CN112239751A

CN112239751A - Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application

Info

Publication number: CN112239751A
Application number: CN202011090171.XA
Authority: CN
Inventors: 吴康; 沈飞; 章雪明; 方强; 陈旭晋
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2021-01-19
Anticipated expiration: 2040-10-13
Also published as: CN112239751B

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 regulated in plain texts in many countries including China, which means that the residual quantity of chloramphenicol must be lower 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; the florfenicol inhibits the embryo development, and the like, which are not inconstant, so that the highest residual standard of the two drugs in the edible parts of animal sources specified in China is less than 100-1000 ppb.

For more than a decade, after the German Baifa (R-biopharm) company brought the chloramphenicol ELISA special detection kit to the Chinese market, the Shenjia loyalty academy team of the Chinese agricultural university and the Beijing WeideWeikang company also put out corresponding quick check products, in particular to a teaching team from xu of Jiangnan university, the teaching team is provided with chloramphenicol quick-detection products such as chloramphenicol, thiamphenicol, florfenicol triple colloidal gold test strips and the like, although the products play an irreplaceable role in the high-throughput screening of chloramphenicol single target or/and residues of various targets in livestock and poultry, because the self limitation of the immune rapid detection product, particularly the immune cross property among similar targets, each single target of the targets cannot be completely and clearly defined and accurately quantified, and the immune rapid detection product cannot be used as the law basis of administrative punishment after banning or exceeding the standard and only plays a role in negative exclusion.

In view of this, the departments such as food security inspection at all levels in China pay more attention to the mechanical analysis. 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 interference of a matrix is difficult to completely eliminate by the purification of the solid-phase extraction column;

4. the recovery rate of the target substance purified by the solid-phase extraction column is very low, and the actual residual quantity of the target substance of the sample to be detected can be accurately calculated only by correcting the pretreatment recovery rate by using the corresponding isotope standard substance 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 column SPE (solid Phase Extraction cartridge) relies on import, is affected by new crown epidemics and has a risk of supply chain disruption.

Based on the technical bottleneck of the physicochemical pretreatment of the to-be-measured sample, the basic principle that a chloramphenicol specific antibody has strong specificity (selectivity), high affinity and antigen-antibody reaction reversibility is adopted, the chloramphenicol strong specificity immunoaffinity chromatography gel is prepared and is used as a mode of the pretreatment in the machine measurement, the strong selectivity and the high enrichment of the immunochromatography technology on a target can be perfectly combined with the effective chromatographic separation of a chromatographic column on 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 florfenicol₅₀1.35ppb) and higher working titer (working dilution 1: 40000) (ii) a

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 gel prepared by the monoclonal antibody and the pretreatment reagent for machine measurement in pretreatment and/or detection of a chloramphenicol antibiotic residue 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 test, so that the immunoaffinity chromatography reagent has various excellent effects of excellent detection limit, quantitative limit, recovery rate, purification capacity, matrix interference resistance and the like after pretreatment for machine test.

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

the florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain has the preservation number of CCTCC No. C201945 and is named as 9D₄-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 screening₄And (3) detecting the cross reaction rate of the induced monoclonal antibody, thiamphenicol and chloramphenicol, wherein when the CR value of florfenicol is determined to be 100%, the cross reaction rate of thiamphenicol is higher, the CR values are 69.69% respectively, 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)₂After 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 albumin₂Compared with the artificial antigen prepared by taking succinic anhydride (four-carbon chain connecting arm) as a modifier in the florfenicol reported in the prior art, the novel artificial antigen has the advantages that:

(1) lengthening the connecting arm: from original C₄Chain to C₅And 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, and can be finished in a common laboratory without high-grade instruments and equipment.

(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 invention₄The steps of-FF are specifically as follows:

firstly, aiming at the synthesis and physicochemical characterization analysis of florfenicol artificial antigen-immunogen;

synthesis of florfenicol artificial antigen-coating antigen;

(iii) Balb/C mouse immunization and selection of qualified immunized mice;

cell fusion, and screening and cloning aiming at the florfenicol monoclonal antibody hybridoma cell strain;

acquiring and evaluating induced ascites in vivo;

through the steps, the florfenicol monoclonal hybridoma cell-9D is obtained₄-FF, which induces in vivo ascites samples, analyzed by an indirect competitive ELISA assay, said samples having an extremely high detection sensitivity (IC) for florfenicol₅₀1.35ppb) and higher working titer (working dilution 1: 40000) The immunological cross-over rate with thiamphenicol is about 69.69%, and the immunological cross-over rate with chloramphenicol is less than 0.001%. 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/immunoassay of a chloramphenicol residual sample machine, or in the preparation of a chloramphenicol residual sample machine pretreatment/immunoassay 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 for 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 the China center for type culture Collection in 2018, 3 months and 14 days, and is addressed to Wuhan university at Lojia mountain of Wuchang city.

Preferably, the chloramphenical 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 81 ppb; aiming at the detection limit and the quantitative limit of thiamphenicol, 0.3ppb, 0.9ppb and an effective adsorption concentration domain: 0.3 to 81 ppb; aiming at the chloramphenicol detection limit and the quantification limit, 0.03,0.1ppb, effective adsorption concentration domain: 0.01 to 81 ppb; can meet the basic requirements of preparing the chloramphenicol mixed immunoaffinity chromatography adhesive.

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

The immunoaffinity chromatography gel provided by the invention is used as a core reagent for purifying chloramphenicol residual samples, three drug mixtures with equal doses of series concentrations are respectively doped into shrimp meat/natural lake water to serve 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 treatment/immunodetection product before detection of a chloramphenicol specific immunoaffinity chromatography machine, which comprises immunoaffinity chromatography gel or an immunoaffinity chromatography column pre-filled by the gel, wherein the immunoaffinity chromatography gel is formed by purifying monoclonal antibody secreted by a hybridoma cell strain with the preservation number of CCTCC No. C201945 and chemically crosslinking the purified monoclonal antibody with CNBr-activated Sepharose-4B gel.

Preferably, the pre-machine-test/immunoassay product further comprises: 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.

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

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 chromatographic gel prepared by secreting monoclonal antibody from hybridoma cell strain with the preservation number of CCTCC No. C201945 and/or chromatographic gel prepared by secreting monoclonal antibody from hybridoma cell strain with the preservation number of CCTCC No. C201870, and the volume ratio of the chromatographic gel to the chromatographic gel is preferably 1: 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 affinity to florfenicol 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 standard operation procedures of GE company, and detects the obtained chromatography gel/column product by analytical equipment such as LC-MS/MS and the like, thereby having better detection limit and quantitative limit, and simultaneously having better matrix interference resistance, standard recovery rate and purification treatment capability compared with the national standard SPE method.

Biological preservation information description

(1) Hybridoma cell line 9D for preservation₄The classification of-FF is named:

the preservation unit is 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: year 2019, month 3, day 18;

the preservation number is: CCTCC No. C201945;

(2) the classification of hybridoma cell strain 2D2-C1 for preservation is named as:

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

the preservation date is as follows: 3, 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 a chloramphenicol standard;

FIG. 4 shows the 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 a series of target mixture standard solution mixed gel processing sample detection signals;

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 graph showing the detection peak patterns of three target mixed standard samples of natural lake water after two kinds of purification treatments;

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: preparation and evaluation of 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, 0.1mmol), adding into 2mL of pyridine solution containing 11.4mg (0.1mmol) of glu (glutaric anhydride), and stirring at room temperature for reaction overnight; after completion, the pyridine was blown dry with nitrogen; dissolving FF-glu semialdehyde with 4mL of solvent (DMF and 1, 4-dioxane are mixed according to the volume ratio of 1: 1); adding 26.2 μ L (about 0.1mmol) of n-tributylamine, stirring in ice bath for 10min, adding 14.4 μ L (about 0.1mmol) of isobutyl chloroformate, stirring at room temperature for 1 h;

the activated FF solution was added dropwise to 5mL of a 10mg mL solution treated in an ice-water bath^-1Sodium borate solution (0.1mol L) of BSA (bovine serum Albumin)^-1pH8.5) over 1h, and the reaction was stirred at room temperature overnight. Dialyzing with 0.1mol sodium borate solution (pH8.5) overnight, dialyzing with 0.01M PBS (pH7.4) 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 as BSA-glu-FF.

A synthetic circuit diagram:

2. synthesis of artificial antigen-coating antigen against 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 5 days, with the dialysate 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^-1And respectively adding the two to A-G lines of an enzyme label plate; h line as negative control: 3. mu.g mL^-1OVA/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 ELISA plate, spin-drying, washing with PBS for 2+1(1 means adding PBS for spin-drying for the first time, 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) for several times (each time, each hole is 300 mu L), and patting to dry;

(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: diluting the serum of an antibody-free mouse to 1:3000 as a negative control at the same time by five dilutions of 1:1000, 3000, 9000, 27000 and 81000 to obtain a primary incubation antibody, and performing pre-treatment at room temperature for one hour;

(5) washing: taking out the enzyme label plate, spin-drying, washing for 2+1 times by PBST, and patting to dry;

(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 2 h;

(7) and (3) secondary antibody dilution: horseradish peroxidase-labeled secondary antibody was diluted with antibody diluent to 1:500, a step of;

(8) washing: taking out the enzyme label plate, spin-drying, washing for 4+1 times by PBST and patting to dry;

(9) adding a secondary antibody: adding 100 μ L of second antibody diluent into each well, and incubating at 37 deg.C for 80 min;

(10) washing: taking out the ELISA plate, spin-drying, washing with PBST for 3+1 times, washing with PBS for 1+1 times, and patting to dry;

(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 well after the color development is finished;

(13) OD measurement with microplate reader₄₉₀The value is obtained.

Indirect competitive ELISA qualitative calibration of antiserum affinity 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: OVA/CBS with the same concentration; 100 μ L was added to each well. 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^-1The 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^-1Six 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. h line plus PBS buffer diluted competitor-free antiserum (dilution is the nominal antiserum titer); adding 100 mu L of the culture medium into each well, and carrying out wet incubation for 2h at 37 ℃;

(5) wash-add secondary antibody-wash-develop-terminate the same checkerboard ELISA as above.

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

(1) Culturing mouse myeloma cell line-Sp 2/0 cells: the cell strain is given by a monoclonal antibody room of Jiangsu province blood research institute, cultured and passaged in a 10% FBS (Earl Bio products)/RPMI-1640 normal culture medium, and a cell subcloning test proves that the cell strain is regarded as a qualified culture system after being completely sensitive to HAT by a single cell colony forming rate of more than 70% and a 1XHAT selection culture test; 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 several 96-well culture plates at 4/piece and 100 μ l/well, and culturing at 37 deg.C and 5% CO₂Culturing in an incubator;

(3) acquisition of spleen cells: performing immunization shock for another 28 days after the seven-immunization, and taking blood from the eyeball after 3d of shock; after the neck is cut off and the rat is died, soaking the rat in 75% alcohol for 1-2 min; 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, then transferring into a centrifuge tube, centrifuging at 1000rpm for 3min, removing supernatant, adding RPMI-1640 culture solution to 30mL, and counting for later use;

(4) cell fusion: uniformly mixing splenocytes myeloma cells at a ratio of about 8:1, centrifuging at 1000r/min for 10min, removing the culture medium, and gently beating to relax the precipitate (facilitating 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 with 1mL of PEG1500, twice with 1mL of RPMI-1640 culture medium, twice with 1.5mL of RPMI-1640 culture medium and 5mL of RPMI-1640; centrifuging at 1000r/min for 5min, removing culture medium, and slightly beating 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) Cell culture: the fused cell suspension was dropped into the above-mentioned feeder cells-containing 96-well plate (100. mu.L per well) and incubated at 37 ℃ with 5% CO₂Continuously culturing in an incubator; respectively performing half-liquid exchange for 4d and 7d with RPMI-1640 medium containing 1XHAT (i.e. removing half volume with Papanicolaou dropper after blowing and mixing uniformly, and then supplementing volume with RPMI-1640 medium containing 1 XHAT); 10d, carrying out one-time half-liquid change by using RPMI-1640 medium containing 1 × HT; when the fused cells grow to the area of 1/10-1/4 of a 96-well plate, a little bit of the fused cells can be selected for primary screening, and the rest part of the fused cells are still at 37 ℃ and 5% CO₂And continuing culturing in the incubator.

6. Screening and evaluation of hybridoma cells

Coating with coating concentration calibrated by chessboard ELISA experiment, washing and sealing as 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 the above 24-hole culture plate with holes in pairsDiluting the stock solution with PBS buffer solution to obtain stock solution and 1/10 diluent, dividing each diluent into two parts, adding equal volume of PBS solution into one part, and adding equal volume of 2 μ g mL into the other part^-1FF 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, carrying out oscillation incubation for one hour at room temperature, adding the centrifuge tubes into an ELISA plate for primary antibody incubation, and carrying out ELISA (enzyme-linked immunosorbent 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 dilution₄₉₀Value comparison, and the candidate clones maintained strong hybridization signals after expanded culture in 24-well plates (i.e., 1/10 dilution OD₄₉₀Still strong), the strong signal can be effectively inhibited by FF standard sample (the same group of FF treatment has obvious inhibition), positive holes of the antibody specific to FF are screened from the candidate clone sample.

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 until the positive rate is 100%, and then selecting a plurality of wells with strongest signals and best cell growth for amplification, culture, freezing 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 by 1X10⁶Inoculating 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 fluid was 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 chessboard and indirect competition ELISA evaluation of the antiserum, and the cross-immunoassay with thiamphenicol and chloramphenicolThe assay was the same as an indirect competitive ELISA using flubenomycins as inhibitors.

10. Results

(1) Artificial immunogens against florfenicol: through the chemical synthesis, about 41mg (calculated by protein) of artificial antigen is recovered, 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 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 of FF to BSA can be roughly calculated to be 18:1 according to the formula [ FF-glu-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: wherein the working dilution of two antiserums is more than 1:10000 (OD)₄₉₀1.5) with half-inhibitory concentration IC against florfenicol standard solution₅₀(50% Inhibition Concentration) < ═ 5ng/ml, and the mice were judged as qualified antiserum, and the corresponding mice were used as candidate mice, and the spleens were harvested at 4-week intervals for fusion.

(4) Screening, cloning and evaluating the florfenicol monoclonal antibody hybridoma cell strain: the candidate mouse splenocytes are fused with Sp2/0 cells and then cultured in 10 culture plates with 96 holes, the supernatants of the cultured wells for 10 days are in one-to-one correspondence with 10 ELISA holes, and the indirect ELISA detection is carried out by 3ug/ml OVA-FFA coating: in total, 10-well suspected positive wells (OD) were obtained₄₉₀1.5); 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) … … \ 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; 10xDiluting the supernatant, adding 2 mu g/ml of florfenicol with the same volume, adding each sample to be detected into 10 detection units (each processing 2 multiple wells) in a one-to-one correspondence manner, and finally diluting the anti-blood sample by 1000x for the two detection units to serve as a synchronous positive control, and 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 coordinate₄After two rounds of single cell subcloning, the positive rate is 100% by detection; the second round of subcloning totally obtains 28 single cell clone detections which are all positive: A1-C4 have stronger immune hybridization signals; c5-8 is a negative control of Sp2/0 culture supernatant; c9-12 is a 1:5000 antiserum positive control. The result shows that the positive cloning rate reaches 100 percent (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 simultaneously, the clone is sent to the China center for type culture collection for seed preservation and is subjected to the collection 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: 1:40000 dilution; 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 of cells for FF

From table 2 and fig. 2, it can be derived: the system isThe system is directed to the florfenicol valid detection domain: 0.556-3.153ng mL^-1(ppb，IC₂₀-IC₈₀) (ii) a Detection sensitivity: 1.354ng mL^-1(ppb，IC₅₀) The results of immunological cross-over rate detection of chloramphenicol and thiamphenicol with the same parameters are shown in Table 3 below;

TABLE 39D₄-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 with high affinity.

Example 2: preparation and evaluation of ascites sample of monoclonal antibody 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 competition ELISA detection method aiming at chloramphenicol antiserum evaluation and chloramphenicol monoclonal antibody secretion fusion cell clone screening and evaluation; the quantitative or semi-quantitative determination of chloramphenicol standards in the supernatant of cultured cells of monoclonal antibody clones of interest is described in the literature "Development of a monoclonal antibody based-ELISA for the detection of chloramphenicol in-shock," feed and batch samples and evaluation by LC-MS/MS couppled with immunological definition-up "(animal. methods.2019.11.507-516). A hybridoma cell strain 2D2-C1 with high chloramphenicol titer and strong immunoaffinity is selected by evaluation to prepare a cell strain for the following experiments.

2. Large scale preparation and evaluation of monoclonal antibodies against chloramphenicol: large amounts of logarithmic phase-growing cells of hybridoma cell line 2D2-C1 were collected and 10 male Balb/C-nuke mice of 10 weeks of age were injected in vivo as mentioned in example 1. After obtaining the corresponding induced ascites sample, a small sample is diluted to 1 by 0.1 percent OVA/PBS: 5000. 10000, 20000, 40000 and 80000, the checkerboard and indirect competitive ELISA assessment of the antiserum is the same as the indirect competitive ELISA assessment of fluorophlomycin and thiamphenicol by using the immunoassay method and chloramphenicol as an inhibitor.

3. Results

(1)2D2-C1 cell culture supernatant to chloramphenicol standard sample quantitative determination and standard inhibition curve drawing: the fused cells are screened and subcloned for two times to obtain a hybridoma cell strain aiming at the chloramphenicol monoclonal antibody: 1B1 and 2D 2; selecting a cell strain 2D2-C1 with higher affinity to chloramphenicol, taking culture supernatant of the cell strain in 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)₄₉₀The mean value is B, OD of control (12 duplicate wells)₄₉₀The average value is B₀Inhibition per concentration point was expressed as (B)₀-B)/B₀100% calculation; the inhibition curves of the samples against chloramphenicol standards were plotted on the abscissa and the ordinate on the concentration of the inhibitor, and the results are described in the "Development of a monoclonal antibody based-ELISA for the detection of chloramphenicol in-shock, feed and milk samples and evaluation by LC-MS/MS coated with immunological clearance-up" (anal. methods.2019.11.507-516).

(2)2D2-C1 induced ascites is plotted against chloramphenicol standard inhibition curves and their homologs are assayed for immunological cross-reactivity: 10 Balb/C-nuke mice were co-injected with 2D2-C1 cells, and about 67ml induced ascites were collected at 11D and 12D; chessboard ELISA calibration, ascites working dilution: 1:40000 dilutedReleasing; 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-IC₈₀) And detection sensitivity: 0.51ng/ml (IC)₅₀) The immunological cross-over rates with florfenicol and thiamphenicol are shown in table 4:

TABLE 42D 2-C1 Cross-over rates of inducing ascites with chloramphenicol drugs

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

the monoclonal antibody secreted by the first and second 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 development of immunoadsorption products with excellent selective performance on complex samples to be detected containing chloramphenicol 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 advantage.

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. And then, respectively filling Protein G Resin (product of Kinserin) with the same volume as the ascites stock solution into 2 small columns (30ml) for chromatography, purifying the two monoclonal antibody proteins according to a standard method provided by Kinserin, collecting purified Protein eluates of the two monoclonal antibodies in parts, neutralizing the eluates with 1/10 volumes of 1M Tris-Cl (pH8.5), placing the neutralized eluates into Protein dialysis bags (product of 7500 + 14000MW solarBio), and dialyzing the neutralized eluates in 0.01M PBS (pH7.4) pre-cooled by ice water bath for 24 hours (changing the solution once every 4-6 hours). 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^TM4B, crosslinking:

(1) pretreatment of monoclonal antibody protein before crosslinking: respectively taking the two monoclonal antibody purified monoclonal antibody protein liquids with calibrated protein content, respectively placing the two monoclonal antibody purified monoclonal antibody protein liquids in two dialysis bags (7500 + 14000MW) and 200 × 0.1M NaHCO in 100-₃0.5M NaCl solution (pH 8.3) is dialyzed at 4 deg.C for 24hr (changing solution every 4-6 hr), and the dialyzed protein is filtered with 0.22 μ M microporous membrane, and stored at 4 deg.C for 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 pearl powder is 1: 3.5; and then, the swelling coefficient of the purified monoclonal antibody protein is 5 mg: 1 ml) to obtain CNBr-activated Sepharose^TM4B (a product of healthcare company) dry powder is placed in 2-piece 50ml centrifuge tubes (a product of burning company), the steps of peptizing and swelling, washing, crosslinking with purified protein, washing, blocking with activated groups, alternate washing of two buffers with different pH values and the like are carried out according to the method provided by healthcare company, small samples of protein liquid 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 ascites samples are purified by a protein G resin one-step method, 9D₄-FF strain obtained 35ml 1.35mg/ml total 47.25mg purified protein; the 2D2-C1 strain obtained 28ml 1.65mg/ml total 46.20mg purified protein, and the results of SDS-PAGE electrophoresis (see figure 4) show that both antibody purifications contain two distinct protein bands of 50Kd (antibody heavy chain) and 25Kd (antibody light chain), have no distinct other protein bands, and are consistent with the calibration molecular weights of the heavy chain and the light chain of the mouse IgG molecule, which indicates that the invention has obtained two high-purity monoclonal antibody proteins and can completely and completely complete obtain the monoclonal antibody with high purityThe method meets the requirements of the preparation of practical immunoaffinity chromatography gel on the amount and purity of the single-antibody protein.

(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 drops sharply to 0.05mg/ml, which is a good indication that both monoclonal antibodies are able to cross-link well with the solid phase gel, and this result gives: 9D₄-crosslinking ratio of FF strain: (1.25-0.04)/1.25 × 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 target substances treated by the mixed immunoaffinity chromatography gel and by the conventional solid Phase Extraction column SPE (solid Phase Extraction cartridge), the present example was measured 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 1290 model 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 50mm) 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: 35 psi; 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 multi-component mixed glue aiming at chloramphenicol: 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 (combination solutions), leacheate (washing solution), eluent and different immunological binding times, and the standards of higher recovery rates of three target substances are used to obtain the optimal immunoaffinity condition.

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

selecting a leaching solution: 0.01mol mL of pure water^-1PBS + Water, 0.01mol mL^-1Three buffers of PBS + 10% methanol water;

eluent is selected as follows: 0.1% formic acid (pH 2.5), 100% methanol solution, 80% methanol aqueous solution, MeOH: HCOOH (9:1), MeOH: NH₃·H₂0(9:1) five buffers;

the immunoaffinity time is selected from the following steps: 10, 15, 30 and 45 min. After immunoaffinity chromatography, each group of elution liquid nitrogen is blown (60 ℃), 1mL pure water is redissolved, filtered and tested on a prototype.

(3) Analysis of the adsorption properties of the mixed gel 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 are prepared by three target object mother liquor with 0.01M PBS (pH7.4) and respectively used as solutions to be detected, 1ml of solution to be detected and 100 mu l of mixed gel in each group are subjected to an immunochromatography test (three independent repeated tests are set for each group, n is 3), each group is subjected to nitrogen blowing, redissolution, filtration and 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 molar ratio of the mouse IgG to the target (MW 300D) of 1:1, the maximum theoretical adsorption capacity of the 100 mul mixed gel is about 500ng for each component), an immunoadsorption test is carried out according to the 1ml single target saturated solution and the 100 mul mixed gel (each target is subjected to six independent repeated tests, n is 6), supernatant liquid is obtained for detection after immunoadsorption, the maximum adsorption capacity of the reaction is calculated according to (the detection value after 500-immunoadsorption) ng/ml of 1ml, and 6 calculated values of each target are obtained, and the average value and the standard deviation between the 6 calculated values are calculated.

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-grade rod liquid-mass tandem mass spectrometer LC-MS matched with a ZORBAX Stable Bond C18(1.8 mu m,3.0 multiplied by 50mm) chromatographic column show that:

each concentration of each target is typically concentration-dependent on the peak area of the corresponding received signal, and the fitting rate R of the recovery curve is plotted²Are all larger than 0.99;

each target in the mixture sample had an exact time to peak, chloramphenicol: 4.618 min; thiamphenicol: 3.548 min; florfenicol: 4.059min (see Table 6 below), the retention time errors of different concentrations of the same target are all 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 spectrum peak pattern (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 chromatographic column selection and the LC-MS technical parameter setting are easy, and the simultaneous determination of the three target mixture 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) + H₂O; the eluent was methanol + ammonia (9:1 v/v). Thus, the following are selected: 0.01M PBS (pH7.4) as binding solution; 0.01M PBS (pH7.4) + H₂O is used as a washing liquid; methanol + ammonia (9: 1v/v) as eluent; the immunological combination time of the mixed gel and the sample to be tested is 40min, and the mixed gel and the sample to be tested are used as the optimal immunochromatographic 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 1 to 32ng/ml of each component, namely the characteristic peak area of each target, is in typical concentration dependence, and the fitting rate R of a concentration-to-corresponding signal intensity correlation curve plotted by the 6 concentration points²All greater than 0.99, three targets: the respective detection curves of the chloramphenicol, the thiamphenicol and the 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 established by the invention can accurately confirm and accurately quantify each target for the mixed standard sample of three targets 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 the three targets 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 steric hindrance interference is not generated for respective immunoadsorption target objects of the chloramphenicol solid-phase immunoadsorbent and the 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 (500ng/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, 109 ng; the average adsorption capacity and standard deviation between 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 practical data of the purification effect of the glue which is comprehensively superior to the purification effect of the national standard method is an important basis for the glue to replace a conventional solid phase extraction column to be applied to a sample to be detected actually, so that freshwater shrimps (purchased from a local farmer market) with the body length of about 12cm and natural lake water (collected from a water sample 50cm below the surface of a single villa lake near a single villa lake school district of Suzhou university) are selected as model matrixes to be used for subsequent tests after the quality of agricultural products and safety monitoring center of Suzhou proves that no chloramphenicol, thiamphenicol and florfenicol are left.

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, shearing, homogenizing at 4 deg.C 12000r/min for 5min, and collecting shrimp meat homogenate or directly preparing standard 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^-1After mixing the standard solution, the sample isVortex for 5min and mix well, at 4 ℃ refrigerator overnight. The next day, each centrifuge tube was vortexed for 5min, then 20mL ethyl acetate and 0.5mL ammonia were added, vortexed and mixed for 5min, shaken for 10min, centrifuged at 5000r/min for 10min at room temperature, 15mL of supernatant was transferred to another centrifuge tube, and dried at 60 ℃ with nitrogen. Adding 3mL of pure water, vortexing for 30min for redissolving, adding 2mL of normal hexane, vortexing and uniformly mixing for 3min, centrifuging at 5000r/min for 10min, and taking 1mL of lower-layer water phase as mixed gel for immune purification. 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, placing into four 50mL polypropylene centrifuge tubes with covers, sequentially adding 0, 20, 40, and 80 μ L of three target substances with concentration of 1 μ g mL^-1The 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 mixed target substance of three target substances mixed with the shrimp meat/natural Taihu lake water sample by using an SPE column:

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

4. Purifying and sample introduction detecting of the SPE column to a sample to be detected: counting Oasis^@HLB (300mg, 6mL product from Waters) columns, adding 5mL of methanol to each column for leaching activation and 5mL of water for leaching balance, slowly adding a 5mL of water phase sample to be purified to one SPE column, adding 5mL of pure water and 5mL of 5% methanol aqueous solution for leaching after the sample solution is drained, adding 5mL of methanol solution for elution, and drying the eluent in a water bath at 60 ℃ by nitrogen. 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. Results

(1) And (3) calibrating the detection standard curve of the three target mixed external standard samples of the four treatment matrixes: to correct the substrate pair LC-MS system pairThree target detection results in systematic deviation. This example uses four matrices: namely, the mixed glue is used for re-dissolving water phase (two types) after the chloramphenical antibiotic blank shrimp meat/natural lake water is purified; 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 R²The 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 signals²The concentration ranges of the three target substances are all larger than 0.99, so that the accurate quantification of the three target substances in the concentration ranges by the four matrixes is fully shown, and the result lays a firm foundation for the reliability of the accurate quantification of the purified sample of the three target substance matrixes doped with the target substances by the LC-MS system at the level of the equivalent matrixes in the next step.

TABLE 8 quantitative regression equation and correlation coefficient for target in water-phase external standard sample after two-mode purification of natural lake water

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 target substances, and the respective retention times are adjacent to the substantially non-impurity peak;

comparing the peak amplitudes of three target objects of the two processed samples: the samples treated by the mixed gel are obviously higher than those treated by SPE, and are respectively CAP:0.7> 0.23; TAP 0.3> 0.15; FF is 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 12min), 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 kinds of purification media have certain retention capacity for three kinds of target objects in the mixed standard sample, the characteristic peak amplitudes of the three kinds of target objects are taken as qualitative indexes, and the retention capacity of the mixed gel for the three kinds of target objects is far higher than that of the SPE small column for the three kinds of target objects;

although the matrix effect generated by the natural lake water mode matrix does not substantially interfere the display of the characteristic peaks of the three target objects in the sample to be detected, the noise level of the mixed gel processing sample is obviously lower than that of the SPE processing sample by taking the 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% > 67.2%, difference 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 shrimp muscle sample purified with mixed glue (blank control) (see fig. 9) also exhibited only a very low peak amplitude of the peak of the miscellaneous peak without interfering with the characteristic peak of the target, the peak profile of the three targets obtained from the LC-MS test of two purified samples of shrimp muscle mixed with the three targets at the same concentration (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 generate respective characteristic peaks in specific retention time of three target objects after being treated by two kinds of samples, the mixed glue treated sample can keep no interference of the impurity peaks in the vicinity of the respective retention time of the three target objects; 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 objects of the two processed samples: the samples treated by the mixed gel are also obviously higher than those treated by SPE, and respectively have CAP:1.2> 0.25; TAP 0.4> 0.07; FF is 0.7>0.08.

Baseline (background) comparisons beyond 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 object is eliminated as a qualitative index, and the mixed gel treatment is compared with SPE treatment: 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.

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

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 and generated 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 a chloramphenicol residual sample machine or the preparation of a chloramphenicol residual sample machine pretreatment/immunoassay product.

4. The use of claim 3, wherein the hybridoma cell line with the preservation number of CCTCC No. C201945 or the monoclonal antibody secreted by the hybridoma cell line with the preservation number of CCTCC No. C201870 or the monoclonal antibody secreted by the hybridoma cell line is used in the pretreatment/immunoassay of a chloramphenicol residual sample machine, or the preparation of a chloramphenicol residual sample machine pretreatment/immunoassay product.

5. The use of claim 3 or 4, wherein the pre-treatment/immunoassay product further comprises a binding solution, a washing solution and an elution solution; the combined solution is 0.01mol mL^-1PBS, the washing solution is 0.01mol mL^-1PBS and water, the eluent is methanol: mixed eluent with the volume ratio of ammonia water being 9: 1.

6. A chloramphenicol specific immunoaffinity chromatography pretreatment/immunodetection product is characterized by comprising immunoaffinity chromatography gel or immunoaffinity chromatography column pre-filled by the gel, wherein the immunoaffinity chromatography gel is formed by purifying monoclonal antibody secreted by hybridoma cell strain with the preservation number of CCTCC No. C201945 and chemically crosslinking with CNBr-activated Sepharose-4B gel.

7. The pre-test/immunoassay product of claim 6, 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.

8. The pre-machine/immunoassay product of claim 6 or 7, further comprising:

binding solution, washing solution and eluent; the combined solution is 0.01mol mL^-1PBS, the washing solution is 0.01mol mL^- ¹PBS and water, the eluent is methanol: mixed eluent with the volume ratio of ammonia water being 9: 1.

9. A method for pretreating a chloramphenical residual sample is characterized in that a sample to be tested is subjected to mechanical test pretreatment by adopting the mechanical test pretreatment/immunodetection product of any one of 6-8.

10. A method for detecting a chloramphenical residual sample is characterized in that a sample to be detected is subjected to mechanical test pretreatment by adopting any one of the mechanical pretreatment/immunodetection products of 6-8, and the treated sample is detected by LC-MS/MS.