CN110205302B - Cell strain secreting monoclonal antibody against mycophenolic acid, monoclonal antibody and application thereof - Google Patents
Cell strain secreting monoclonal antibody against mycophenolic acid, monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a cell strain secreting monoclonal antibody against mycophenolic acid and application thereof. The preservation number of the cell strain secreting the anti-mycophenolic acid monoclonal antibody is CGMCC NO: 17590. the monoclonal antibody secreted by the cell strain has the advantages of high titer, good specificity and strong affinity with antigen, and the indirect competitive ELISA detection method for mycophenolic acid based on the monoclonal antibody has good sensitivity and specificity, and can be used for detecting mycophenolic acid residue in food and animal feed.
Description
Technical Field
The invention relates to a cell strain secreting monoclonal antibody against mycophenolic acid, a monoclonal antibody secreted by the cell strain and application of the monoclonal antibody in the field of immunodetection, and belongs to the technical field of immunochemistry.
Background
Mycophenolic acid (mycophenolic acid) is a mycotoxin produced by penicillium moulds. Mycophenolic acid can contaminate not only corn, silage, grain crops and products thereof, but also daily foods such as fruits, dairy products, nuts, dried fruits, meat products and the like. In animal bodies, mycophenolic acid can block T, B lymphocyte proliferation reaction, inhibit antibody formation and cytotoxic T cell generation, and further inhibit immune system function. The adverse effect not only directly harms the health of livestock and poultry and reduces the quality of animal-derived food, but also threatens the health of human beings. Therefore, the content of mycophenolic acid in the livestock and poultry feed and the animal derived food needs to be detected and monitored.
The detection method for the mycophenolic acid mainly comprises a liquid chromatography-mass spectrometry combined detection method (LC-MS), an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS), a High Performance Liquid Chromatography (HPLC) and the like, some detection methods are complex in operation, some samples are complex in processing, some samples depend on professionals and all need professional instruments, the detection process is long in time consumption and high in cost, and the detection method is not beneficial to actual production and application. Therefore, the establishment of a detection method with high sensitivity and simple operation has great significance for detecting the mycophenolic acid residue in food and feed. Enzyme-linked immunosorbent assay (ELISA) is widely applied to mycotoxin and veterinary drug residue detection. The method has the advantages of strong specificity, high detection efficiency, simple operation, high sensitivity and suitability for mass detection. The invention lays a foundation for research and development and popularization of the mycophenolic acid residue detection indirect competition ELISA kit and the colloidal gold test strip.
Disclosure of Invention
The invention aims to provide a cell strain secreting monoclonal antibodies against mycophenolic acid, monoclonal antibodies secreted by the cell strain, and application of the cell strain and the monoclonal antibodies secreted by the cell strain in the field of immunoassay.
The monoclonal antibody against mycophenolic acid is obtained by immunizing mice with mycophenolic acid immunogen. The immunogen is synthesized by a carbodiimide method, and the detecting antigen is synthesized by an active ester method.
The invention provides a cell strain secreting monoclonal antibody against mycophenolic acid, which has the preservation number of CGMCC NO: 17590.
the cell strain is registered and preserved in China general microbiological culture Collection center (CGMCC) at 5 and 8 months in 2019, and the preservation number is CGMCC NO: 17590, categorical designation: the anti-mycophenolic acid monoclonal antibody cell strain 9C 2.
The invention provides an anti-mycophenolic acid monoclonal antibody in a second aspect, which is prepared from the following components in a preservation number of CGMCC NO: 17590 or a subcultured cell line thereof.
The basic steps for preparing the cell strain 9C2 provided by the invention are as follows:
1. complete antigen synthesis: coupling carboxyl of the mycophenolic acid with amino of a carrier protein BSA by using a carbodiimide method to prepare immunogen; the active ester method is utilized to couple the carboxyl of the mycophenolic acid with the amino of the carrier protein OVA to prepare the detecting antigen. After the reaction is finished, the complete antigen is separated and purified by dialysis, and is identified by an ultraviolet spectrum scanning method and an SDS-PAGE Coomassie brilliant blue staining method.
2. Immunizing a mouse: after the antigen is emulsified with Freund's adjuvant, BALB/c mice are immunized by intraperitoneal injection in combination with dorsal, axillary and inguinal injection. Freund's complete adjuvant is adopted for the first immunization; freund's incomplete adjuvant was used for booster immunization; the adjuvant is not used during impact immunization, the antigen and normal saline are mixed evenly and then are directly injected into the abdominal cavity, and the immunization interval is two weeks. One week after the third immunization, tail vein blood sampling is carried out to detect the serum titer and the inhibition rate of the mice.
3. Cell fusion and cell line establishment: fusion was induced by polyethylene glycol (PEG-1450) to connect mouse splenocytes to myeloma cells according to a 5: 1, culturing the fused cells by using an HAT culture medium, performing half-liquid exchange on a cell plate by using the HAT culture medium on the fifth day after fusion, observing cell clusters with good and obvious growth states on the tenth day after fusion, sucking supernatant for detection, detecting the inhibition effect of cell pores by using an indirect competitive ELISA method, performing three times of subcloning by using a limiting dilution method after screening, and finally screening the anti-mycophenolic acid monoclonal cell strain 9C2 with high titer and good inhibition effect.
4. Characterization of cell line Properties: detecting the antibody subtype by using a mouse antibody subtype determination kit; purifying the antibody by using a Hitrap protein G immunoaffinity chromatography column; IC50 values, cross-reactivity assays, antibody affinity assays were performed using indirect ELISA.
The invention has the beneficial effects that: (1) the anti-mycophenolic acid monoclonal antibody cell strain obtained by the invention can achieve the purpose of detecting mycophenolic acid. (2) Two coupling methods are selected to synthesize immunogen and detection antigen, and different carrier proteins and methods are used for coupling to achieve higher detection sensitivity.
Biological material sample preservation: the cell strain 9C2 of the invention is classified and named as anti-mycophenolic acid monoclonal antibody cell strain, is preserved in China general microbiological culture Collection center (CGMCC) for short, and has the address as follows: the collection number of the microbial research institute of Chinese academy of sciences, No. 3 Xilu No.1 of Beijing, Chaoyang, and the collection date is CGMCC No.17590, and 2019, 05 and 08 days.
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FIG. 1 shows the results of Coomassie blue staining of the gel electrophoresis of immunogen (A) and detector (B).
FIG. 2. immunogen UV scanning spectrum.
FIG. 3 is a graph showing the original UV absorption spectrum.
FIG. 4 serum titers and inhibition rates of mice.
FIG. 5 Coomassie Brilliant blue staining of purified antibody gels.
FIG. 6 shows a standard competition curve for mycophenolic acid.
FIG. 7 shows the results of monoclonal antibody subtype identification.
Detailed Description
The following examples of the present invention are provided as further illustration of the present invention and are not intended to limit the scope or content of the present invention. The invention is further illustrated by the following examples.
The preparation method comprises the steps of immunizing a mouse with the prepared mycophenolic acid immunogen, selecting the mouse with the best titer and inhibition effect for cell fusion, screening cells by using an HAT selective culture medium, screening cell supernatants by using indirect ELISA and indirect competitive ELISA to obtain a cell hole with the best mycophenolic acid inhibition effect, and finally obtaining a cell strain capable of secreting a monoclonal antibody with better affinity and sensitivity to mycophenolic acid by performing three times of subcloning on the cells in the hole.
Example 1: preparation of anti-mycophenolic acid monoclonal cell line 9C2
1. Synthesis of complete antigens
(1) Preparation of immunogen: MPA was coupled to BSA (bovine serum albumin) by the carbodiimide method. 24mg of MPA is weighed and dissolved in DMF, 17.4mg of NHS and 29mg of EDC are weighed and dissolved in PBS solution, and the two solutions are mixed and stirred to react for 12 hours, thus obtaining solution A. 50mg of BSA was weighed and dissolved in PBS solution to obtain solution B. Dropwise adding the A solution into the B solution, and stirring for reaction for 12 hours. Dialyzing at 4 deg.C for 3 days, subpackaging at-20 deg.C, and freezing.
(2) Preparation of a detection source: detecting the original synthesis: MPA and OVA (ovalbumin) were coupled by the active ester method. Weighing 12mg MPA, dissolving in DMF, weighing 8.75mg NHS and 6.4mg DCC, dissolving in DMF, mixing, stirring, and reacting for 12h to obtain solution A. 27g OVA was weighed out and dissolved in 0.13mol/L NaHCO3And (4) obtaining a solution B. Dropwise adding the A solution into the B solution, and stirring for reaction for 12 hours. Dialyzing at 4 deg.C for 3 days, subpackaging at-20 deg.C, and freezing.
(3) Complete antigen identification: the synthesized immunogen and the detection antigen are identified by SDS-PAGE electrophoresis Coomassie brilliant blue staining, and the results are respectively shown in figure 1A and figure 1B. The identification results of the synthesized immunogen and the detection antigen by ultraviolet scanning are shown in figures 2 and 3.
2. Animal immunization
Selecting 6-8 weeks old female BALB/c mice for immunization, immunizing each mouse with 100 mu g immunogen MPA-BSA, diluting the immunogen with a certain volume of normal saline, uniformly mixing the diluted immunogen with an isovolume of Freund's adjuvant, fully emulsifying, and injecting the mixture at multiple points in abdominal cavity, back subcutaneous region, axilla and groin. The Freund complete adjuvant is adopted for the first immunization, the Freund incomplete adjuvant is adopted for the boosting immunization, the impact immunization is only diluted by normal saline, the four weeks after the first immunization are twice immunized, and the boosting immunization is separated for 4 times in two weeks. One week after the third immunization, the serum of the mice is collected, and the titer and the inhibition rate of the serum of the mice are detected by indirect ELISA and indirect competitive ELISA, and the detection result is shown in figure 4. And detecting the titer and the inhibition rate of the mouse serum by the same method one week after the fifth immunization, and screening the abdominal cavity impact immunization of the mouse with high titer and good inhibition effect three days before cell fusion.
3. Cell fusion
Three after impact immunizationAnd (3) performing cell fusion by adopting 50% PEG-1450, aseptically picking the spleen of the mouse, and enabling the ratio of the spleen cells of the mouse to the myeloma cells to be 5: 1 to form cells. The fusion operation was as follows: (1) collecting SP/20 cells which are cultured in advance and grow to a logarithmic phase, suspending the cells by using an incomplete DMEM culture solution after centrifugation, and placing the cells in a 37 ℃ water bath for later use after counting; (2) picking up the spleen of a mouse in a sterile operation, grinding the spleen, filtering to obtain a spleen cell suspension, removing fat and other tissues through an electric pipettor, centrifuging, repeating the operation once, re-suspending the spleen cell suspension by using incomplete DMEM culture solution, counting, and placing the spleen cell suspension in a water bath kettle at 37 ℃ for later use; (3) spleen cells and SP/20 cells were mixed as 5: mixing according to the proportion of 1, centrifuging, removing culture solution, slightly shaking and uniformly mixing, adding 1mL of preheated PEG, finishing adding within 1min, firstly slowing, then quickly dripping, simultaneously shaking and uniformly mixing, standing and fusing for 1 min; slowly dripping 50mL of DMEM incomplete culture solution while rotating, finishing adding within 5min, slowly adding at first, quickly adding at second, and standing for 10 min. (4) Centrifuging, discarding supernatant, resuspending cells with HAT culture medium, spreading in 96-well cell culture plate at 200 μ L/well, standing at 37 deg.C and 5% CO2Culturing in a constant temperature incubator, and regularly observing.
4. Cell screening and cell line establishment
And on the fifth day after fusion, half liquid exchange is carried out on the cell plate by using HAT culture solution, on the tenth day after fusion, a cell mass with a good and obvious growth state is observed, supernatant is sucked for detection, a cell strain with high titer and good mycophenolic acid identification is screened out through indirect ELISA and indirect competitive ELISA for subcloning, a positive monoclonal cell strain 9C2 is obtained by performing subcloning for 3 times by adopting a gradient dilution method, and the corresponding CGMCC (China general microbiological culture collection center) preservation number is No. 17590.
5. Preparation of monoclonal antibodies
A large number of monoclonal antibodies were prepared by in vivo induction in mice. Taking 10-12-week-old male BALB/c mice, and injecting 0.5mL of Freund's incomplete adjuvant into the abdominal cavity of each mouse; intraperitoneal injection of 0.5X 10 per mouse after one week6After 7 days, when the abdomen of the mouse is enlarged, ascites is collected by a syringe needle. After ascites collection, centrifuging to remove upper fat and lower sediment, and taking clear liquid for later use; miningAfter the subtype of the antibody is identified by using the mouse antibody subtype identification kit, the subtype of the antibody is IgG2bPurifying with Protein G purifying column, subpackaging, and storing at-20 deg.C. The purified antibody was identified by Coomassie blue staining on SDS-PAGE, and the results are shown in FIG. 5.
6. Monoclonal antibody titer and inhibition rate determination
Diluting the detection source to a certain concentration by using a coating buffer solution, coating an enzyme label plate by 100 mu L per hole, and standing overnight at 4 ℃; PBST is washed for 3 times, 200 mu L of blocking solution is added into each hole, and incubation is carried out for 2h at 37 ℃; diluting the purified antibody by multiple times, incubating for 1h at 37 ℃ and 100 mu L per well; washing with PBST solution for 6 times, adding enzyme-labeled secondary antibody diluted by 5000 times, incubating at 37 deg.C for 1h, wherein each well is 100 μ L; after PBST solution washing, 100. mu.L of the prepared color developing solution is added into each hole, and the reaction is carried out for 15min at 37 ℃ in a dark place. 50 μ L of 2mol/L H per well2SO4The solution stops the reaction. And (5) placing the monoclonal antibody in a microplate reader for detecting the titer of the monoclonal antibody under the wavelength of 450 nm.
The titer and the inhibition rate of the monoclonal antibody against the mycophenolic acid are detected by adopting indirect competition ELISA, and the IC50 of the monoclonal antibody against the mycophenolic acid is calculated by drawing a standard curve as shown in figure 6: 0.59ng/mL, can be used for detecting the residue of mycophenolic acid in food and feed.
Example 2: properties of monoclonal antibody against mycophenolic acid
1. Cross reaction
And detecting whether the prepared monoclonal antibody has cross reaction with other different mycotoxins by adopting indirect competition ELISA. The assay source was diluted in carbonate buffer at 100. mu.L/well for 2h at 37 ℃. The dilution of the detection source is 2000 times, and the dilution of the monoclonal antibody is 32000 times determined by a chessboard test. The plate was discarded, patted dry and washed three times with PBST solution for 3min each. After patting dry, 200. mu.L of blocking solution was added thereto at 37 ℃ for 2 hours. Washing with the same method, and drying. 50 μ L of standard solution and 50 μ L of antibody were added to each well, after incubation for 1h at 37 ℃, PBST solution was washed 6 times and blotted dry, 100 μ L of enzyme-labeled secondary antibody diluted 5000-fold was added to each well, and incubation for 1h at 37 ℃. PBST solution is washed for 6 times and patted dry, 100 mu L of prepared color development liquid is added into each hole, and the reaction is carried out for 15min at 37 ℃ in a dark place. 50 μ L of 2mol/L H per well2SO4Solution finishingStopping the reaction. And (5) placing the sample in a microplate reader for detection at a wavelength of 450 nm.
Several mycotoxins were detected by cross-reaction, see table 1: mycophenolate mofetil (mycophenolate mofetil), zearalenone (zearalenone), deoxynivalenol (deoxynivalenol), rapamycin (rapamycin), and cyclosporin A (cyclosporine A). The cross-reactivity rate was determined by the ratio of mycophenolic acid IC 50/other reactant IC50 × 100% to determine the specificity of the monoclonal antibody prepared. The cross reaction rate with mycophenolate mofetil is 88%, and the cross reaction rate with other substances is not cross reaction.
TABLE 1 Cross-reactivity of monoclonal antibodies with other mycotoxins
| Name of drug | Median inhibitory concentration IC50(ng/mL) | Cross reaction Rate CR (%) |
| Mycophenolic acid | 0.59 | 100 |
| Mycophenolic acid morpholine ethyl ester | 0.67 | 88 |
| Zearalenone | >10000 | 0.006 |
| Deoxynivalenol | >10000 | 0.006 |
| Rapamycin | >10000 | 0.006 |
| Cyclosporin A | >10000 | 0.006 |
2. Addition recovery test
The recovery rate was calculated by detecting mycophenolic acid in milk samples with monoclonal antibodies, see table 2.
TABLE 2 results of determination of recovery of mycophenolic acid addition in milk base
Solution preparation:
phosphate buffer (0.01mol/L, pH 7.4): weighing NaCl 4g and KH2PO4 0.135g，KCl 0.1g，Na2HPO40.71g, adding 500mL of UPW, mixing, adjusting pH to 7.4, and storing at 4 deg.C for no more than two months.
Carbonate buffer (0.05mol/L, pH 9.6): weighing Na2CO3 0.848g，NaHCO31.428g, adding UPW 500mL, mixing, adjusting pH to 9.6, storing at 4 deg.C for less than one month.
Substrate buffer (0.2mol/L, pH 5.2): weighing Na2HPO4·12H2O 1.058g，KH2PO413.2005g, adding UPW to 500mL, adjusting pH to 5.2, and storing at 4 deg.C.
TMB storage solution: 21mg of TMB was dissolved in 5mL of absolute ethanol and stored at 4 ℃ in the dark.
UHP storage solution: 165mg of urea hydrogen peroxide is dissolved in 10mL of UPW, fully and uniformly mixed, subpackaged and stored at the temperature of minus 20 ℃.
TMB color development liquid: measuring UPW 4.75mL, substrate buffer 4.95mL, UHP 50 μ L and TMB 250 μ L, mixing well, preparing for use, and storing at 4 deg.C in dark place.
Stop solution (2 mol/L): UPW 178.3mL was measured, and 21.7mL of concentrated sulfuric acid (98%) was added dropwise thereto and stored at room temperature.
Wash buffer (0.01mol/L PBST solution): weighing 0.5mL of Tween-20, adding into 1L of PBS solution, mixing well, and storing for no more than 7 days.
3. Antibody subtype identification
The mouse Ig class antibody subtype identification kit is used for identifying the ascites antibody subtype prepared by 9C2 cells, and the result is shown in figure 7, and the antibody prepared by 9C2 ascites is IgG2bSubtype, Kappa light chain.
Claims (3)
1. A cell strain secreting monoclonal antibody against mycophenolic acid is disclosed, wherein the preservation number of the cell strain is CGMCC NO: 17590.
2. an anti-mycophenolic acid monoclonal antibody secreted by the cell strain or the subcultured cell strain of claim 1.
3. The use of the cell line of claim 1 secreting monoclonal antibodies to mycophenolic acid for detecting mycophenolic acid residues in food and animal feed.
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| US20050176080A1 (en) * | 2004-02-10 | 2005-08-11 | Vani Bodepudi | Hapten, immunogens and derivatives of ascomycin useful for preparation of antibodies and immunoassays |
| JP2012510468A (en) * | 2008-11-28 | 2012-05-10 | アボット・ラボラトリーズ | Stable antibody composition and method for stabilizing the same |
| ES2708565T3 (en) * | 2013-03-15 | 2019-04-10 | Atyr Pharma Inc | Conjugates of Fc-histidyl-tRNA synthetase |
| DK3442580T3 (en) * | 2016-04-11 | 2020-12-21 | Genfit | PROCEDURES FOR THE TREATMENT OF CHOLESTATIC AND FIBROTIC DISEASES |
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| CN101518653A (en) * | 1999-06-25 | 2009-09-02 | 基因技术股份有限公司 | Methods of treatment using anti-erbb antibody-maytansinoid conjugates |
| CN1767836A (en) * | 2003-04-01 | 2006-05-03 | 诺瓦提斯公司 | Parenteral formulation of mycophenolic acid, a salt or prodrug thereof. |
| WO2017049120A1 (en) * | 2015-09-17 | 2017-03-23 | Medical Service Consultation International Llc | Methods and compositions for detecting mycotoxins |
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