonamides, oxytetracycline and the like, the antibacterial spectrum of the TML is obviously increased and the treatment effect of the TML is obviously enhanced. Its antibacterial mechanism mainly acts on ribosome to inhibit the synthesis of its protein. The product is only used as veterinary drug, avoids the mutual influence of common drugs between human beings and livestock and poultry, and well controls the appearance of human cross drug-resistant strains. TML has been widely used around the world and is recommended to swine experts as it has a good effect in treating mycoplasma diseases in animals.
In recent years, the demand of people for animal-derived foods is increasing, and the development of animal husbandry in China is also potentially driven. Most animal derived foods come from livestock and poultry farming, where TML as a veterinary antibiotic is commonly used as a feed additive and for the treatment or prevention of diseases. The use of antibiotics in the livestock and poultry industry properly will maintain the good therapeutic effect of the existing antibiotics, however, the abuse of antibiotics will lead to the rapid spread of antibiotic resistance in bacteria and the development of various drug-resistant pathogens, and also spread to human through the food chain, thereby producing an enrichment effect, posing a great threat to the health of human, so that the antibiotics are gradually losing its antibacterial value. The use of antibiotics in food production animals is strictly controlled by the european union, and veterinary uses are regulated to prevent the spread of resistance. At present, the maximum residual quantity of TML in food-borne animals is regulated in multiple countries in the world, and the improper use and residue of tiamulin can harm the health of human beings, so that the strengthening of the detection and monitoring of the tiamulin in animal products is very important.
At present, TML detection and analysis methods in animal derived foods are various, and commonly used methods mainly comprise chromatographic detection, such as GC, HPLC, UPLC-MS/MS, LC-MS/MS and the like. In addition, immunological detection methods such as ELISA and immunochromatographic strip technology have been rapidly developed in recent years. The chromatographic detection has the disadvantages of complex sample pretreatment and determination process, high cost, requirement of professional operation and unsuitability for screening of a large number of samples. The ELISA has the characteristics of simple and convenient sample pretreatment, simple operation, rapidness, sensitivity, high flux and the like, is known as the rapid detection technology which has the most competition and challenge in the 21 st century, and has wide application prospect in the field of food safety. However, in the case of immunoassays, antibodies are used as a core material, and the effect of antibodies depends greatly on the structure of the antigen that causes an immune response in the corresponding animal. The patent with the publication number of CN108330102 discloses a tiamulin monoclonal antibody hybridoma cell strain, and the IC50 value of the monoclonal antibody secreted by the cell strain for detecting tiamulin is only 0.73ng/mL, which cannot meet the requirements of sensitive and accurate detection of tiamulin. Therefore, the method for preparing the tiamulin-containing antibody with stable antigen structure and high specificity has great significance for quickly, sensitively and accurately detecting the tiamulin.
Disclosure of Invention
The invention aims to overcome the defects of the existing tiamulin detection method and provide a tiamulin hapten TMLO, an artificial antigen, an antibody, a preparation method and application thereof. The synthesis method of the tiamulin hapten TMLO utilizes carbonyl on a five-membered ring of a tiamulin molecule to generate oximation reaction with carboxymethyl hydroxylamine, so that the tail end of the tiamulin hapten TMLO has an active group-carboxyl.
The first purpose of the invention is to provide a tiamulin hapten TMLO.
The second purpose of the invention is to provide a preparation method of the tiamulin hapten TMLO.
The third purpose of the invention is the application of the tiamulin hapten TMLO in preparing a tiamulin artificial antigen or antibody.
The fourth purpose of the invention is to provide a tiamulin artificial antigen.
The fifth purpose of the invention is to provide the application of the tiamulin artificial antigen in preparing a tiamulin antibody.
The sixth purpose of the invention is to provide a tiamulin antibody.
The seventh purpose of the invention is to provide a composition of immunogen and coating antigen for enzyme-linked immunosorbent assay.
The eighth purpose of the invention is to provide the application of the tiamulin hapten TMLO, the tiamulin artificial antigen or the composition of the immunogen and the coating antigen in the detection of tiamulin or the preparation of a tiamulin detection kit.
The above purpose of the invention is realized by the following technical scheme:
the invention provides tiamulin hapten TMLO, which has a structural formula shown in a formula (I):
Figure BDA0002938810580000031
the tiamulin hapten TMLO and the molecular skeleton and side chain structure of the tiamulin are completely overlapped, so that the immune induction of the full molecular specificity of the tiamulin is favorably generated, and the high-specificity identification of the tiamulin is realized.
The invention also provides a preparation method of the tiamulin hapten TMLO, which is prepared by reacting tiamulin with carboxymethyl hydroxylamine.
The reaction formula for the preparation of hapten TMLO is shown below:
Figure BDA0002938810580000032
more preferably, the preparation method of the tiamulin hapten TMLO specifically comprises the following steps:
s1, dissolving tiamulin in anhydrous pyridine, and adding carboxymethyl hydroxylamine to perform oximation reaction;
s2, extracting the reaction solution, and collecting an organic phase; extracting the water phase with ethyl acetate, and collecting the organic phase; the organic phases were combined and washed with saturated NaCl solution and then with anhydrous Na 2 SO 4 Drying and filtering;
s3, removing the solvent from the filtrate obtained in the step S2 through reduced pressure distillation, and purifying the residue through silica gel column chromatography to obtain a dark brown solid, namely the tiamulin hapten TMLO.
Preferably, the molar ratio of the tiamulin to the carboxymethyl hydroxylamine in the step S1 is 1.
The invention claims the application of the tiamulin hapten TMLO in preparing artificial antigen or antibody of tiamulin.
The invention also provides a tiamulin artificial antigen which is obtained by coupling the tiamulin hapten TMLO and carrier protein, and the structural formula of the tiamulin artificial antigen is shown as the formula (II):
Figure BDA0002938810580000041
preferably, the carrier protein is chicken ovalbumin, hemocyanin, bovine serum albumin, lactoferrin, or concanavalin.
When the tiamulin hapten TMLO is coupled with carrier Protein (Protein), the specific structure of the tiamulin hapten TMLO protrudes out of the surface of the carrier Protein and is used as an epitope of a carrier to be exposed to an animal immune system, and a foundation is laid for obtaining an antibody with high specificity and high quality.
Preferably, the preparation method of the tiamulin artificial antigen is to couple the hapten TMLO and carrier protein by an active ester method, and the specific steps are as follows:
s21, dissolving tiamulin hapten TMLO in DMF, adding NHS and EDC, and reacting at 4 ℃;
s22, dissolving carrier protein in a phosphate buffer solution;
s23, dropwise adding the solution obtained in the step S21 into the solution obtained in the step S22, reacting at 4 ℃ for 8 hours, dialyzing the reaction solution at 4 ℃ for 3 days, and replacing the dialyzate for 2 times every day to obtain the tiamulin artificial antigen.
Preferably, the molar ratio of the haptens TMLO, NHS and EDC in step S21 is 1:1 to 2:1 to 2.
More preferably, the molar ratio of haptens TMLO, NHS and EDC in step S21 is 1:1.5:1.5.
preferably, the molar ratio of carrier protein to tiamulin hapten TMLO is 1:60.
the invention claims the application of the tiamulin artificial antigen in preparing a tiamulin antibody.
Preferably, the tiamulin antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a nano antibody.
The invention also claims a tiamulin antibody which is prepared by taking the tiamulin artificial antigen as an immunogen.
Preferably, the tiamulin antibody is a monoclonal antibody.
Preferably, the immunogen is an artificial antigen obtained by coupling hapten TMLO and one of hemocyanin KLH, bovine serum albumin BSA, lactoferrin LF, concanavalin ConA or chicken ovalbumin OVA.
Preferably, the preparation method of the tiamulin monoclonal antibody comprises the following steps:
immunizing a mouse by taking the tiamulin antigens TMLO-OVA, TMLO-KLH, TMLO-BSA, TMLO-LF or TMLO-ConA as immunogens, fusing mouse spleen cells and SP2/0 myeloma cells to obtain hybridoma cells, culturing the hybridoma cells in a female Balb/c mouse to obtain ascites containing high-concentration monoclonal antibodies, and purifying the ascites to obtain the high-specificity anti-tiamulin monoclonal antibodies.
The invention claims a composition of immunogen and coating antigen for enzyme-linked immunosorbent assay of tiamulin, wherein the immunogen is a conjugate of a tiamulin hapten TMLO and lactoferrin, and the coating antigen is a conjugate of the tiamulin hapten TMLO and chicken ovalbumin.
The application of the tiamulin hapten TMLO, the tiamulin artificial antigen or the composition of the immunogen and the coating antigen in detecting tiamulin or preparing a tiamulin detection kit also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
the invention firstly provides a tiamulin hapten TMLO, the overlapping degree of the skeleton structure of the tiamulin hapten TMLO and a substance to be detected, namely tiamulin is high, and the immunogenicity of the tiamulin hapten TMLO is effectively improved; further using an artificial antigen obtained by coupling the tiamulin hapten TMLO and carrier proteins KLH, BSA, LF and ConA as an immunogen to immunize a mouse, obtaining hybridoma cells through cell fusion, preparing ascites through in vivo inoculation of the hybridoma cells, and preparing the tiamulin monoclonal antibody after purification. The antibody prepared by the invention has better titer, specificity and affinity, the minimum detection limit of the antibody on tiamulin is 0.09ng/mL, and IC (integrated Circuit) 50 0.53ng/mL, and the linear range is 0.21-1.35 ng/mL; the method has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity, and has good application prospect and wide development space in the rapid and effective detection of tiamulin.
Drawings
FIG. 1 is a graph of indirect competition ELISA standards established based on monoclonal antibodies.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 preparation of tiamulin hapten TMLO
1. Experimental methods
Weighing 4.05mmol of tiamulin, dissolving the tiamulin in 5-10 mL of anhydrous pyridine, adding 5.61mmol of carboxymethyl hydroxylamine, and carrying out reflux reaction at 60 ℃ for 7h. After the reaction was stopped, the solvent was evaporated by evaporation, 50mL of ice water was added, pH =3 was adjusted with 1mol/L hydrochloric acid solution, and extraction was performed 3 times with 100mL of ethyl acetate. Combining organic phases, washing the organic phases by using 50mL of saturated salt solution for 3 times, drying the organic phases by using anhydrous sodium sulfate, separating the organic phases by using a 200-300-mesh silica gel column chromatography (a developing agent is petroleum ether: ethyl acetate = 1), collecting product point components, and removing an organic reagent by rotary evaporation to obtain a dark brown solid product, namely the tiamulin hapten TMLO, wherein the reaction formula is as follows:
Figure BDA0002938810580000061
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2. results of the experiment
Spectroscopic data for tiamulin hapten TMLO were as follows: ESI-MS (m/z) 567.34[ 2 ], [ M + H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ5.77(dddt,J＝16.7,9.9,1.8,1.0Hz,1H),5.10(dd,J＝16.7,2.4Hz,1H),5.01(dd,J＝10.1,2.4Hz,1H),4.83–4.73(m,2H),4.67(tq,J＝7.1,1.6Hz,1H),3.49(ddt,J＝8.6,3.1,1.6Hz,1H),3.46–3.37(m,2H),2.87–2.71(m,3H),2.70–2.54(m,5H),2.39–2.26(m,2H),2.09(dd,J＝12.5,7.0Hz,1H),1.91(p,J＝7.0Hz,1H),1.81(dd,J＝12.4,7.1Hz,1H),1.77–1.65(m,2H),1.65–1.32(m,4H),1.10–1.06(m,3H),1.06–0.97(m,11H),0.94(d,J＝6.8Hz,3H). 13 C NMR(125MHz,DMSO-d 6 ) Delta 171.83,171.07,161.56,141.83,114.77,79.11,77.66,65.42,55.69,50.92,47.30,46.83,46.67,41.81,40.65,38.41,35.98,35.35,34.20,30.92,30.10,28.10,27.53,22.00,16.54,16.37,13.44,11.05. It can be seen by mass spectrometry that 567.35 is the positive ion molecular peak of hapten TMLO, which is consistent with the actual relative molecular mass; in addition, NMR spectrum peak shift and division can be assigned to target molecules one by one, which indicates that the tiamulin hapten TMLO is successfully prepared. The structural formula of the tiamulin hapten TMLO hapten is shown as the formula (I):
Figure BDA0002938810580000071
example 2 preparation of tiamulin Artificial antigen
The tiamulin hapten TMLO prepared in example 1 was coupled with chicken ovalbumin OVA (albumin), hemocyanin KLH (keyhole limpet hemocyanin), bovine serum albumin BSA (Bovine albumin), lactoferrin LF (lactoferrin) and concanavalin ConA (concanavalin A) by an active ester method. The method comprises the following specific steps:
dissolving tiamulin hapten TMLO in DMF, adding 1.5 times (molar ratio) of NHS and EDC (molar ratio TMLH: NHS: EDC = 1.5) to react in solution at 4 ℃ overnight, and marking as solution A; dropwise adding the solution A into a PBS buffer solution of carrier protein, and continuously reacting for 8 hours at 4 ℃; dialyzing the reaction solution at 4 deg.C for 3 days, changing dialysate twice a day, dialyzing for 6 times, and collecting the solution in dialysis bag to obtain tiamulin artificial antigen (tiamulin complete antigen TMLO-OVA, TMLO-KLH, TMLO-BSA, TMHL-LF, TMLO-ConA). The structural formula of the tiamulin artificial antigen is shown as a formula (II):
Figure BDA0002938810580000072
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wherein the carrier protein is hemocyanin KLH, bovine serum albumin BSA, lactoferrin LF, concanavalin ConA or chicken ovalbumin OVA.
Example 3 evaluation of Artificial antigen immunization and antiserum Effect of tiamulin
1. Experimental methods
1. Animal immunization
Healthy 6-week-old Balb/c female mice were used as experimental animals, and the complete antigens TMLO-KLH, TMLO-BSA, TMHL-LF and TMHL-ConA were used as immunogens, and were injected subcutaneously into the neck, back and abdominal cavity of the mice at an immunization dose of 0.5mL (containing 0.5mg of immunogen) each time. The first immunization is carried out by emulsifying 0.5mL of complete Freund's adjuvant with antigen and then using the emulsified complete Freund's adjuvant for immunization, after 4 weeks, 0.5mL of incomplete Freund's adjuvant is used for enhancing immunization after being emulsified with antigen and then is immunized once every 2 weeks, a small amount of blood is taken from tail vein during the immunization for antibody quality identification, after the antibody is stabilized, the mouse with the best performance is selected for cell fusion, and the mouse with 0.5mg of immunogen is directly injected for additional immunization once in abdominal cavity 3 days before the cell fusion.
2. Evaluation of antiserum Effect
The tiamulin complete antigen TMLO-OVA prepared in example 2 was used as a coating antigen, the collected mouse serum was used as a detection antibody, and the antiserum titer and inhibition rate of the mouse serum were measured by an indirect competitive ELISA method, and the titer and inhibition rate of each antiserum were evaluated in consideration of the whole titer and inhibition rate. The specific operation steps are as follows:
(1) Wrapping a plate: tiamulin complete antigen TMLO-OVA was diluted to 1000ng/mL with 0.05M carbonate buffer (pH = 9.6) and coated overnight at 100 μ L/well at 4 ℃; discarding the coating solution, washing with PBST for 2 times, adding 120 μ L of blocking solution (5% skimmed milk) into each well, and blocking at 37 deg.C for 3 hr; removing the sealing liquid, drying at 37 ℃ for 60min, and packaging with a sealing bag at 4 ℃ for later use to obtain the wrapped ELISA plate.
(2) Serum titer and inhibition detection: in the enzyme label plate coated in the step (1), the titer is listed as follows: add 50. Mu.L PBS and 50. Mu.L serum diluted by gradient multiple (1K, 2K, 4K, 8K, 16K, 32K, 64K) separately to each well; inhibition column: each well was added 50. Mu.L of diluted 1000ng/mL drug (tiamulin) and 50. Mu.L of serum diluted in gradient multiples (1K, 2K, 4K, 8K, 16K, 32K, 64K) to make 2 replicates. Incubate 40min at 37 ℃, wash five times with PBST, pat the well dry, add 1: incubating enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted by 5000 at 37 ℃ for 30min, washing the secondary antibody with PBST for five times, patting the liquid in the hole dry, adding 100 mu L of TMB substrate solution, and developing the color for 10min at 37 ℃ in a dark place; add 50. Mu.L of stop solution (2M H) 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
2. Results of the experiment
Antiserum obtained by immunizing Balb/c female mice generates immune response reaction, but the titers of TMLO-KLH, TMLO-BSA and TMLO-ConA are not more than 8k, the effect is poor, the antiserum is not suitable for further preparing antibodies, the immunization of 5 parallel animals of TMLO-LF has good immune response effect, wherein the titer and inhibition rate of the antiserum of the No. 4 mouse are optimal (table 1), which indicates that the immune response effectiveness of antigen TMLO-LF is good, and the antiserum can be used for preparing antibodies such as tiamulin monoclonal antibodies, polyclonal antibodies and nano antibodies and establishing an immunoassay method.
TABLE 1 potency and inhibition of antisera
Figure BDA0002938810580000091
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Example 4 preparation of tiamulin monoclonal antibody, polyclonal antibody, single chain antibody and Nanobody
1. Preparation of monoclonal antibodies
Cell fusion and screening of positive hybridomas: and (3) fusing the spleen cells of the No. 4 Balb/c mice which produce specific antibodies after immunization in the example 3 with myeloma cells SP2/0, measuring cell supernatants by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Carrying out subcloning on the positive hole by using a limiting dilution method, screening single cells capable of stably secreting uniform antibodies, and carrying out amplification culture, wherein the method comprises the following specific steps:
(1) Recovering myeloma cells: taking out myeloma cells from liquid nitrogen, quickly putting the myeloma cells into a water bath at 37 ℃ for unfreezing, centrifuging for 7min at l000r/min after thawing, pouring out supernatant in an ultra-clean workbench, adding about 1mL of complete culture solution into cell precipitates, blowing off the cells, taking out the cells by using a pipette, uniformly mixing the cells with the complete culture medium, putting the cells into a 9cm culture dish, expanding the culture dish to 5-6 dishes, changing the culture solution for 1-2 times, and when the cells of each culture dish are fully paved at the bottom, fusing the cells.
(2) Preparing feeder cells: the Kunming mouse is killed by picking eyeballs 1 day before cell fusion, soaked in 75% alcohol for 5min, and then moved to an ultra-clean workbench for dissection. Cutting off the abdomen, peeling off the skin of the abdomen, exposing the abdominal muscle, then picking up the peritoneum by using a small forceps, opening a small opening on the peritoneum of the mouse, injecting 3mL HAT complete culture medium, repeatedly sucking and flushing by using a pipette, taking out the culture solution containing the feeder layer cells by using the pipette, and repeating the operation for 2-3 times to ensure that enough feeder layer cells are obtained.
(3) Preparation of splenocytes: the orbit of an immunized Balb/c mouse No. 4 is bled, serum is collected, and then the mouse is soaked in 75% alcohol for 5min for disinfection and is moved into an ultra-clean workbench for dissection. Taking out spleens aseptically, washing the spleens with PPMI-1640 basic culture medium, and putting the spleens in a culture dish for later use. The medium was aspirated with a disposable syringe, the removed spleen was grasped with forceps on the left hand, and the medium was slowly injected into the spleen after the syringe was inserted into the spleen to wash out the cells in the spleen. This was repeated until the spleen turned from deep red to colorless and transparent, and the spleen was discarded. Collecting the mixed culture medium, sealing in a 50mL centrifuge tube, and centrifuging at a rotation speed of l000r/min for 8min; centrifuging, and discarding the upper residue solution.
(4) Cell fusion: centrifuging the bone marrow tumor cells of step (1) to remove supernatant, and mixing the bone marrow tumor cells with the immune spleen cells of step (3) in a ratio of 1: and 5, mixing the mixture in a centrifugal tube, adding 25mL of basic culture medium, sealing the centrifugal tube, centrifuging at the rotating speed of 1000r/min for 8min, and centrifuging and then discarding the supernatant for later use. The bone marrow tumor cells and immune spleen cells mixed by centrifugation were discarded from the supernatant, and the centrifuge nozzle was directed downward to aspirate the excess medium with a gun. Loosening the precipitated cells with fingers, placing the centrifuge tube in 37 deg.C warm water, sucking 0.8mL of PEG preheated to 37 deg.C with a gun head, slowly adding PEG into the precipitated cells within 1min, stirring with the gun head gently for mixing every addition of one drop of PEG, standing for 1min, adding 10mL of preheated complete culture medium within 2min, and adding PEG along the wall with gentle stirring to separate PEG. Centrifuging for 10min at 1000r/min in a centrifugal tube, draining off the supernatant, adding HAT culture medium, sucking liquid and discharging liquid gently by using an elbow suction tube, stirring gently, taking out the HAT culture medium in the centrifugal tube, mixing with about 75mL of HAT fresh culture medium, and uniformly adding into 10 feeder cell-containing 96-well culture plates prepared in the previous day. The volume of HAT medium containing feeder cells per well and the volume of HAT medium containing fused cells were kept the same, and about 24mL of HAT medium was used in each plate.
(5) Screening of positive hybridomas: and (4) using HAT culture medium within 7-10 days after the cell fusion in the step (4), then replacing and using HT culture medium replacement solution, and using complete culture solution according to proliferation conditions after 14 days. When the cells adhere to 1/3 of the wells (about 12 days for general cell fusion), the upper parts of the wells in the multi-well culture plate are extracted, the specific antibodies in the culture solution are detected by indirect ELISA, the positive hybridoma cells with high titer and strong drug inhibition are selected, and the positive wells with the best fusion effect are screened and marked. Under aseptic condition, picking the cells growing in clusters in the positive holes by a microscope, transferring the cells to a 96-hole culture plate plated with feeder cells in advance, cloning each original hole into 8 holes, taking supernatant after the cells grow to the bottom of 1/2-1/3 of the holes by adherence, carrying out an ICELISA detection, taking the positive strong person as a measurement index, carrying out subcloning by using a limiting dilution method, repeating the steps for 3-4 times (paying attention to the fact that the cells of the positive holes picked out in each round need to be expanded and cultured, freezing and storing for later use) until each hole of each plate is positive and the titer and inhibition are similar after detection, and successfully establishing the hybridoma cell line capable of stably secreting uniform antibodies. Picking single cell clone, transferring the detected full positive to a 24-hole cell culture plate or a cell culture dish for expanding culture, and freezing and storing in time.
(6) Large-scale preparation of monoclonal antibodies: after obtaining a hybridoma cell secreting a specific monoclonal antibody, a large amount of monoclonal antibody is usually prepared by an in vitro culture method and an in vivo induced monoclonal antibody method in animals. The conventional method is as follows: injecting liquid stone 30863 into the abdominal cavity of more than ten Balb/c mice aged for more than 8 weeks in advance, wherein the dosage is 0.5mL per mouse, and injecting hybridoma cells into the abdominal cavity of the mice after 1-2 weeks. Observing the state of the mouse every day after inoculating the cells, particularly observing the abdominal cavity of the mouse to swell from the seventh day, aseptically collecting ascites by using a disposable syringe before the death of the mouse, centrifuging the collected ascites at 12000r/min for 10min, removing upper-layer fat and lower-layer fibrin, collecting the middle layer, measuring the titer and the inhibition rate by using an icellSA method, and storing the mouse at-20 ℃ for later use after purification.
2. Preparation of polyclonal antibodies
The immunogen TMLO-LF prepared in example 3 is mixed and emulsified with an equal amount of adjuvant (complete Freund's adjuvant is used for the first time, and then incomplete Freund's adjuvant is used for the second time), and a plurality of injection modes of dorsal subcutaneous, leg muscle and marginal ear vein are adopted to immunize New Zealand white rabbits with the weight of 2.5-3 kg, and two corresponding immunogens are injected. The first immunization was boosted every three weeks after four weeks, the third one week after the peripheral venous blood draw, and the serum titer was determined by indirect ELISA, and when the titer no longer increased, the peripheral venous boosting was used. After two days, the heart is sampled, kept stand for 0.5 to 1 hour at room temperature, centrifuged for 10min at 12000r/min at 4 ℃, and the supernatant is taken and separately loaded into a centrifuge tube and stored for use at-20 ℃.
3. Preparation of Single chain antibodies
Extracting RNA of tiamulin monoclonal cells or spleen cells of mice immunized by tiamulin immunogen, carrying out reverse transcription to obtain cDNA, designing an antibody light and heavy chain amplification primer, amplifying light and heavy chain genes of the antibody by using PCR technology, inserting into an expression plasmid TCI strain, expressing in escherichia coli, purifying by using an immunoaffinity method to obtain a genetic engineering antibody, identifying the purity by SDS-PAGE electrophoresis, subpackaging small bottles, and storing at-20 ℃.
4. Preparation of Nanobodies
(1) Animal immunization: the four immunogens prepared in example 3 are mixed and emulsified with the same amount of adjuvant (complete Freund's adjuvant is used for the first time, and incomplete Freund's adjuvant is used for the later time), the camel is subjected to interval immunization, interval immunoassay detection is carried out, and the peripheral blood of the camel is obtained for the subsequent construction of a nano antibody library.
(2) Preparing and purifying a nano antibody: separating lymphocytes from peripheral blood, extracting RNA, performing reverse transcription to obtain cDNA, amplifying VHH gene by using a nested-PCR technology, connecting with a pComb3XSS carrier, transferring into electrotransformation competent Ecoli.TG1 to construct a VHH gene library, rescuing by using an auxiliary phage M13K07 to obtain a phage display nano antibody library, and obtaining the anti-tiamulin nano antibody by using a solid-phase affinity elutriation technology.
EXAMPLE 5 establishment of an Indirect competitive ELISA Standard Curve based on monoclonal antibodies
1. Coating and sealing
TMLO-OVA artificial antigen prepared in example 2 was used as a coating antigen, and the TMLO-OVA coating antigen was diluted to 62.5ng/mL with a coating solution and coated overnight at 37 ℃. Washing twice with PBST (0.01M PBS, 0.06%; tween-20 (v/v)) on the next day, adding 2% skimmed milk powder, sealing at 37 deg.C for 3h and 120 μ L per well, discarding the sealing solution, oven drying at 37 deg.C for 60min, packaging in a sealed bag at 4 deg.C,
2. establishment of a Standard Curve
(1) Experimental method
Adding 50 muL of anti-tiamulin monoclonal antibody with the concentration of 3.5ug/mL and a series of 50 muL of tiamulin standard substances with different concentrations into each well of the coated enzyme label plate, incubating for 40min at 37 ℃, washing for five times by PBST, patting dry wellsInternal liquid, adding 1: incubating enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted by 5000 at 37 ℃ for 40min, washing the secondary antibody with PBST for five times, patting the liquid in the hole dry, adding 100 mu LTMB substrate liquid, and developing the color for 10min at 37 ℃ in a dark place; add 50. Mu.L of stop solution (2M H) 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader. Taking concentration of tiamulin standard substance to abscissa, B/B 0 (OD of wells to which tiamulin was added 450 OD450 of wells without tiamulin) as ordinate, an indirect competition standard curve was established.
(2) Results of the experiment
An indirect competition ELISA standard curve graph established based on the monoclonal antibody is shown in figure 1, and can be seen that the standard curve is S-shaped, the linear correlation is good, the minimum detection limit of tiamulin is 0.09ng/mL, and IC (integrated Circuit) is 50 0.53ng/mL, and the linear range is 0.21-1.35 ng/mL; high detection sensitivity and wide linear range.
Example 6 specific detection of tiamulin monoclonal antibody
1. Experimental methods
The tiamulin monoclonal antibody prepared in example 4 was used to detect roxithromycin, kanamycin, arsenopyrite, enrofloxacin, ofloxacin, clarithromycin, sarafloxacin, fleroxacin, and salbutamol, in the same manner as in example 5.
2. Results of the experiment
The specific experimental results are shown in table 2 below, and the results show that the tiamulin monoclonal antibody prepared in example 4 has no cross (less than 0.05%) to roxithromycin, kanamycin, arsenic anilide, enrofloxacin, ofloxacin, clarithromycin, sarafloxacin, fleroxacin and salbutamol, which indicates that the tiamulin monoclonal antibody prepared in example 4 has good specificity and is not easy to cause false positive.
TABLE 2 tiamulin structural and functional analogs Cross-reaction results
Figure BDA0002938810580000121
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Figure BDA0002938810580000131
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims (2)

1. The composition of immunogen and coating antigen for enzyme-linked immunosorbent assay (ELISA) detection of tiamulin is characterized in that the immunogen and the coating antigen have structural formulas shown as a formula (II):
Figure FDA0003974691620000011
the carrier protein on the immunogen is lactoferrin, and the carrier protein on the coating antigen is chicken ovalbumin.
2. The use of the composition of immunogen and coatingen as claimed in claim 1 in detecting tiamulin or preparing a tiamulin detection kit.
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