CN112239751B - Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses a florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, a monoclonal antibody secreted by the same and application of the monoclonal antibody. The hybridoma cell strain is prepared from florfenicol artificial antigen, the secreted monoclonal antibody has higher cross reaction to florfenicol and thiamphenicol, and has extremely high detection sensitivity and higher working titer to florfenicol; the prepared immunoaffinity chromatography adhesive is mixed with an immunoaffinity chromatography adhesive of chloramphenicol to serve as a purification reagent for pretreatment of mechanical measurement, and the immunoaffinity chromatography adhesive is combined with an LC-MS/MS detection technology, so that a chloramphenicol residual sample can be efficiently detected, and the immunoaffinity chromatography adhesive has various excellent effects of excellent detection limit, quantitative limit, recovery rate, purification capacity, matrix interference resistance and the like.

Description

Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application

Technical Field

The invention relates to the technical field of biology, in particular to a florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, a monoclonal antibody secreted by the same and application of the monoclonal antibody.

Background

Chloramphenicol antibiotics (including chloramphenicol, thiamphenicol and florfenicol) are widely applied to prevention and treatment of animal bacterial diseases due to the advantages of wide antibacterial spectrum, easy absorption by organisms, low medication cost and the like, and are increasingly highlighted in toxic and side effects on human bodies through food chains after years of use, especially the chloramphenicol causes aplastic anemia in human bodies, so that attention is paid. Therefore, chloramphenicol cannot be detected in meat, poultry, egg and milk products in clear text regulations in many countries including China, which means that the residual quantity of chloramphenicol must be less than 0.1ppb, which is the highest detection limit of the existing detection technology.

In view of the banned use of chloramphenicol, the derivatives replacing the nitro group on the phenyl ring of the chloramphenicol molecule with a methylsulfonyl group are the second generation of chloramphenicol antibiotics: the toxic and side effects of thiamphenicol and florfenicol on animals and human bodies are greatly reduced, the thiamphenicol and florfenicol are still used as chloramphenicol substitute drugs to be rapidly popularized and used in livestock raising and aquaculture bacterial disease treatment at home and abroad by the characteristics of broad spectrum, high efficiency, rapid absorption, wide distribution, safety, special use for animals and the like, and certain toxic and side effects on organisms such as inhibition of thiamphenicol on the development of organism immune systems are still achieved although the toxic and side effects are far smaller than those of chloramphenicol; florfenicol inhibits embryonic development and the like in a non-trivial way, and the highest residual standard of two drugs in edible parts of animal sources specified in China is less than 100-1000ppb.

In more than ten years, after the chloramphenicol ELISA-specific detection kit is introduced to the market in china by R-biopharm (R), the shenzhou shi team at the university of china in combination with beijing wedgeukang, and particularly the professor team at the university of south jiang has introduced chloramphenicol rapid detection products such as chloramphenicol, thiamphenicol, florfenicol triple colloidal gold test strips, although these products have irreplaceable effects on high-throughput screening of chloramphenicol single targets or/and multiple target residues in livestock and poultry, and have no completely definite and precise quantification of each single target due to the limitations of the immunological rapid detection products themselves, particularly the immunological crossability among similar targets, and have no negative exclusion effect as a legal basis for illegal or overproof use.

In view of this, the mechanical analysis is more concerned by domestic food security inspection departments and the like. In recent years, the departments are equipped with powerful analysis equipment with strong functions such as GC-MS, HPLC-MS/MS, UPLC-MS/MS and the like of international well-known companies such as Waters, agilent and the like, and the technical parameters such as chromatographic separation level, detector signal response level and the like can meet the national standards of the residue limits of the three target substances, but the actual demand of a sample to be detected is difficult to meet due to low detection flux, high technical requirements, high detection cost and the like; more importantly, the authenticity, reliability and reproducibility of detection data are overwhelmingly determined by the sample pretreatment effect, for the pretreatment of chloramphenicol antibiotic residue samples in machine testing, including various test schemes such as the national standards of chloramphenicol residue detection revised in 2008, the sample pretreatment adopts a conventional physicochemical treatment mode, wherein the purification link of the solid phase extraction column is a key step and various technical bottlenecks exist:

1. the loss of multiple targets is large or even lost in complicated operation links;

2. although the three targets belong to the same class, the physical and chemical properties of the targets are greatly different, so that the purification by a single solid phase extraction column is difficult to obtain a satisfactory recovery rate;

3. the purification of the solid-phase extraction column is difficult to completely eliminate the interference of the matrix;

4. the recovery rate of the target object is very low after being purified by the solid-phase extraction column, and the actual residual quantity of the target object of the sample to be detected can be accurately calculated only by correcting the pretreatment recovery rate by using the corresponding isotope standard product as an internal standard, so that the detection cost is very high, and the storage and treatment of radioactive waste are tired.

5. Solid Phase Extraction Cartridge SPE (Solid Phase Extraction Cartridge) relies on import, is affected by new crown epidemics and has a risk of interruption of its supply chain.

Based on the technical bottleneck of the physicochemical pretreatment of the to-be-detected sample, the chloramphenicol strong specificity immunoaffinity chromatography gel is prepared by using the basic principle that the chloramphenicol specific antibody has strong specificity (selectivity), high affinity and antigen-antibody reaction reversibility, and the mode of the chloramphenicol strong specificity immunoaffinity chromatography gel is used for the pretreatment of the detection by a machine, so that the strong selectivity and the high enrichment of the immunochromatography technology to a target can be perfectly combined with the effective chromatographic separation of a chromatographic column to the target and the strong molecular information analysis capability of a detector, and the combined technology is the target pursued by related scholars.

Although a large number of corresponding antibody preparations aiming at chloramphenicol exist in China in more than ten years, and a few sporadic reports aiming at the preparation of florfenicol/thiamphenicol antibodies exist in recent years, the application reports of the antibody preparations in the immunoaffinity chromatography technology are few, particularly, the three target substances in a sample to be detected are simultaneously enriched by a one-step immunochromatography method, and are not formally reported, so that the strong technical support effect of the technology on HPLC-FLD or LC-MS/MS detection is limited.

Disclosure of Invention

In view of the above, the present invention aims to provide a florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain and a monoclonal antibody produced by the same, such that the monoclonal antibody produced by the hybridoma cell strain has high cross-reaction to florfenicol and thiamphenicol, and has extremely high detection sensitivity (IC) to florfenicol ₅₀ 1.35 ppb) and higher working titer (working dilution 1:40000 );

the invention also aims to provide an immunoaffinity chromatography reagent consisting of the immunoaffinity chromatography gel prepared by the monoclonal antibody and a machine-test pretreatment reagent, so that the immunoaffinity chromatography gel has various excellent effects of excellent detection limit, quantitative limit, recovery rate, purification capacity, matrix effect resistance and the like after the immunoaffinity chromatography gel is subjected to machine-test pretreatment;

the invention also aims to provide the application of the immunoaffinity chromatography glue prepared from the monoclonal antibody and the pretreatment reagent for machine detection in pretreatment and/or detection of a chloramphenicol antibiotic residual sample.

The invention also aims to provide an immunoaffinity chromatography reagent consisting of the immunoaffinity chromatography gel prepared by the monoclonal antibody, the immunoaffinity chromatography gel prepared by the chloramphenicol monoclonal antibody and a pretreatment reagent for machine detection, so that the immunoaffinity chromatography reagent has excellent effects of detection limit, quantification limit, recovery rate, purification capacity, matrix interference resistance and the like after pretreatment by the machine detection.

In order to achieve the purpose, the invention provides the following technical scheme:

the florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain has a preservation number of CCTCC No. C201945 and is named as 9D ₄ -FF, and deposited in the China center for type culture Collection at 18 months 3 and 2019, at Wuhan university in Wuchang Lojia mountain.

The invention takes the florfenicol novel artificial antigen as immunogen to immunize mice, and the 9D is obtained by screening ₄ -FF hybridoma cell strain, cross reaction rate of induced monoclonal antibody with thiamphenicol and chloramphenicolWhen the CR value of florfenicol is determined to be 100%, the cross-reaction rate of thiamphenicol is found to be larger, the CR values are 69.69%, and the CR value of chloramphenicol is less than 0.001%.

The novel artificial antigen is modified into florfenicol glutaric acid semialdehyde, N, by dissolving florfenicol (hapten) in pyridine and reacting with glutaric anhydride (five-carbon-chain connecting arm) ₂ After air drying, the mixture is transferred into a dimethylformamide/1, 4-dioxane mixed solvent, activated by isobutyl chloroformate/n-tributylamine and then mixed with free-NH in bovine serum albumin ₂ Compared with the prior report that the florfenicol is prepared by taking succinic anhydride (four-carbon chain connecting arm) as a modifier, the novel artificial antigen has the advantages that:

(1) The length of the connecting arm is lengthened: from original C ₄ Chain to C ₅ And the chain enables the florfenicol epitope to be exposed more fully and is convenient for an organism to recognize.

(2) The florfenicol chemical modification, the activation of the modification group and the crosslinking with the protein carrier are all finished in a mild reaction system, do not need advanced instruments and equipment and can be finished in a common laboratory.

(3) The reaction system has no extraction step: not only ensures the product yield, but also can realize the micro-quantification of the reaction system.

The specific reaction process is as follows:

hybridoma cell strain-9D obtained by the invention ₄ The steps of-FF are specifically as follows:

(1) aiming at the synthesis and physicochemical characterization analysis of florfenicol artificial antigen-immunogen;

(2) synthesizing an artificial antigen-coating antigen aiming at florfenicol;

(3) immunizing Balb/C mice and selecting qualified immunized mice;

(4) cell fusion, and screening and cloning of florfenicol hybridoma cell strains;

(5) obtaining and evaluating induced ascites in vivo;

through the steps, the florfenicol monoclonal hybridoma cell-9D is obtained ₄ -FF, which induces in vivo ascites samples, analyzed by an indirect competitive ELISA assay, said samples having an extremely high detection sensitivity (IC) for florfenicol ₅₀ 1.35 ppb) and higher working titers (working dilution 1:40000 The immunological cross-over rate with thiamphenicol is about 69.69 percent, and the immunological cross-over rate with chloramphenicol is less than 0.001 percent. The result provides a core raw material with stable supply, reliable quality and low price for preparing the florfenicol/thiamphenicol specific immobilized immunoadsorbent.

Based on the excellent technical effects, the invention provides the application of the hybridoma cell strain or the monoclonal antibody secreted by the hybridoma cell strain in the pretreatment/immunodetection of a chloramphenicol residual sample machine, or in the preparation of a chloramphenicol residual sample machine pretreatment/immunodetection product; wherein the chloramphenical is florfenicol and/or thiamphenicol.

More preferably, the invention further provides an application of the hybridoma cell strain with the preservation number of CCTCC No. C201945 or the monoclonal antibody secreted by the hybridoma cell strain in combination with the preservation number of CCTCC No. C201870 or the monoclonal antibody secreted by the hybridoma cell strain in pretreatment/immunoassay of a chloramphenicol residual sample machine, or an application in preparation of a chloramphenicol residual sample machine pretreatment/immunoassay product.

Monoclonal antibodies secreted by hybridoma cell strains with the preservation number of CCTCC NO. C201870 have strong specificity (one-to-one) and high affinity for chloramphenicol, but have no immunological cross-over property on two important substitutes of chloramphenicol, thiamphenicol and florfenicol; the hybridoma cell strain has a preservation number of CCTCC NO. C201870, is named as 2D2-C1, is preserved in China center for type culture collection in 2018, 3 months and 14 days, and is addressed to Wuhan university in Wuchang Lojia mountain.

Preferably, the chloramphenicol is one or more of florfenicol, thiamphenicol and chloramphenicol.

In a specific embodiment of the invention, the pre-machine detection/immunoassay product is an immunoaffinity chromatography gel or an immunoaffinity chromatography column pre-filled with the gel.

After parameters such as a binding solution, a washing solution, an eluent and the like are optimized, an immunochromatography gel enrichment sample of two target substances of florfenicol/thiamphenicol series concentration standard solutions is quantitatively determined by HPLC-MS/MS: for florfenicol detection limit and quantification limit, 0.1,0.3ppb, effective adsorption concentration domain: 0.1 to 81ppb; for thiamphenicol detection limit and quantification limit, 0.3,0.9ppb, effective adsorption concentration domain: 0.3 to 81ppb; for chloramphenicol detection limit and quantification limit, 0.03,0.1ppb, effective adsorption concentration region: 0.01 to 81ppb; can meet the basic requirements of preparing the chloramphenicol mixed immunoaffinity chromatography gel.

Preferably, the pretreatment/immunodetection product further comprises a binding solution, a washing solution and an eluent; the combined solution is 0.01mol mL ^-1 PBS, the washing solution is 0.01mol mL ^-1 PBS and water (alternate wash), the eluent was methanol: and the volume ratio of the ammonia water is 9.

The immunoaffinity chromatography gel provided by the invention is used as a core reagent for purifying chloramphenicol residual samples, three drug mixtures with series concentrations and equal doses are respectively doped into shrimp meat/natural lake water to be used as mode samples, the mode samples are purified before being detected by a chromatography gel machine, the processed samples are subjected to LC-MS/MS qualitative and quantitative analysis, and a comparative test is carried out with a solid phase extraction SPE (solid phase extraction) purification method of a national standard method. The results show that: the indexes of treatment flux, matrix interference resistance, detection limit, recovery rate of doped target substances and the like are used for evaluation, and the mode is comprehensively superior to the national standard solid phase extraction purification mode.

According to the application, the invention provides a chloramphenicol specific immunoaffinity chromatography pretreatment/immunodetection product, which comprises immunoaffinity chromatography chromatographic gel or an immunoaffinity chromatography chromatographic column pre-filled by the gel, wherein the immunoaffinity chromatography chromatographic gel is formed by purifying monoclonal antibodies secreted by hybridoma cell strains with the preservation number of CCTCC No. C201945 and then chemically crosslinking the monoclonal antibodies with CNBr-activated Sepharose-4B gel.

Preferably, the pre-machine-test/immunoassay product further comprises: the monoclonal antibody is secreted and produced by a hybridoma cell strain with the preservation number of CCTCC No. C201870, purified and chemically crosslinked with CNBr-activated Sepharose-4B gel to form immunoaffinity chromatography chromatographic gel or an immunoaffinity chromatography chromatographic column pre-filled by the gel.

More preferably, it further comprises: binding solution, washing solution and eluent; the combined solution is 0.01mol mL ^-1 PBS, the washing solution is 0.01mol mL ^-1 PBS and water (alternate washes), the eluent was methanol: and the volume ratio of ammonia water is 9.

In the preparation of the immunoaffinity chromatography gel, the invention is obtained by chemically crosslinking a purified monoclonal antibody protein and a CNBr-activated Sepharose-4B gel to form a solid-phase antibody protein, and the immunoaffinity chromatography column is obtained by filling the immunoaffinity chromatography gel in a column bed. According to the detection requirement, the chromatographic column can be added with a chromatographic gel prepared by secreting and producing monoclonal antibodies from hybridoma cell strains with the preservation number of CCTCC No. C201945 and/or a chromatographic gel prepared by secreting and producing monoclonal antibodies from hybridoma cell strains with the preservation number of CCTCC No. C201870, and the volume ratio of the two is preferably 1.

According to the performance of the immunoaffinity chromatography chromatographic gel/column, the invention also provides a method for pretreating a chloramphenicol residual sample, and the pretreatment/immunodetection product for the mechanical test of the sample to be detected is adopted.

Meanwhile, the invention also provides a method for detecting the chloramphenicol residual sample, the sample to be detected is subjected to machine pretreatment by adopting the machine pretreatment/immunodetection product, and the treated sample is detected by LC-MS/MS.

The invention selects monoclonal antibody hybridoma cell strain source with better florfenicol affinity and thiamphenicol immunological cross reaction to prepare purified antibody protein, prepares immunoaffinity chromatography gel by using the antibody protein and chloramphenicol monoclonal antibody secreted by specific hybridoma cell according to GE standard operation procedures, and detects the obtained chromatography gel/column product by LC-MS/MS and other analytical equipment, thereby having better detection limit and quantification limit, and simultaneously having better matrix interference resistance, standard recovery rate and purification treatment capacity compared with the national standard SPE method.

Biological preservation information description

(1) Hybridoma cell line 9D for preservation ₄ The classification of-FF is named:

the preservation unit is fully called as follows: china center for type culture Collection;

the preservation unit is abbreviated as: CCTCC (China center for type communication);

the address of the depository: wuhan university in Wuchang Lojia mountain;

the preservation date is as follows: 2019, month 3, 18;

the preservation number is as follows: CCTCC No. C201945;

(2) The classification and designation of the hybridoma cell strain 2D2-C1 for preservation is as follows:

the preservation unit is called as follows: china center for type culture Collection;

the preservation unit is abbreviated as: CCTCC;

the address of the depository: wuhan university in Wuchang Lojia mountain;

the preservation date is as follows: 2018, month 3, and day 14;

the preservation number is: CCTCC NO. C201870;

drawings

FIG. 1 shows the mass spectrum of FF-glu-BSA macromolecule;

FIG. 2 is a graph showing competitive inhibition of FF by induced ascites in cells;

FIG. 3 is a graph showing competitive inhibition of ascites due to induction of cells against chloramphenicol standards;

FIG. 4 is a protein electrophoresis identification chart of two purified products of two monoclonal antibody induced ascites samples purified by protein G;

FIG. 5 is a visual chart showing the detection of protein content before and after crosslinking of two monoclonal antibody protein purifiers with gelatin;

FIG. 6 is a characteristic peak pattern diagram of a single target measured by LC-MS/MS of three target mixed standard solutions;

FIG. 7 is a three-dimensional bar graph of the recovery of mixed standard for mixed gum under three-factor multi-level combination conditions;

FIG. 8 shows a regression equation and a fitting curve of the series of concentration regression equations of the detection signals of three target mixture solutions mixed with glue;

FIG. 9 is a graph showing the peak patterns detected by LC-MS/MS of a sample purified from natural lake water/shrimp muscle by mixed gel (Blank control); the two matrixes only have impurity peaks with extremely low peak amplitude after being purified by mixed glue, and the impurity peaks are presumed to be caused by residual inorganic salt in the purification process and do not interfere the presentation of the characteristic peak of the doped target substance;

FIG. 10 is a comparison chart showing the detection peak patterns of three target mixed sample samples of natural lake water after two kinds of purification treatment;

FIG. 11 is a graph showing a comparison of detection peak patterns of three target compound mixed samples of shrimp muscle after two kinds of purification treatment.

The specific implementation mode is as follows:

the invention discloses a florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, a monoclonal antibody secreted by the same and application thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the hybridoma cells and monoclonal antibodies and uses thereof of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced and used with modification, or with appropriate modification, and combination of the hybridoma cells and monoclonal antibodies and uses thereof, without departing from the spirit, scope, and spirit of the invention.

The florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain provided by the invention, the monoclonal antibody secreted by the same and application thereof are further described below.

Example 1: the preparation and evaluation of the monoclonal antibody hybridoma cell strain for florfenicol/thiamphenicol

1. Synthesis of artificial antigen-immunogen against florfenicol: weighing 35.8mg of FF (florfenicol) standard sample (mixed extinction type, 0.1 mmol), adding into 2mL of pyridine solution containing 11.4mg (0.1 mmol) of glu (glutaric anhydride), and stirring at room temperature for reaction overnight; after the completion, the pyridine is dried by nitrogen; dissolving FF-glu semialdehyde with 4mL of solvent (DMF mixed with 1, 4-dioxane at a volume ratio of 1); adding 26.2 μ L (about 0.1 mmol) of n-tributylamine, stirring in ice bath for 10min, adding 14.4 μ L (about 0.1 mmol) of isobutyl chloroformate, stirring at room temperature for 1h;

the activated FF solution was added dropwise to 5mL of a 10mg mL solution treated in an ice-water bath ^-1 Sodium borate solution (0.1 mol L) of BSA (bovine serum Albumin) ^-1 pH 8.5) over 1h, and the reaction was stirred at room temperature overnight. Dialyzing with 0.1mol sodium borate solution (pH 8.5) overnight, dialyzing with 0.01M PBS (pH 7.4) for 2d, centrifuging at 3000rpm for 20min, and collecting supernatant; taking a little for macromolecular mass spectrometric identification, preparing the rest part into lyophilized powder, and naming the lyophilized powder as BSA-glu-FF.

A synthetic circuit diagram:

2. synthesis of artificial antigen-coating antigen for florfenicol: weighing 6mgFFA (florfenicol amine) and dissolving in 100 muL DMF (dimethylformamide) to obtain solution A; weighing 10mg of OVA (ovalbumin) and dissolving in 1.5mL of PBS to obtain solution B; slowly dropping the solution A into the solution B, and then adding 4mg of EDC and 3mg of NHS; reacting at room temperature overnight; the reaction was packed into dialysis bags and dialyzed against PBS buffer at 4 ℃ for 5d, the dialysate was changed 3 times a day and named: FFA-OVA. Synthesizing a circuit diagram:

3. animal immunization and selection of qualified immunized mice: 6 female Balb/c mice, six weeks old, were purchased from Schleck and carefully housed according to strict SPF criteria, each mouse being immunized simultaneously according to the immunization schedule of Table 1 below.

TABLE 1 immunization plan

Description of the drawings: F-B represents FF-BSA conjugate, F represents FF, CFA represents Freund's complete adjuvant, and IFA represents Freund's incomplete adjuvant;

4. evaluation of serum samples from immunized mice

Qualitative calibration of titer of immune mouse antiserum by chessboard ELISA

(1) Coating: the coating antigen FFA-OVA is diluted to 9, 3, 1, 0.33, 0.11, 0.03 and 0.01 mu g mL by using the coating buffer solution respectively ^-1 And respectively adding the two to A-G lines of an enzyme label plate; h line as negative control: 3. Mu.g mL ^-1 OVA/CBS; add 100. Mu.L to each well. After coating, carrying out wet incubation at 37 ℃ for 1.5h, and then carrying out wet incubation at 4 ℃ overnight;

(2) Washing: taking out the enzyme label plate and spin-drying, washing with PBS 2+1 (1 means adding PBS for spin-drying for the first time and then not oscillating on a shaking table for 3min,2 means oscillating on the shaking table for 3min and then spin-drying after adding PBS for the last two times) and patting to dry (each time, each hole is 300 mu L);

(3) And (3) sealing: adding 350 mu L of sealing solution into each hole, and carrying out wet incubation for 2h at 37 ℃;

(4) Primary anti-dilution: each serum was diluted with antibody dilutions to: 1, 1000, 3000, 9000, 27000, 81000, while diluting the antibody-free mouse serum to 1, 3000 as a negative control, as a primary incubation antibody, pre-acting for one hour at room temperature;

(5) Washing: taking out the enzyme label plate, drying, washing with PBST for 2+1 times, and drying;

(6) Adding a primary antibody: adding 100 mu L of primary antibody incubation solution into each well, and carrying out wet incubation at 37 ℃ for 2h;

(7) And (3) secondary antibody dilution: horseradish peroxidase-labeled secondary antibodies were diluted with antibody diluent to 1:500;

(8) Washing: taking out the enzyme label plate, drying, washing with PBST for 4+1 times, and drying;

(9) Adding a secondary antibody: adding 100 mu L of secondary antibody diluent into each hole, and carrying out wet incubation at 37 ℃ for 80min;

(10) Washing: taking out the enzyme label plate and drying, washing with PBST for 3+1 times, then washing with PBS for 1+1 time and drying;

(11) Color development: adding 100 μ L of color development liquid into each well, and developing for 3min in dark;

(12) And (4) terminating: immediately adding 50 mu L of stop solution into each hole after the color development is finished;

(13) OD measurement with microplate reader ₄₉₀ The value is obtained.

Indirect competitive ELISA qualitative calibration of affinity of antiserum to FF standard

(1) Coating: using the coating concentration calibrated by the chessboard ELISA as a standard, respectively coating the FFA-OVA two coating antigens on the A-G lines of the two enzyme label plates; h line as negative control: the same concentration of OVA/CBS; each well was filled with 100. Mu.L. After coating, carrying out wet incubation at 37 ℃ for 1.5h, and then carrying out wet incubation at 4 ℃ overnight;

(2) Wash-block same checkerboard ELISA as above;

(3) Treatment of primary antibody and competitor:

a: diluting the antiserum to two times of the calibrated antiserum titer by using PBS buffer solution, and using the antiserum as a working primary antibody for standby;

b: FF standard sample is weighed and diluted to 1mg mL with methanol ^-1 The solution is used as a storage solution for standby;

c: stock solutions were diluted with methanol to concentrations of FF standards of 0.2, 1, 5, 25, 125, and 625ng mL, respectively ^-1 Six dilutions were made and equal volumes of working primary antibody were added to each dilution after treatment to give final concentrations of 0.1, 0.5, 2.5, 12.5, 62.5 and 312.5ng mL ^-1 。

D: the treated antibody is used as a primary incubation antibody, and shaking is carried out for 45min at room temperature.

(4) Adding a primary antibody: adding the treated incubation primary antibody to the A-F line of the ELISA plate; G. line plus PBS buffer diluted competitor-free antiserum (dilution is the nominal antiserum titer); adding 100 mu L of the solution into each hole, and carrying out wet incubation for 2h at 37 ℃;

(5) Wash-Add secondary antibody-Wash-color development-stop with the above chessboard ELISA.

5. Preparation and evaluation of florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain

(1) Culturing mouse myeloma cell strain-Sp 2/0 cells: the cell strain is given by the monoclonal antibody chamber of Jiangsu provincial blood institute, cultured and passaged in 10% FBS (Earl Bio products)/RPMI-1640 normal culture medium, and the cell strain is regarded as a qualified culture system after the cell strain is completely sensitive to HAT (Haemophilus influenzae) through a cell subcloning test, wherein the colony forming rate of single cells is more than 70 percent, and a 1XHAT selection culture test proves that the cell strain is completely sensitive to HAT; culturing the cell strain with a normal culture medium to logarithmic phase, wherein the cell shape and size are uniform, and the viable cell rate is more than 98%, lightly tapping the bottle wall of an Sp2/0 cell bottle to collect cells, centrifuging at 1000r/min for 5min to remove the culture medium, then adding serum-free RPMI-1640 culture medium to resuspend the cells, and counting for later use;

(2) Obtaining feeder cells: one day before fusion, taking male small Balb/c mice with the age of 10 weeks, and soaking the mice in 75% alcohol for 1-2min after killing; after the epidermis of the abdomen is cut open, alcohol cotton is pressed on the abdomen for disinfection; cutting off peritoneum, then washing abdominal cavity of mouse with RPMI-1640 culture medium containing 1XHAT by sucking with sterile pipette, removing macrophage in the culture medium, and blowing to suspend; planting in blocks of 96-well culture plates at a rate of 4 blocks/cell and 100 μ l/well, at 37 deg.C, 5% ₂ Culturing in an incubator;

(3) Obtaining spleen cells: performing immunization shock again 28 days after the seven-immunization, and taking blood from the eyeball 3d after shock; after the neck is cut off, soaking the rat in 75% alcohol for 1-2min; cutting skin and muscle of chest and abdomen, taking out spleen with forceps, and cutting off non-spleen connective tissue; washing in RPMI-1640 culture solution, transferring into a centrifuge tube, centrifuging at 1000rpm for 3min, discarding supernatant, adding RPMI-1640 culture solution to 30mL, and counting for use;

(4) Cell fusion: uniformly mixing splenocytes and myeloma cells according to a ratio of about 8, centrifuging at 1000r/min for 10min, removing the culture medium, and lightly tapping to relax the precipitate (facilitate the dissolution of the precipitate); the subsequent drops of reagents were kept at a constant temperature of 37 ℃, slowly added and continuously stirred, and were added in the order: respectively culturing in 1mL of PEG1500, twice in 1mL of RPMI-1640 culture medium, twice in 1.5mL of RPMI-1640 culture medium and 5mL of RPMI-1640; centrifuging at 1000r/min for 5min, removing culture medium, and gently tapping to relax precipitate; adding a small amount of RPMI-1640 medium containing 1XHAT, shaking gently, and adding to the RPMI-1640 medium containing 1XHAT at 37 ℃ after the precipitate is completely dissolved to prepare a fused cell suspension.

(5) And (3) cell culture: the suspension of the fused cells was dropped into the above-mentioned feeder cell-containing 96-well plate (100. Mu.L per well), and the content of CO was 5% at 37 ℃ ₂ Continuously culturing in an incubator; 4d and 7d at RPM of 1XHATRespectively carrying out half-liquid replacement on the I-1640 culture medium (namely, uniformly mixing by blowing, removing half volume by using a Pasteur dropper, and then complementing the volume by using RPMI-1640 culture medium containing 1 XHAT); 10d, carrying out one-time half-liquid change by using RPMI-1640 medium containing 1 × HT; when the fused cells grew to 1/10-1/4 of the area of the 96-well plate, one point was selected for preliminary screening, and the remaining part was still at 37 ℃ and 5% CO ₂ And continuing culturing in the incubator.

6. Screening and evaluation of hybridoma cells

Coating with the coating concentration calibrated by a chessboard ELISA experiment, and washing-sealing the same as the above;

transferring 100 mu L of culture supernatant of the fused cells to a new 96-well plate in a one-to-one correspondence manner; taking a 96-well plate, taking Sp2/0 cell culture supernatant as a negative control, diluting antiserum to 1 10000 as a positive control, and oscillating for one hour at room temperature to serve as a primary incubation antibody;

transferring 100 mu L of the primary antibodies into an ELISA plate in a one-to-one correspondence manner, and performing the other steps of the ELISA plate ELISA. After the color development is terminated, the wells with strong signals are selected as suspected wells, and one of the suspected wells is amplified into a 24-well plate correspondingly for further culture.

7. Confirmation of positive clones for FF-specific antibodies

Coating-washing-sealing-washing method is the same as above;

taking hybridoma cell culture supernatant in logarithmic phase in the wells of the 24-well culture plate, diluting the stock solution with PBS buffer solution to obtain stock solution and 1/10 diluent solution, dividing each diluent solution into two parts, adding PBS solution with equal volume into one part, and adding 2 μ g mL of PBS solution with equal volume into the other part ^-1 FF standard/PBS solution, i.e. one suspected positive clone sample, was treated 4 times, each with two replicate wells; placing the prepared centrifuge tubes on a shaking table, oscillating and incubating for one hour at room temperature, adding the centrifuge tubes into an ELISA plate for primary antibody incubation, and performing ELISA (enzyme-linked immuno sorbent assay) on the other steps as the chessboard; after color development was terminated, OD was measured between the FF standard treatment and the non-FF standard treatment of each sample at the same dilution ₄₉₀ Value comparison, and the candidate clone can maintain strong hybridization signal (i.e., 1/10 dilution OD) after expanded culture in 24-well culture plate ₄₉₀ Value is still strong), the strong signal can be represented by FF standardAnd (4) screening positive holes of FF-specific antibodies from the candidate clone sample by taking effective inhibition (obvious inhibition of FF treatment in the same group) as a standard.

8. Subcloning of FF-specific antibody positive clones: the selected positive wells were subcloned (limiting dilution method) and subjected to a 9-12d microscopic examination, and then supernatants from single colony wells with good cell status and a large number of cells (about 128 cells) were selected as test samples for the indirect ELISA. And then subcloning is carried out until the positive rate is 100%, and then the cells with the strongest signals and the best cell growth are selected for amplification, culture, frozen storage and seed preservation.

9. Large-scale preparation and evaluation of monoclonal antibodies against florfenicol: 10 male Balb/C-nuke mice of 10 weeks old purchased from Shanghai Schleger company, were sensitized by injecting pristine into the abdominal cavity at a rate of 0.5 ml/mouse for 7 days, and then sensitized at a rate of 1X10 ⁶ Inoculating hybridoma cells into abdominal cavity, feeding according to SPF animal feeding standard, collecting ascites after the abdomen is obviously enlarged, centrifuging at 4 deg.C for 10min at 1000r/min, and collecting supernatant; centrifuging at 12000r/min at 4 deg.C for 20min, collecting supernatant, collecting small sample for evaluation, and freezing at-20 deg.C for use. The ascites were diluted with 0.1% ova/PBS to 1: 5000. 10000, 20000, 40000 and 80000 are respectively used as incubation primary antibody, and the evaluation method is the same as the checkerboard and indirect competitive ELISA evaluation of antiserum, and the immunological cross test method of thiamphenicol and chloramphenicol is the same as the indirect competitive ELISA method using fluorobenzmycin as an inhibitor.

10. Results

(1) Artificial immunogens against florfenicol: through the chemical synthesis, about 41mg (calculated by protein) of artificial antigen is recovered together, and the recovery rate is about 82%; the results of mass spectrometry of macromolecules are shown in FIG. 1, which shows: the molecular weight of the BSA standard sample is 66592, and the molecular weight of the FF-glu-BSA conjugate is 72991. This indicates that FF has been successfully coupled to BSA and that the molar ratio FF to BSA can be calculated approximately as 18 according to the formula [ FF-glu-BSA molecular weight-BSA molecular weight ]/FF molecular weight ]. Meets the requirement of mouse immunity.

(2) Artificial coating antigen against florfenicol: through the chemical synthesis, about 8.5mg (calculated by protein) of artificial antigen is recovered, and the recovery rate is about 85%; the following test requirements were met.

(3) Selection of the florfenicol immunogen qualified immunized mice: after 7 days after six immunizations of six immunized mice, 6 parts of antiserum obtained after tail cutting are respectively used as primary antibodies to be tested, and the test is carried out by the chessboard and indirect competitive ELISA with the OVA-FFA coating of 3 mu g/ml as a standard: two of the antisera working dilutions were > 1 10000 (OD) ₄₉₀ > = 1.5) with semi-inhibitory concentration IC against florfenicol standard solution ₅₀ (50% >, inhibition Concentration) < =5ng/ml, and the mice were judged as qualified antisera, and the mice were used as candidate mice, and the spleens thereof were removed at intervals of 4 weeks for immunization shock and used for fusion.

(4) Screening, cloning and evaluating the florfenicol monoclonal antibody hybridoma cell strain: and (3) fusing the candidate mouse splenocytes with Sp2/0 cells, culturing in 10 culture plates with 96 wells, wherein supernatants of each well after 10 days of culture correspond to 10 ELISA wells one by one, and performing indirect ELISA detection by using OVA-FFA coating of 3 ug/ml: in total, 10-well suspected positive wells (OD) were obtained ₄₉₀ > = 1.5); then, corresponding to the amplification in the wells of the 24-well culture plate until the logarithmic phase, taking the supernatant as a primary antibody to be detected, and dividing the 96-well ELISA plate into 4 rows and 2 columns (8 wells in total) per detection unit: (1-2) x (A-D) \ (3-4) x (A-D) \8230;, (11-12) x (E-H) 12 detection units: each supernatant was subjected to four treatments: adding PBS with equal volume to the supernatant; adding equal volume of 2 mu g/ml florfenicol into the supernatant; diluting the supernatant by 10x, and adding PBS with the same volume; diluting the supernatant by 10x, adding isovolumetric 2 mug/ml florfenicol, adding each sample to be tested into 10 detection units (each processing 2 multiple wells) one by one, and using 1000x diluted antihemorrhagic request as a synchronous positive control by the last two detection units, detecting: the control of the two dilutions has a certain inhibition value to florfenicol, which indicates that the detection system works normally; of 10 clones detected, 6 clone samples lose immune hybridization signals, the other 4 clones retain immune hybridization signals, only one of the clones has obvious competitive inhibition price for florfenicol, the clone is confirmed for florfenicol monoclonal antibody, and the clone is named as-9D according to the original coordinate ₄ After two rounds of single cell subcloning, the positive rate is 100% by detection; a total of 28 single-cell clones obtained from the second round of subcloning were positive: A1-C4 have stronger immune hybridization signals; c5-8 is Sp2/0 onA clear negative control; c9-12 is 1. The result shows that the positive cloning rate reaches 100% (28/28) after two rounds of single cell subcloning, which indicates that the cell strain is fully purified and secretes the monoclonal antibody stably, one clone is taken for expanding culture and frozen storage, and is simultaneously sent to the China center for type culture collection for seed preservation and is subjected to the preservation number: CCTCC No. C201945.

(5) Obtaining and evaluating ascites samples for florfenicol monoclonal antibody: 10 Balb/C-nuke mice are injected into the hybridoma cells, and about 65ml induced ascites is collected at 10d and 11 d; ascites is measured by a chessboard ELISA method to obtain the optimal calibration: ascites working dilution: 1, diluting with 40000; FFA-OVA coating concentration: the results of indirect competitive ELISA assay for florfenicol at 3. Mu.g/ml, using these two dilutions as the main technical parameters, are shown in Table 2 and FIG. 2.

TABLE 2 affinity of induced ascites by cells for FF

From table 2 and fig. 2, it can be derived: the system aims at the effective detection domain of florfenicol: 0.556-3.153ng mL ^-1 (ppb，IC ₂₀ -IC ₈₀ ) (ii) a Detection sensitivity: 1.354ng mL ^-1 (ppb，IC ₅₀ ) The immunological cross-over rate detection results of chloramphenicol and thiamphenicol with the same parameters are shown in the following table 3;

TABLE 3 9D ₄ -FF induced ascites cross-over rate to chloramphenicol drugs

The result shows that the monoclonal antibody aiming at the florfenicol has high titer and high affinity, in addition, the antibody has excellent immunological cross-over property (the immunological cross-over rate reaches 69.69%) on thiamphenicol which is another important member in chloramphenicol, and the result provides a controllable test raw material with easily obtained source, low cost and stable quality for preparing the immunoadsorbent aiming at the florfenicol/thiamphenicol high affinity.

Example 2: preparation and evaluation of ascites samples of monoclonal antibodies against chloramphenicol

1. Screening of hybridoma cell strains aiming at chloramphenicol monoclonal antibodies and evaluation of supernatants of corresponding cells: preparing a chloramphenicol artificial antigen: comprises chemical crosslinking of chloramphenicol succinic acid and KLH (keyhole limpet hemocyanin) (used as immunogen), chemical crosslinking of chloramphenicol and BSA (bovine serum albumin) (used as coating antigen) through diazo reaction after reduction; balb/C mice are immunized; establishing and optimizing a checkerboard ELISA and indirect competitive ELISA detection method aiming at chloramphenicol antiserum evaluation and chloramphenicol monoclonal antibody secretion fusion cell clone screening and evaluation; for the quantitative or semi-quantitative determination of chloramphenicol standards in the supernatant of cultured cells of monoclonal antibody target clones, see the methods in the literature "Development of a monoclonal antibody based-ELISA for the detection of chlorinated in-vitro culture, feed and milk samples and validation by LC-MS/MS coupled with immunological definition in the" up-p "(animal. Methods.2019.11.507-516). A cell-producing strain against a hybridoma cell strain 2D2-C1 having a high chloramphenicol titer and a high immunoaffinity was selected by evaluation and used in the following experiment.

2. Large scale preparation and evaluation of monoclonal antibodies against chloramphenicol: a large number of log phase growing cells of the hybridoma cell line 2D2-C1 were collected and 10 male Balb/C-nuke mice aged 10 weeks were injected in vivo as mentioned in example 1. After obtaining the corresponding induced ascites sample, the small sample is diluted to 1: 5000. 10000, 20000, 40000 and 80000, the checkerboard and indirect competitive ELISA evaluations of the antiserum are the same as the indirect competitive ELISA evaluation method using chloramphenicol as an inhibitor and fluorobenzothromycin and thiamphenicol.

3. Results

(1) Quantitatively detecting a chloramphenicol standard sample by using 2D2-C1 cell culture supernatant and drawing a standard inhibition curve: the fused cells are screened and twiceThe hybridoma cell strain for the chloramphenicol monoclonal antibody is obtained by subcloning: 1B1 and 2D2; selecting a cell strain 2D2-C1 with higher affinity to chloramphenicol, taking a culture supernatant of the cell strain in the logarithmic growth phase as a primary antibody to be detected, and calibrating by chessboard ELISA: the best coating is: 5 μ g/ml BSA-CAP, optimal primary antibody dilution: 1:400, respectively; then the technical parameters and chloramphenicol with concentration points of 0.01,0.03,0.09,0.27,0.81,2.43 and 7.29ng/ml are respectively used as competitors, and a competitor-free control group is used for indirect competition ELISA determination, and the determination is carried out as follows: OD of each concentration point (12 wells) ₄₉₀ The mean value is B, OD of control (12 duplicate wells) ₄₉₀ The average value is B ₀ Inhibition per concentration point was expressed as (B) ₀ -B)/B ₀ * Calculating by 100%; the inhibition curves of the samples against chloramphenicol standard solutions were plotted on the abscissa and on the ordinate as the concentration of the inhibitor, and the results are described in the "Development of a monoclonal antibody based-ELISA for the detection of chromatographic in-shock, fed and mil samples and evaluation by LC-MS/MS coupled with immunological definition-up" (anal. Methods.2019.11.507-516).

(2) 2D2-C1 induced ascites is drawn on a chloramphenicol standard inhibition curve and the immunological cross assay of homologues thereof is carried out: 10 Balb/C-nuke mice are injected into the 2D2-C1 cells, and about 67ml induced ascites is collected at 11D and 12D; chessboard ELISA calibration, ascites working dilution: 1, 40000 dilution; CAP-OVA coating concentration: standard inhibition curves for chloramphenicol standards using this parameter are shown in fig. 3, which shows: effective detection threshold for chloramphenicol: 0.033-2.53ng/ml (IC) ₂₀ -IC ₈₀ ) And detection sensitivity: 0.51ng/ml (IC) ₅₀ ) And the immunological cross-over rate with florfenicol and thiamphenicol is shown in a table 4:

TABLE 4 crossing rate of 2D2-C1 induced ascites to chloramphenicol drugs

As can be seen from fig. 3 and table 4:

the monoclonal antibody secreted by the first and the 2D2-C1 cell strains has extremely high immunoaffinity to chloramphenicol, and lays a foundation for the next step of research of immunoadsorption products with excellent enrichment performance on samples with extremely low content of chloramphenicol targets.

Secondly, monoclonal antibodies secreted by the 2D2-C1 cell strain have extremely strong specificity (one-to-one) on chloramphenicol, and the method provides possibility for further research and development of immunoadsorption products with excellent selective performance on complex samples to be detected of chloramphenicol-containing target substances.

Thirdly, compared with the culture supernatant, the immunoaffinity of the cell induced ascites sample to chloramphenicol is not reduced, but the antibody titer is increased by nearly one hundred times. The result provides a test raw material with rich target antibody content and low cost for the next step of research and development of a chloramphenicol solid phase immunoadsorbent product with certain price competitive advantages.

Example 3: preparation of two monoclonal antibody corresponding solid-phase immunoadsorption glue

1. And (3) large-scale purification of two monoclonal antibody proteins: the two monoclonal antibodies, 8ml of ascites samples, were diluted 3X with 0.01M PBS (pH 8.0) chilled in an ice water bath for use. Then, the Protein G Resin (product of Kinserin) with the same volume as the ascites stock solution was loaded into 2 chromatography columns (30 ml), the two monoclonal antibody proteins were purified according to the standard method provided by Kinserin, and the purified Protein eluents of the two monoclonal antibodies were collected in portions, neutralized with 1M Tris-Cl (pH 8.5) in 1/10 volume, placed in Protein dialysis bags (product of 7500-14000MW solarBio), and dialyzed in 0.01M PBS (pH 7.4) pre-cooled in ice-water bath for 24hr (changing every 4-6 hr). Respectively taking a small sample: protein content was measured using BCA kit (available from Biyuntian corporation) according to the instructions; the purity of the protein is identified by SDS-PAGE electrophoresis, and the rest part is subpackaged at-80 ℃ for freezing storage for later use.

2. Two monoclonal antibody purified protein and CNBr-activated Sepharose ^TM 4B crosslinking:

(1) Pretreatment of monoclonal antibody protein before crosslinking: respectively placing the above purified monoclonal antibody protein solutions with calibrated protein content in two dialysis bags (7500-14000 MW) respectively, and adding 100-200 × 0.1M NaHCO ₃ 0.5M NaCl solution (pH 8.3) dialyzed at 4 deg.C for 24hr (changing solution every 4-6 hr)) After dialysis, the protein was filtered through a 0.22 μ M microfiltration membrane and stored at 4 ℃ for further use.

(2) Crosslinking the protein liquid and the solid gel: the two monoclonal antibody samples are respectively weighed according to the milligrams of the monoclonal antibody protein to be crosslinked and purified/5/3.5 (note: firstly, the swelling coefficient of the small gelatin bead powder is 1 ^TM 4B (a product of Healthcare company) dry powder is placed in 2 50ml centrifuge tubes (a product of burning company), steps of peptizing and swelling, washing, protein purification crosslinking, washing, activated group blocking, two buffer solutions with different pH values and the like are carried out according to the method provided by Healthcare company, small samples of the protein solution before crosslinking and after crosslinking are respectively taken for content measurement, and the crosslinking rate is calculated.

3. Results

(1) Obtaining and identifying two monoclonal antibody purified proteins: 8ml of each of the two monoclonal antibody abdominal water samples are purified by the protein G resin one-step method 9D ₄ Strain FF obtained 35ml 1.35mg/ml total 47.25mg of purified protein; the 2D2-C1 strain obtains 28ml 1.65mg/ml purified protein accounting for 46.20mg, and the purified protein is identified by SDS-PAGE electrophoresis (see figure 4), and the result shows that the two antibody purifications both contain two obvious protein bands of 50Kd (antibody heavy chain) and 25Kd (antibody light chain), have no obvious other impurity protein bands, and are consistent with the calibration molecular weights of the heavy chain and the light chain of a mouse IgG molecule, which shows that the invention has obtained two high-purity monoclonal antibody proteins and can completely meet the requirements of preparing the single antibody protein in practical immunoaffinity chromatography gel and on purity.

(2) Evaluation of the crosslinking rate of the two monoclonal antibody purified proteins with the immobilized carrier: according to the Healthcare company, reasonable crosslinking degree ranges are recommended: 2-10mg antibody protein: 1ml swelling glue, aiming at the two monoclonal antibodies, the invention adopts the intermediate value of the crosslinking degree: 5mg of antibody protein: 1ml of swelling glue, and the concentration is determined before and after the cross-linking of two monoclonal antibody purified proteins by combining the formula: (protein concentration before crosslinking-protein concentration after crosslinking)/protein concentration before crosslinking 100% calculate the respective crosslinking rate, two monoclonal antibody purification protein treatment before and after concentration determination results are shown in figure 5: the protein concentration of the two monoclonal antibody purified products is respectively and steeply reduced from 1.25mg/ml to 0.04mg/ml by solid phase gel crosslinking; 1.57mg/ml is steeply reduced to 0.05mg/ml and filledThe results show that the two monoclonal antibody proteins can be excellently crosslinked with the solid phase glue, and the results show that: 9D ₄ -crosslinking ratio of FF strain: (1.25-0.04)/1.25 x 100% =96.8%; crosslinking ratio of 2D2-C1 Strain: (1.57-0.05)/1.57 × 100% =96.81%; the results show that: the invention obtains two solid-phase immunoadsorbents with excellent monoclonal antibody crosslinking rate, and lays a solid foundation for the next test.

Example 4: preparation and binding performance determination of chloramphenical mixed type immunoaffinity chromatography adhesive

In order to confirm and accurately quantify the targets treated by the mixed immunoaffinity chromatography gel and by the conventional Solid Phase Extraction Cartridge (Solid Phase Extraction Cartridge), this example was carried out by LC-MS/MS method.

1. Establishment and optimization of chloramphenicol multi-component LC-MS (liquid chromatography-mass spectrometry) determination method

(1) Reagent:

chloramphenicol, florfenicol, was purchased from dr. Thiamphenicol, purchased from alatin (shanghai, china, all chromatographically pure). Ethyl acetate, methanol, n-hexane, formic acid (Fisher HPLC grade), and other reagents were analytically pure.

(2) Instruments and accessories:

chromatograph: agilent model 1290 high performance liquid chromatograph

Mass spectrometry: agilent 6545 (QTOF-MS) type mass spectrometer

A chromatographic column: in a ZORBAX Stable Bond C18 (1.8 μm, 3.0X 50 mm) column

(3) The measurement conditions were as follows:

column temperature: 30 ℃; sample introduction: 20 mu L of the solution; mobile phase: methanol +0.1% formic acid water; flow rate: 0.3mL min ^-1 (ii) a Gradient elution procedure: table 5 below; an ion source: negative ion ESI; the drying temperature is 200 ℃; gas flow rate of 12L min ^-1 (ii) a Air pressure: 35psi; temperature of sheath gas: 375 ℃; sheath gas flow rate of 12L min ^-1 (ii) a The capillary voltage is 4000V; nozzle voltage 2000V; the segment voltage is 110V.

TABLE 5 gradient elution procedure

A is methanol; b-0.1% formic acid water.

(4) And (3) LC-MS (liquid chromatography-mass spectrometry) for standard mixed standard substance detection and standard detection curve drawing of three target substances of chloramphenicol: 1mg of chloramphenicol, thiamphenicol and florfenicol are respectively and accurately weighed and prepared into 1mg/ml by methanol and are stored as mother liquor, a little of the three solutions are respectively taken, mixed in equal volumes, and diluted by the mobile phase to each target substance containing: and (3) respectively preparing a test solution from three target substance mixed solutions with seven concentration points of 0.1, 0.5, 2.5, 12.5, 62.5, 312.5 and 1562.5ng/ml, and detecting each concentration point three times to verify the reliability of the LC-MS detection system.

2. Aiming at the preparation, optimization and absorption performance determination of the chloramphenicol multi-component mixed glue:

(1) Preparing a chloramphenicol multi-component mixed glue: after mixing the two immunoaffinity chromatography gels mentioned in example 3 in equal volumes, 100. Mu.l (total gel volume)/tube were dispensed into 1.5ml EP tubes, and the PBS was washed twice by centrifugation with 0.01M PBS (pH 7.2) and aspirated as dry as possible.

(2) The mixed gel (IAC) of the invention optimizes the immunochromatography conditions of multi-component target substances: in order to allow the mixed IAC to adsorb three targets simultaneously and achieve high recovery rate, the concentration of each of the three targets in this embodiment is 20ng mL ^-1 (the middle value of the maximum column capacity) mixed standard solution is used as a mode sample, four-index multi-level combination tests are carried out by using different sample loading solutions (binding solution), leacheate (washing solution), eluent and different immunological binding time, and the standards of higher recovery rates of three target substances are used to obtain the optimal immunoaffinity condition.

And (3) selecting a sample loading solution: 0.01mol mL of pure water ^-1 PBS，0.01mol mL ^-1 PBS +10% aqueous methanol (1) three buffers;

leaching solution separationThe following steps are used: 0.01mol mL of pure water ^-1 PBS + Water, 0.01mol mL ^-1 Three buffers of PBS +10% methanol water;

eluent is selected and used as follows: 0.1% formic acid water (pH = 2.5), 100% methanol solution, 80% methanol aqueous solution, meOH: HCOOH (9), meOH: NH ₃ ·H ₂ 0 (9;

the immunoaffinity time is selected from: 10 15, 30 and 45min. Each group was subjected to immunoaffinity chromatography, eluted with liquid nitrogen (60 ℃ C.), redissolved with 1mL of pure water, filtered, and tested on a prototype.

(3) Analysis of the adsorption properties of the mixed glue on three target single components: after the optimal condition of the gel for the immunochromatography of a multi-component target object is selected, six groups of mixed liquor of 1,2, 4, 8, 16 and 32ng/ml of single target object are prepared by using 0.01M PBS (pH 7.4) for three target object mother liquor and are respectively used as solutions to be detected, the immunochromatography test is carried out on 1ml of solution to be detected and 100 mu l of mixed gel (three independent repeated tests are set for each group, n = 3), each group is subjected to nitrogen blowing, redissolution and filtration-sample loading machine test according to the method after the immunochromatography, and the recovery rate of each component passing through each concentration point is used as a regression curve; then, the three target mother solutions are respectively prepared into 500ng/ml single target saturated solution (the maximum theoretical adsorption capacity of the 100 mul mixed gel is calculated according to the mole ratio of 1 of mouse IgG to the target (MW 300D) to 1.

3. Results

(1) And (3) verifying the LC-MS detection system by three target substance standards: the detection results of three target mixed liquid with seven concentration points of 0.1, 0.5, 2.5, 12.5, 62.5, 312.5 and 1562.5ng/ml for three targets by using an Agilent one-weight two-level rod liquid-mass cascade instrument LC-MS matched with a ZORBAX Stable Bond C18 (1.8 mu m,3.0 multiplied by 50 mm) chromatographic column show that:

of each objectThe fitting rate R of each concentration and the peak area of the corresponding received signal are typically concentration-dependent, and a recovery curve is drawn ² Are all larger than 0.99;

each target in the mixture sample had an exact time to peak, chloramphenicol: 4.618min; thiamphenicol: 3.548min; florfenicol: 4.059min (see table 6 below), the retention time errors of different concentrations of the same target are less than 5%;

the molecular weights of parent ions of three targets are respectively analyzed by mass spectrometry: chloramphenicol > 322.0123; thiamphenicol > 355.0048; florfenicol > 357.0005 (see Table 6 below); matching with the molecular weights marked by the three target object standards;

spectral patterns of three target mass spectral signals: all are sharp peak-type single peaks and have no any impurity peak. See FIG. 6 for mass peak patterns (mass spectrum of mixed solution of 20ng/ml for each target).

TABLE 6 molecular weight and retention time of single target parent ion calibrated by three target mixed standard solutions LC-MS

Note: CAP is chloramphenicol; TAP is thiamphenicol; FF florfenicol

The above results show that: the detection system can confirm and accurately quantify single target in three target mixed standard samples with single component concentration ranges of 0.1-1562.5ng/ml, and can meet the requirement of subsequent sample determination. The selection of the chromatographic column and the setting of various technical parameters of LC-MS are easy, and the simultaneous determination of the three target mixtures does not generate mutual interference. The molecular weight of the parent ion for each target is clear and can be used as direct evidence for confirmatory determination.

(2) Selecting mixed immunoaffinity chromatography gel affinity chromatography conditions: three target substance concentrations are mixed with a standard substance of 20ng/ml as a sample, the single target substance recovery rate is more than 60 percent as a gold standard, and the three factors are comprehensively optimized under the following conditions: binding liquid: 0.01M PBS (pH7.4); washing solution 0.01M PBS (pH7.4) + H ₂ O; eluent methanol + ammonia (9. Thus, the following are selected: 0.01M PBS (pH7.4) as binding solution; 0.01MPBS(pH7.4)+H ₂ O is used as a washing liquid; methanol + ammonia (9; the immunological combination time of the mixed gel and the sample to be detected is 40min, and the mixed gel and the sample to be detected are used as the optimal immunochromatography test condition combination to be applied to the subsequent tests. The recovery of each target obtained for each set of experimental conditions is shown in FIG. 7.

(3) The mixed immunoaffinity chromatography gel has the following adsorption performance on three target mixed standard liquids and the maximum adsorption capacity of a single target:

the mixed immunoaffinity chromatography gel is used for calibrating the adsorption performance of three target substance mixed standard solutions: the optimal reaction conditions are selected, and the results of LC-MS measurement of elution samples of three target objects, which contain six groups of mixed liquid of 1,2, 4, 8, 16 and 32ng/ml, are shown in the specification: the signal response intensity obtained from 6 concentration points of each component from 1 to 32ng/ml, i.e., the characteristic peak area of each target, is typically concentration-dependent, and the fitting rate R of the concentration plotted by the 6 concentration points to the corresponding signal intensity correlation curve ² All greater than 0.99, three targets: the respective detection curves of chloramphenicol, thiamphenicol and florfenicol are respectively shown in figure 8;

the results in FIG. 8 show that:

the combined detection system of the mixed immunochromatographic gel and the LC-MS, which is established by the invention, can accurately confirm and accurately quantify each target object for the mixed standard sample of three target objects within the concentration range of 1-32ng/ml, and provides possibility for the detection requirement of the actual sample to be detected of the mixed residue of three target objects with low or even extremely low content.

Aiming at three target substance mixed standard samples in the concentration domain, the mixed gel immunoadsorption effect has no mutual interference: the method is characterized in that steric hindrance interference is not generated for respective immunoadsorption targets of a chloramphenicol solid phase immunoadsorbent and a florfenicol/thiamphenicol solid phase immunoadsorbent; aiming at the florfenicol/thiamphenicol solid phase immunoadsorbent, under the condition that the antibody adsorption epitope of the solid phase immunoadsorbent is supersaturated, the florfenicol and the thiamphenicol do not generate an energy-efficiency competitive effect.

The mixed immunoaffinity chromatography gel is used for calibrating the maximum adsorption capacity of a single component: after the fact that the mixed gel has excellent enrichment property on a mixed sample of three target substances with low or even extremely low content is verified, another important technical parameter of the gel, aiming at the maximum adsorption capacity of a single target substance, is considered, namely the maximum adsorption capacity of the gel for the single target substance is determined by accurately and quantitatively measuring the residual content of three saturated standard solutions (500 ng/ml) of the single target substance and the gel after adsorption respectively according to the difference value between the content of the single target substance before adsorption and the content of the single target substance after adsorption. The three single components of the target object are respectively averaged by 6 times of repeated tests, and are determined as follows: the average adsorption capacities of chloramphenicol, thiamphenicol and florfenicol are respectively as follows: 81 140, 109ng; the average adsorption capacities and standard deviations among groups for the three targets are shown in table 7 below;

TABLE 7 maximum adsorption Capacity and Standard test error of the Mixed glue for three targets

	CAP	TAP	FF
				Maximum column capacity (ng)	81	140	109
RSD(％)	1.6	4.1	4.7

Example 5: comparison test for purifying chloramphenicol multi-component doped standard sample by mixed gel and conventional solid phase extraction column

After the mixed glue has excellent immunoadsorption performance on the standard mixed liquid of three target substances, the matrix interference resistance of the glue is a key index for determining whether the glue can be introduced into practical application. Furthermore, a parallel comparison test is carried out with the national standard method, and the obtained actual data of the purification effect of the gel, which is comprehensively superior to the purification effect of the national standard method, is an important basis for replacing a conventional solid phase extraction column with the gel to be applied to an actual sample to be detected, so that the embodiment selects freshwater shrimps (purchased from a local farmer market) with the body length of about 12cm and natural lake water (water samples which are acquired from 50cm below the surface of a villa lake near a Suzhou villa lake school zone) to serve as a mode matrix for subsequent tests after the quality of agricultural products and safety monitoring center of Suzhou proves that no chloramphenicol, thiamphenicol and florfenicol are remained.

1. And (3) purifying and detecting the mixed target substance mixed with the shrimp meat sample by the mixed glue: removing shell and viscera of live shrimp, cutting, homogenizing at 4 deg.C 12000r/min for 5min, and collecting shrimp meat homogenate or directly preparing doped sample or freezing at-20 deg.C for use. Respectively weighing four parts of 4g shrimp meat homogenate into four 50mL polypropylene centrifuge tubes with covers, and sequentially adding 0, 20, 40 and 80 μ L of three target substances with the concentration of 1 μ g mL ^-1 After mixing the standard solutions, the samples were vortexed for 5min and mixed thoroughly overnight in a refrigerator at 4 ℃. The next day, each centrifuge tube was vortexed for 5min, then 20mL of ethyl acetate and 0.5mL of ammonia were added, vortexed and mixed for 5min, extracted by shaking for 10min, centrifuged at 5000r/min for 10min at room temperature, 15mL of supernatant was transferred to another centrifuge tube, and dried at 60 ℃ under nitrogen. Adding 3mL of pure water, swirling for 30min for redissolution, adding 2mL of normal hexane, swirling and mixing uniformly for 3min, centrifuging at 5000r/min for 10min, and taking 1mL of lower-layer water phase for immune purification by using the mixed gel. The sample purification-elution sample detection and other methods are the same as the steps. The experiment was independently repeated three times.

2. And (3) purifying and detecting the mixed target substance of the three target substances mixed with the natural lake water by using the mixed glue: centrifuging natural lake water at 4 deg.C 12000r/min for 10min, respectively weighing four parts of 4mL supernatant in four 50mL polypropylene centrifuge tubes with coversSequentially adding three target substances with the concentrations of 1 mu g mL of 0, 20, 40 and 80 mu L ^-1 The standard solution is mixed and then the subsequent treatment is the same as the above method. Three experiments were also independently repeated.

3. And (3) purifying and detecting the shrimp meat/natural Taihu lake water sample doped with the three target mixed substances by using an SPE (solid phase extraction) column:

pretreatment of a sample to be detected: the preparation method comprises the steps of doping three target substances into a shrimp meat/natural lake water sample, extracting the target substances with ethyl acetate, drying extraction liquid with nitrogen, redissolving dried substances, degreasing with n-hexane and the like, and obtaining a water phase sample to be purified for later use.

4. Purifying and sampling detection of the SPE column on a sample to be detected: counting Oasis ^@ HLB (300mg 6mL of a product of Waters company) small columns, 5mL of methanol is added into each small column for elution activation and 5mL of water for elution balance, a 5mL of water phase sample to be purified is slowly added into one SPE small column, 5mL of pure water and 5mL of 5% methanol aqueous solution are added for elution after the sample solution is drained, 5mL of methanol solution is added for elution, and the eluent is dried by nitrogen in a water bath at 60 ℃. And (3) redissolving 1mL of pure water, filtering the solution by using a filter membrane, detecting by using LC-MS (liquid chromatography-mass spectrometry), and calculating the recovery rate.

5. As a result, the

(1) And (3) calibrating the detection standard curve of the three target mixed external standard samples of the four treatment matrixes: in order to correct the systematic deviation generated by the detection of three target objects by the matrix pair LC-MS system. This example uses four matrices: the re-dissolved water phase (two kinds) of the purified mixed glue to chloramphenicol antibiotic blank shrimp meat/natural lake water; oasis ^@ The HLB column takes six-level mixed standard liquid of 1,2, 4, 8, 16 and 32ng/ml as an upper computer sample for three target objects of re-soluble aqueous phases (two phases) after the purification of the chloramphenical antibiotic blank shrimp meat/natural lake water. After detection, respectively making standard detection curves of different standard adding levels of single target object processed in the same way, making regression equation and calculating fitting rate R ² The results of external standard detection of the four matrices are shown in tables 8 and 9 below. From tables 8-9, three target external standard mixed solutions with four treated water phases as diluents can be obtained, and the detection signals obtained from the targets have good correlation with the concentrations thereof, and the correlation coefficient R of the detection signals ² Are all bigAt 0.99, the accurate quantification of the three targets in the concentration domain by the LC-MS system is fully shown to be not interfered by the four matrixes, and the result lays a firm foundation for the reliability of the accurate quantification of the purified sample of the three target matrix doped with the targets by the LC-MS system at the equivalent matrix level in the next step.

TABLE 8 quantitative regression equation and correlation coefficient for target in water-phase external standard sample after natural lake water is purified by two modes

TABLE 9 quantitative regression equation and correlation coefficient of target in water phase external standard sample after shrimp meat is purified by two modes

(2) Comparison of purification effects of two treatments of three target mixed targets of natural lake water:

and (3) qualitative comparison: in order to visually show the purification comparison effect of the mixed gel and the SPE column on the three target mixed targets, after a detection peak pattern (see a natural lake water sample in figure 9) obtained by detecting a mixed gel treated natural lake water purification sample (blank control) through LC-MS shows only a hybrid peak with extremely low peak amplitude and does not interfere the display of a target characteristic peak, three target peak pattern patterns (see figure 10) obtained by detecting two kinds of purification samples (LC-MS) of the three target mixed targets with lake water at the concentration of 20ng/ml are shown:

the two processed samples can respectively generate characteristic peaks at specific retention times of the three targets, and the respective retention times are adjacent to the substantially non-impurity peak;

comparing the peak amplitudes of three target characteristic peaks of two treatment samples: samples treated by the mixed gel are obviously higher than those treated by SPE, and are respectively CAP:0.7 >; TAP 0.3 >; FF 0.3> -0.12;

and (3) continuously measuring peaks throughout the whole process of three target objects (the flow phase is continuously eluted and detected for about 12 min), and comparing the baseline (background) outside the characteristic peaks of the three target objects: the mixed gel treated samples outperformed the SPE treated samples.

The above results show that:

the two purification media have certain retention capacity for three targets in the mixed standard sample, the characteristic peak amplitudes of the three targets are used as qualitative indexes, and the retention capacity of the mixed gel for the three targets is far higher than that of the SPE small column for the three targets;

although the matrix effect generated by the natural lake water mode matrix does not form substantial interference on the display of three target object characteristic peaks in a sample to be detected, the noise level of a mixed gel processing sample is obviously lower than that of an SPE processing sample by taking a hybrid peak-matrix noise signal peak corresponding to the three characteristic peaks as a qualitative index, and the filtering capability of the mixed gel to the matrix is fully shown to be superior to that of the SPE column;

the two indexes are integrated: the IAC purification effect is obviously superior to that of the SPE small column

Quantitative comparison: the detection and statistical results of the three-level mixed standard two treatment groups with the concentrations of the three targets respectively being 5, 10 and 20ng/ml are shown in the following table 10, the table 10 shows that the target recovery rate of the mixed gel purified sample is generally higher than that of the SPE purified sample target, and the results show that the intermediate value obtained by the difference value of the same target added with the same standard level is taken as a representative: CAP:78% >38%, difference 40%; TAP:84% is larger than 67.2%, and the difference is 16.8%; FF:79.2% >45.6%, difference 33.6%; therefore, the mixed gel is far better than the purification treatment of SPE small columns in the purification effect of target substance doped samples.

TABLE 10 recovery rate of single target from each group of three target concentration mixed standard samples of natural lake water after two purification treatments

(3) Comparison of purification effects of two treatments of three target compound mixed with shrimp muscle:

and (3) qualitative comparison: after the peak profile of the test obtained from the gel-mixed purified shrimp muscle sample (blank control) (see fig. 9) also showed only very low peak amplitude of the peaks without interfering with the characteristic peaks of the target, the peak profile of the three targets obtained from the LC-MS test of two purified samples of the shrimp muscle mixed with the three targets at the same concentrations (see fig. 11) showed that:

compared with the treatment effect of the natural lake water mixed standard mode sample, although the shrimp meat mixed standard mode sample can also generate respective characteristic peaks in the specific retention time of three targets after being treated by two kinds of samples, the mixed glue treated sample can keep no interference of the mixed peaks in the vicinity of the respective retention time of the three targets; the three targets of the SPE processed sample have different degrees of interference of the hybrid peaks near the retention time, and the hybrid peaks of the FF \ TAP two targets are more obvious.

Comparing the peak amplitudes of three target characteristic peaks of two treatment samples: the mixed gel treated samples were also significantly higher than the SPE treated samples, CAP:1.2>, 0.25, respectively; TAP 0.4 >; FF:0.7 >.

Baseline (background) comparisons beyond the three target characteristic peaks: the mixed gel treated samples were substantially free of contaminating peaks, while the SPE treated samples all had a large number of contaminating peaks.

The above results show that:

in the face of a sample to be purified with a more complex matrix, the two purifying media can still keep certain retention performance on three targets in a mixed standard sample, the characteristic peak amplitude is taken as a qualitative index, and the retention capacity of the mixed gel on the three targets is far higher than that of the SPE column on the three targets.

The interference of matrix effect on the characteristic peak of the target is eliminated as a qualitative index, and compared with SPE (solid phase extraction) treatment, the mixed gel treatment comprises the following steps: the mixed gel purification treatment not only filters out the matrix directly interfering with the characteristic peak, but also largely removes other matrixes. The detection cost is greatly eliminated.

The two indexes are integrated: the IAC purification effect is obviously superior to that of the SPE small column

Quantitative comparison: the detection and statistical results of the three levels of mixed standard substance with the concentrations of the three target substances of 5, 10 and 20ng/g are shown in the following table 11, the table 11 shows that the recovery rate of the target substance of the mixed gel purified sample is generally higher than that of the target substance of the SPE purified sample, the same standard-added level sample of the single target substance with relatively small variation coefficient (CV (%)) in the two treatment groups is selected for two treatments, and the results show that: CAP:63.4% >32.6%, difference 30.8%; TAP:57.9% >32.3%, difference 25.6%; FF:72.5% >39.5%, difference 33%; therefore, the purification effect of the mixed gel on the target substance doped sample is far better than that of the purification treatment of the SPE small column.

TABLE 11 recovery rate of single target from each group of three target concentration mixed sample of shrimp muscle after two purification treatments

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (8)

1. A florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain is characterized in that the preservation number is CCTCC No. C201945.

2. The anti-florfenicol/thiamphenicol monoclonal antibody is characterized by being secreted by a hybridoma cell strain with the preservation number of CCTCC No. C201945.

3. The hybridoma cell strain with the preservation number of CCTCC No. C201945 or the monoclonal antibody secreted by the hybridoma cell strain is applied to the pretreatment/immunoassay of the chloramphenicol residual sample machine, or the application of the hybridoma cell strain in the preparation of the pretreatment/immunoassay product of the chloramphenicol residual sample machine; the machine detection pretreatment/immunodetection product comprises a binding solution, a washing solution and an eluent; the combined solution is 0.01mol mL ^-1 PBS, the washing solution is 0.01mol mL ^-1 PBS and water, the eluent was methanol: mixing ammonia water in a volume ratio of 9And (4) eluting the solution.

4. The use of claim 3, wherein the hybridoma cell line with the collection number of CCTCC No. C201945 or the monoclonal antibody secreted by the hybridoma cell line with the collection number of CCTCC No. C201870 or the monoclonal antibody secreted by the hybridoma cell line is applied to the pretreatment/immunoassay of chloramphenicol residual samples in an machine test, or the application of the hybridoma cell line in the preparation of a product of the pretreatment/immunoassay of chloramphenicol residual samples in a machine test.

5. A chloramphenicol specificity immune affinity chromatography machine before detection/immune detection product, characterized by that, include secreting and producing monoclonal antibody by hybridoma cell strain with CCTCC No. C201945 preserving number, purify and then with CNBr-activated Sepharose-4B glue chemical cross-linking immune affinity chromatography glue or immune affinity chromatography chromatographic column formed by this glue prepacked, and binding solution, washing liquid and eluant; the binding solution is 0.01mol mL-1PBS, the washing solution is 0.01mol mL-1PBS and water, and the eluent is methanol: and the volume ratio of ammonia water is 9.

6. The pre-test/immunoassay product of claim 5, further comprising:

the monoclonal antibody is secreted and produced by hybridoma cell strain with the preservation number of CCTCC No. C201870, purified and chemically cross-linked with CNBr-activated Sepharose-4B gel to form immunoaffinity chromatography gel or immunoaffinity chromatography column pre-filled by the gel.

7. A method for pretreating a sample with chloramphenical residues, characterized in that the sample to be tested is subjected to a pretreatment for mechanical testing using the pretreatment for mechanical testing/immunodetection product according to any of claims 5 to 6.

8. A method for detecting a sample with chloramphenicol residues, which comprises subjecting a sample to be tested to a pretreatment for mechanical testing using the pretreatment for mechanical testing/immunoassay product according to any one of claims 5 to 6, and subjecting the treated sample to LC-MS/MS detection.

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