CN110286240A - A kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban - Google Patents

A kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban Download PDF

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CN110286240A
CN110286240A CN201910645174.6A CN201910645174A CN110286240A CN 110286240 A CN110286240 A CN 110286240A CN 201910645174 A CN201910645174 A CN 201910645174A CN 110286240 A CN110286240 A CN 110286240A
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triclosan
triclocarban
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邵兵
姚凯
张晶
温凯
杨蕴嘉
沈建忠
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Beijing Center for Disease Prevention and Control
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Abstract

The present invention provides a kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban, and a kind of method of trace triclosan and/or triclocarban in test sample is specifically provided, including the use of immune affinity column-ultra performance liquid chromatography tandem mass spectrum detection method.Wherein, the monoclonal antibody of immune affinity column mesostroma and triclosan and triclocarban is coupled.Detection method high sensitivity provided by the invention, precision, the rate of recovery, LOD and LOQ are able to satisfy the requirement of detection and analysis, effectively solve the problems, such as that sample purification is undesirable in pre-treatment, and reduce sample substrate interference, shorten analysis time, improves the rate of recovery, it can be in complex matrices, trace triclosan and/or triclocarban are especially specifically detected in varieties of food items, are not in the testing result of false positive.

Description

A kind of triclosan monoclonal antibody and/or triclocarban monoclonal antibody are in detection three Purposes in chlorine life and/or triclocarban
Technical field
The present invention relates to technical field of immunoassay, and in particular to a kind of triclosan monoclonal antibody and/or triclocarban Purposes of the monoclonal antibody in detection triclosan and/or triclocarban, be more particularly in a kind of food trace triclosan and/ Or the immune affinity column of triclocarban-ultra performance liquid chromatography tandem mass spectrum detection method.
Background technique
Incretion interferent (Environmental endocrine disrupting chemicals, EDCs), is one The existing xenobiotics for interfering human or animal's endocrine system, majority enter biology by food chain to kind in the environment In vivo, cause organism itself hormone disturbed, recycle, accumulated in fat in blood, it is normal so as to cause organism The physiological systems such as reproduction, development, immune, behavioral activity are abnormal.Triclosan (triclosan, TCS) and triclocarban (triclocarban, TCC) belongs to incretion interferent, is made an addition to personal nursing extensively as high-efficiency broad spectrum antibacterial agent In product and daily chemical products, it can be entered in human body by approach such as skin contact, buccal absorptions.It is big that TCS can reduce the pregnancy period The thyroid hormones level of mouse, the bioactivity of the testosterone of male rat can be enhanced in TCC, and can cause human hepatocyte's DNA damage, there are certain genetoxics.
Therefore carrying out Accurate Determining to the residual level of TCS and TCC in food is very important.Triclosan and trichlorine The current detection method of card class mainly has gas chromatography and gas chromatographymass spectrum, liquid chromatography and liquid-mass chromatography method etc., examines The interference of survey process mesostroma is bigger.The purification method detected at present mainly includes liquid-liquid extraction, Solid Phase Extraction, matrix solid phase Dispersion extraction and accelerated solvent extraction etc., complicated for operation, poor selectivity, and clean-up effect is undesirable, impurity interference is very big, recycling Rate and repeatability are poor.The specificity that immunoassay method is immunoreacted based on Ag-Ab analyzes its specificity compared with instrument By force, high sensitivity can not only greatly simplify or even can save the complex process of sample pre-treatments, but also avoid because a large amount of Using solvent to the pollution of environment.It therefore is essential to the immunoassay research of TCS and TCC.Patent CN105085204A discloses a kind of haptens of triclosanArtificial antigen and preparation are corresponding The method of antibody, but the antibody not publicly prepared detects triclosan for immunochromatography.CN109956848A discloses one kind Triclosan haptensThe preparation method and purposes of antigen and corresponding antibodies, general mentioning can be used for preparing Enzyme linked immunological kit, colloidal-gold detecting-card and immune affinity column, and it is only simple be prepared for immune affinity column, do not confirm It can be used for the detection of triclosan.Patent CN109959784A discloses triclocarban haptensAntigen and preparation method thereof and the application in antibody is prepared, general mentioning can To be used to prepare enzyme linked immunological kit, colloidal-gold detecting-card and immune affinity column, and only simple it is prepared for affine in immunity Column does not confirm the detection that can be used for triclosan.Document: Detection of the Antimicrobial Triclosan In Environmental Samples by Immunoassay (Ahn et al., Environ.Sci.Technol, 2016, 4-Cl or 4 '-Cl of replacement TCS 50:3754-3761) are disclosed, coupling carrier albumen prepares haptens, and immune rabbit obtains TCS antibody, and MBP enzyme linked immuno-adsorbent assay TCS is utilized, it is close with the result of LC-MS/MS detection.Document: An immunoassay to evaluate human/environmental exposure to the antimicrobial Triclocarban (Ahn et al., Environ.Sci.Technol, 2012,46:374-381) is disclosed to be exempted from using enzyme-linked The method that epidemic disease adsorption experiment detects TCC, wherein the haptens used is by replacing the derivative obtained to phenyl.But it is above-mentioned The prior art is not met by actual demand in the sensitivity for detecting TCC and/or TCS, accuracy, it is therefore desirable to which exploitation is a kind of high Effect is accurate, the quick TCC and/or TCS detection method of trace.
Summary of the invention
To make up the deficiencies in the prior art, the present invention provides a kind of using immune affinity column and including ultra high efficiency liquid phase color Compose the method combination including tandem mass spectrum, the method for trace triclosan and/or triclocarban in test sample.Wherein, parent is immunized It is coupled with the monoclonal antibody of column mesostroma and triclosan and triclocarban.Detection method high sensitivity provided by the invention, essence Density, the rate of recovery, LOD and LOQ are able to satisfy the requirement of detection and analysis, and sample purification is undesirable effectively in solution pre-treatment asks Topic, and sample substrate interference is reduced, shorten analysis time, improves the rate of recovery.
Meanwhile the present inventor is prepared for triclosan haptens and triclocarban haptens by creative work, and should Haptens and carrier protein couplet prepare triclosan artificial antigen and triclocarban artificial antigen, using both artificial antigen systems Standby obtained monoclonal antibody specificity is strong, is able to achieve to TCC/TCS trace detection.Test proves that being made using the present invention Standby triclosan haptens and triclocarban haptens obtains obtained by triclosan/triclocarban monoclonal antibody of more efficient valence Antibody I C50 is low, is able to achieve to TCC/TCS trace detection.
The first purpose of the invention is to provide triclosan monoclonal antibodies and/or triclocarban monoclonal antibody to detect Purposes in triclosan and/or triclocarban, the triclosan monoclonal antibody are by triclosan haptens system shown in Formulas I Standby to obtain, the triclocarban monoclonal antibody is prepared by triclocarban haptens shown in Formula II,
Wherein the triclosan monoclonal antibody is for detecting triclosan, and the triclocarban monoclonal antibody is for detecting Triclocarban can also use the triclosan monoclonal antibody and triclocarban monoclonal antibody detection triclosan and triclocarban Mixture.
Triclosan artificial antigen used in the triclosan monoclonal antibody is prepared as shown in formula III, is trichlorine shown in Formulas I Raw haptens and carrier protein, which combine, to be obtained；Prepare triclocarban artificial antigen used in the triclocarban monoclonal antibody such as It is that triclosan haptens shown in Formula II and carrier protein are combined and obtained shown in formula IV,
Preferably, the method for the detection includes immunoassay method, biosensor, Surface enhanced Raman scattering, institute The immunoassay method stated includes immunoaffinity chromatography, ELISA, colloidal gold immunochromatographimethod technology, chemiluminescence immunoassay Analytic approach or suspension array technology, further preferably immunoaffinity chromatography.
Preferably, the method for the detection further include with method associated with other detection methods, it is described associated with detection side Method includes but is not limited to ultra performance liquid chromatography, mass spectrum or ultra performance liquid chromatography and mass spectrometry.
A second object of the present invention is to provide triclosan hapten compounds shown in a kind of Formulas I, are given birth to by the haptens The artificial antigen (Formula II) of production, monoclonal antibody and their preparation method.
The present invention also provides the preparation method of compound of formula I, synthetic route is as follows:
Specifically, the preparation method of compound of formula I includes the following steps: for triclosan to be dissolved in organic solvent, 4- is added Bromomethyl-benzoic acid methyl ester, NaH do catalyst, 40-50 DEG C of reaction 4-6h, and separating-purifying hydrolyzes under alkaline condition, by extraction Take, wash, dry after obtain target formula (I) compound.
The organic solvent is at least one of DMF, THF, DMSO, methylene chloride, acetonitrile, methanol, ethyl alcohol.
It is the triclosan half by above-mentioned formula (I) the present invention also provides triclosan artificial antigen shown in formula (III) Antigen and carrier protein, which combine, to be made, and wherein the coupling ratio of triclosan haptens and carrier protein is 0.5-1:1, and preferably 0.7: 1。
The carrier protein is selected from bovine serum albumin(BSA) BSA, oralbumin OVA, human serum albumin HSA, Niu Jia The combination of one or more of shape gland globulin BTG or blood spoon azurin KLH.Preferably, the carrier protein is BSA, coating antigen are preferably OVA.
The preparation method of triclosan artificial antigen shown in formula III is obtained by method comprising the following steps: a) modus ponens I The triclosan haptens of structural formula is dissolved in n,N-Dimethylformamide, and it is sub- that 1- (3- dimethylamino-propyl) -3- ethyl carbon two is added Amine hydrochlorate and n-hydroxysuccinimide, stirring obtain reaction solution I；B) it takes carrier protein to be dissolved in PBS and reaction solution is made II；C) reaction solution I is added in reaction solution II, is stirred at room temperature, obtain triclosan artificial antigen.
A kind of preparation method of triclosan monoclonal antibody, comprising the following steps: 1) artificial using triclosan shown in formula III Antigen-immunized animal；2) antiserum screens, and chooses the high animal of serum titer and is used as preparation monoclonal antibody；3) cell fusion and It screens, after cell fusion, the high positive cell of screening potency；4) cell clone is carried out, it is anti-to obtain triclosan monoclonal after purification Body.
The preparation method of said monoclonal antibody is known in those skilled in the art, it is preferable that the antiserum sieve Choosing and cell screening use indirect competitive ELISA method；The most suitable working concentration of coating antigen, antibody is determined using square matrix titration； And/or cell clone uses limiting dilution assay；And/or monoclonal antibody induces ascites method using internal；And/or monoclonal antibody Purifying use octanoic acid-ammonium sulfate precipitation method.
Third object of the present invention is to provide triclocarban hapten compound shown in a kind of Formula II, half anti-by this The artificial antigen (formula IV) of original production, monoclonal antibody and their preparation method.
The preparation method of the Formula II compound, synthetic route are as follows:
Specifically, the preparation method of the triclocarban haptens, includes the following steps: to weigh isocyanic acid 3,4- dichloro Organic solvent is added in phenyl ester and 4- aminophenyl methyl butyrate, and 40-50 DEG C is reacted 6-8h under nitrogen protection, is spin-dried for, and methanol is molten Solution is added the lye hydrolysis of the amount of 1-2 times of substance of reactant, is adjusted with acid pH to 2-3 after hydrolysis completely；It extracts, in organic phase Anhydrous sodium sulfate is added, stirs, filters, it is dry, obtain triclocarban haptens.
The organic solvent is that the organic solvent is DMF, THF, in DMSO, methylene chloride, acetonitrile, methanol, ethyl alcohol It is at least one；The liquid alkaline liquid is the aqueous solution of NaOH, KOH；The acid is HCl, HNO3, H2SO4At least one of.
Triclocarban artificial antigen shown in the formula IV is that triclosan haptens shown in Formula II and carrier protein combine It obtains, wherein the coupling ratio of triclocarban haptens and carrier protein is 6-7.5 1, preferably 6.8:1.
Preferably, the carrier protein is selected from bovine serum albumin(BSA) BSA, oralbumin OVA, human serum albumins The combination of one or more of HSA, bovine thyroglobulin BTG or blood spoon azurin KLH.In a tool of the invention In body embodiment, the carrier protein is BSA, and coating antigen is preferably OVA.
The preparation method of triclocarban artificial antigen shown in the formula IV includes the following steps: that (1) will be shown in Formula II Triclocarban haptens is dissolved in n,N-Dimethylformamide, and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride is added Reaction solution I is made in salt and n-hydroxysuccinimide, stirring；(2) carrier protein is dissolved in PBS, reaction solution II is made； (3) reaction solution I is added in reaction solution II, is stirred at room temperature, obtain triclocarban artificial antigen.
A kind of preparation method of triclocarban monoclonal antibody, comprising the following steps: 1) using triclocarban shown in formula IV Animal is immunized in artificial antigen；2) antiserum screens, and chooses the high animal of serum titer and is used as preparation monoclonal antibody；3) cell melts It closes and screens, after cell fusion, the high positive cell of screening potency；4) cell clone is carried out, obtains triclosan Dan Ke after purification Grand antibody.
Fourth object of the present invention is to provide a kind of method of triclosan and/or triclocarban in test sample, including Triclosan monoclonal antibody and/or triclocarban monoclonal antibody is used to prepare immune affinity column to detect triclosan and/or three Chlorine card class, wherein prepare that triclosan haptens used in the triclosan monoclonal antibody is shown in formula I, prepare described Triclocarban haptens is as shown in Formula II used in triclocarban monoclonal antibody.
A kind of method of triclosan and/or triclocarban in test sample, comprising the following steps: use triclosan monoclonal Antibody and/or triclocarban monoclonal antibody prepare immune affinity column to detect triclosan and/or triclocarban, wherein preparation Triclosan artificial antigen used in the triclosan monoclonal antibody prepares the triclocarban Dan Ke as shown in formula III Triclocarban artificial antigen is as shown in formula IV used in grand antibody.
The method for preparing immune affinity column includes: the preparation of column material matrix, the Dan Ke of triclosan and/or triclocarban Grand antibody and matrix are coupled, and wash after coupling, dress column is resuspended.
Wherein, the matrix through overactivation, the activation step is activated by NHS.
Preferably, in the method, the filler mesostroma of the immune affinity column and the volume ratio of monoclonal antibody are 1:1~1:1.5.
It is further preferred that the matrix is Ago-Gel, such as Sepharose 4B, Sepharose 6B, glass Glass microballoon and chitosan.In the specific embodiment of the present invention, the Ago-Gel activated by NHS after through HCl It is coupled after elution, coupling buffer balance with the monoclonal antibody of triclosan and/or triclocarban.
Preferably, the sample is solid sample or fluid sample；Preferably, the sample be selected from food, body fluid, Sewage, weaving or daily chemical product；It is furthermore preferred that the sample is food.It is further preferred that the daily chemical product includes But be not limited to shampoo, perfumed soap, shower cream, cosmetics, washing powder, skin care item, bath accessory or kitchen article etc..In the present invention A specific embodiment in, the test sample be food.
Preferably, the detection includes sample pre-treatments, and the solid sample pre-treatment includes by solid sample and second Nitrile mixing, ultrasound, centrifuging and taking supernatant, nitrogen are blown.It is further preferred that the ultrasonic time is 10-30min.It is highly preferred, The ultrasonic time is 15-25min.It is further preferred that the centrifugation is that 10000rpm is centrifuged 5-15min.Further Preferably, remaining liquid after supernatant is taken to mix again with acetonitrile, ultrasound, be centrifuged combined extract after extraction once.It is further excellent Choosing, the nitrogen blowing temp is 30-50 DEG C.
In the specific embodiment of the present invention, the solid sample pre-treatment include weigh sample it is homogeneous after with Acetonitrile mixing；Oscillation, ultrasonic 10-30min, 10000rpm take supernatant after being centrifuged 5-15min；Acetonitrile repeats to extract above-mentioned homogeneous sample Product to the step of centrifuging and taking supernatant, extracts once again, combined extract；30-50 DEG C of nitrogen is blown, and PBS is added and mixes.
Preferably, the fluid sample pre-treatment includes by fluid sample ultrasonic degassing.It is further preferred that described The ultrasonic degassing time is 10-50min.Highly preferred, the ultrasonic degassing time is 20-40min.At of the invention one In specific embodiment, the fluid sample pre-treatment includes 10-50min that sample ultrasonic deaerates；Then ultrasonic degassing is taken 1-1.5:1 dilutes fluid sample and PBS afterwards by volume；PH to 8-10 is adjusted with 1M NaOH solution.
In the specific embodiment of the present invention, the method for the detection triclosan and/or triclocarban, including such as Lower step:
It 1) is that 1:1~1:1.5 prepares immune affinity column according to the volume ratio of filler mesostroma and monoclonal antibody；
The monoclonal antibody is triclosan monoclonal antibody and/or triclocarban monoclonal antibody, the triclosan Monoclonal antibody is made using triclosan artificial antigen shown in triclosan haptens or formula III shown in Formulas I, the triclocarban Monoclonal antibody is made using triclocarban artificial antigen shown in triclocarban haptens or formula IV shown in Formula II；
2) it weighs sample and carries out sample pre-treatments；
Wherein, solid sample pre-treatment includes mixing solid sample with acetonitrile, being ultrasonic, and centrifuging and taking supernatant, nitrogen is blown；Liquid Sample pre-treatments include by fluid sample ultrasonic degassing；
3) by the sample Jing Guo pre-treatment be splined on preparation immune affinity column, elution, by the eluted product after elution into The detection of row triclosan and/or triclocarban.
Wherein ultra performance liquid chromatography and the concatenated detection side of mass spectrum are preferably used in the detection of triclosan and/or triclocarban Method；Wherein, ultra performance liquid chromatography condition: chromatographic column: C18 column, 100mm × 2.1mm, 1.7 μm or other equivalent columns；Flowing Phase: A, methanol, B, water；Eluent gradient elution program: 0.00-6.00min 30%-100% mobile phase A, 6.00-8.00min 100% mobile phase A, 8.00-8.10min 100%-30% mobile phase A, 30% mobile phase A of 8.10-11.00min；Column temperature: 40 ℃；Sample room temperature: 4 DEG C；Sample volume: 10 μ L.The mass spectrographic condition are as follows: ionization source: ESI (-)；Capillary voltage: 2.5kV；Ion source temperature: 150 DEG C；Desolvation temperature: 400 DEG C；Desolventizing gas flow: 900L/h；TCC parent ion 313.0m/z, daughter ion 160.0,126.0m/z；TCS parent ion 286.8m/z, 288.8m/z, daughter ion 35.0m/z.
The present invention also provides a kind of immune affinity column, filled with above-mentioned triclosan monoclonal antibody (TCC mAb) and/or Triclocarban monoclonal antibody (TCS mAb).
Monoclonal antibody of the present invention can be IgG, IgA, IgM, IgD or IgE hypotype.In a tool of the invention In body embodiment, the monoclonal antibody is IgG.
Some terms that more than ten present invention use below are explained: " OPD " is o-phenylenediamine；" DMEM " is Du Shi culture medium (dulbecco's modified eagle medium), be developed on MEM medium base containing various amino acid and The culture medium of glucose；" IAC " is immunoaffinity chromatography purification method；" RSD " is relative standard deviation；" ME " is Matrix Effect, i.e. matrix effect refer to the influence that the co-elute remnants matrix component of mixing sample ionizes target analytes； " TMB " is 3,3', 5,5'- tetramethyl benzidine；" BSA " is bovine serum albumin(BSA)；" NHS " is N- hydroxysuccinimide； " BPA " is bisphenol-A；" LOD " is detection limit；" LOQ " is quantitative limit, i.e., the minimum value that can be quantified when actually detected,
First for the prior art, the present invention achieve it is following the utility model has the advantages that
One, the present invention creatively proposes to use triclosan haptens and/or triclocarban hapten compound, final to make Standby obtained triclosan half and/or triclocarban monoclonal antibody are for detecting triclosan and/or triclocarban, detection method spirit Sensitivity is high, and precision, the rate of recovery, LOD and LOQ are able to satisfy the requirement of detection and analysis, effectively solves sample purification in pre-treatment Undesirable problem, and sample substrate interference is reduced, shorten analysis time, improves the rate of recovery.
Two, using the obtained triclosan monoclonal antibody of triclosan hapten compound shown in formula (I), and formula is used (II) triclocarban monoclonal antibody made from triclosan haptens chemical combination shown in, respectively has triclosan and triclocarban Excellent selectivity, the IC50 value that for triclosan and/or triclocarban detects low with the cross reacting rate of its analogue It is low, trace triclosan and/or triclocarban can be detected to specificity in complex matrices, be not in the detection of false positive As a result.
Detailed description of the invention
Fig. 1: the LC-MS/MS qualification figure of triclosan haptens shown in Formulas I.
Fig. 2: the 1H-NMR qualification figure of triclosan haptens shown in Formulas I.
Fig. 3 A is the MALDI-TOF qualification figure of BSA, and Fig. 3 B is that the MALDI-TOF after triclosan haptens and BSA coupling reflects Fixed figure.
Fig. 4: the standard curve of triclosan monoclonal antibody detection.
Fig. 5: the LC-MS/MS qualification figure of triclocarban haptens shown in Formula II.
Fig. 6: the 1H-NMR qualification figure of triclocarban haptens shown in Formula II.
Fig. 7 is the MALDI-TOF qualification figure after triclocarban haptens and BSA coupling.
Fig. 8: the standard colour chart curve of triclocarban monoclonal antibody detection.
Fig. 9: triclosan and triclocarban standard chromatogram.
Figure 10: influence of the methanol content to analyte (the TCC, TCS) rate of recovery in load liquid.
Figure 11: influence of the content of methanol to analyte (the TCC, TCS) rate of recovery in eluent.
Figure 12: influence of the effluent volume to analyte (the TCC, TCS) rate of recovery.
Figure 13: select nylon or fibrous glass as the filtering before UPLC-MS/MS sample introduction, to analyte (TCC, TCS) Loss.
Figure 14: the purification that affine in immunity column purification is purified with common three kinds of purification styles (C18, GCB, HLB) It can comparison.
Figure 15: the present invention detects the schematic diagram of TCC and/or TCS using immunoaffinity chromatography.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiment is only section Example of the invention, rather than all.Based in the present invention Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all Belong to the scope of protection of the invention.
The source (all chemical reagent or solution are at least analyzed pure) of material used in the present embodiment, reagent:
TCC standard items (purity > 99.0%) are purchased from (Ontario, Canada Toronto Research Chemicals North Yorkshire)；TCS standard items (purity > 99.5%) are purchased from Dr.Ehrenstorfer GmbH (Augsburg, Germany)；Double phenyl Urea (purity > 98%), methyl TCS, bisphenol-A (purity > 99.0%) and 2,2', 4,4'- tetrabromo Biphenyl Ethers are purchased from Sigma- Aldrich (St. Louis)；Sepharose 4B, the 6B gel of NHS activation win intelligent (China purchased from Webster Beijing)；PP column (polypropylene column, 3mL) is purchased from Biocomma (China Shenzhen)；Waters Sep-Pak C18 solid-phase extraction column (200mg, 3mL), Oasis HLB solid-phase extraction column (60mg, 3mL) is purchased from Milford (Massachusetts, United States)； Supelclean ENVI-Carb solid-phase extraction column (500mg, 6mL) is purchased from Supelco (city Li Fangte, the U.S.)；LC-MS Methanol (MeOH), the acetonitrile (MeCN) of purity grade reagent are provided by Merck (Darmstadt, Germany)；Prepare ultrapure water The ultrapure systems buying of Milli-Q is from Millipore (Massachusetts, United States Bedford)；Analyze pure NaOH and hydrochloric acid (36% HCl it) buys from Beijing Chemical Plant (BeiJing, China)；Phosphate buffer (PBS, pH9.5,0.02mol/L) is NaCl (17.6g)、KCl(0.04g)、NaH2PO4·2H2O (5.8g) and Na2HPO4·12H2O (1.18g), which is dissolved in 1L ultrapure water, to be obtained ?；Microwell plate microplate reader is purchased from Sunnyvale (California, USA)；(0.22 μm) of nylon filter purchase rise from saliva (in State Tianjin)；(0.22 μm) of glass fiber filter is bought from U.S. Pall；Food and beverage buy the supermarket from BeiJing, China.
Embodiment 1The preparation of triclosan monoclonal antibody (TCS mAb)
1, the preparation of triclosan haptens compound of formula I
In the n,N-Dimethylformamide that 424mg triclosan is dissolved in 7mL in 50mL round-bottomed flask, ice bath magnetic agitation Under 48mg sodium hydride is added portionwise, ice bath, normal-temperature reaction 40min are removed when emerging there is no bubble.It is slowly added to 229mg4- Bromomethyl-benzoic acid methyl ester, chromatographic sheet contact plate monitor reaction process.It puts it into 40 DEG C of oil baths and reacts, continuous contact plate, When fully reacting, stop heating.Reaction solution is extracted with ethyl acetate twice, organic phase is collected.Dry, the bottle by organic phase revolving Middle addition 12mL ethyl acetate dissolves residue, and the silica gel for weighing 2 times of weight of residue is added thereto, and revolving is dry, prepared silicon Rubber column gel column loading.Silica gel sample that reaction solution is mixed with silica gel is loaded using dry sample sample-adding method, with petroleum ether: acetic acid second The mixed liquor of ester (8:1, v/v) is eluted as eluent, and 10mL centrifuge tube collects leacheate, and chromatographic sheet contact plate is led to It crosses and is compared with material liquid, determine the time of occurrence and extinction time of object.By all thin layers in silica gel column chromatography Liquid of the chromatography contact plate in the centrifuge tube of same target product position, which is all collected into 100mL revolving bottle, to be rotated.Use 7mL Methanol dissolution is added 2M NaOH solution by the amount of 2 times of substances, heats at 40 DEG C, after hydrolysis completely, is adjusted with the HCl of 6mol/L PH to 2.Reaction solution is extracted twice with 50mL ethyl acetate, anhydrous sodium sulfate, magnetic after addition are added into the organic phase of collection Power stirs 1h.Filtering, revolving is dry, obtains triclosan haptens shown in Formulas I after purification.
High performance liquid chromatography tandem mass spectrum (LC-MS/MS) and nuclear magnetic resonance (Nuclear magnetic is respectively adopted Resonance, NMR) haptens of synthesis is identified, identified, haptens is successfully prepared.LC-MS/MS qualification figure is shown in Attached drawing 1,1H-NMR qualification figure is shown in attached drawing 2.
2, the preparation of triclosan artificial antigen (formula III)
With the coupling ratio of triclosan haptens (Formulas I) and carrier protein BSA for 0.7:1, trichlorine stranger shown in formula III is carried out The preparation of work antigen, the i.e. preparation of triclosan immunogene.Specific step is as follows:
Triclosan haptens shown in the Formulas I of 60 carrier protein equivalents is taken to be dissolved in 1mL n,N-Dimethylformamide, respectively 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide of 180 BSA equivalents is added, 2h is stirred under room temperature, reaction solution I is made；It takes a certain amount of BSA 50mg to be completely dissolved in the PBS of 5mL pH7.4 and reaction is made Reaction solution I is slowly added in reaction solution II by liquid II dropwise, and is stirred at room temperature for 24 hours；Reacting final product is packed into and is dialysed Bag is dialysed 3 days with the PBS buffer solution of 0.01M, changes 3 dialyzates daily to remove unreacted small-molecule substance；With 10000rpm is centrifuged 10min, saves backup after collecting supernatant packing in -20 DEG C.
Using Matrix-assisted laser desorption ionization (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, MALDI-TOF) it is identified, it reflects Determine the results show that triclosan artificial antigen synthesizes successfully, BSA and BSA manually resist with the triclosan after triclosan hapten conjugation Former MALDI-TOF qualification result is shown in attached drawing 3A and Fig. 3 B.
3, the preparation of triclosan monoclonal antibody (TCS mAb)
1) animal immune
BALB/c female mice is immunized using low dose of short cycle scheme.With the above-mentioned triclosan immunogene prepared by 100 μ g/ only, are mixed with physiological saline solution triclosan artificial antigen with Freund's complete adjuvant in equal volume, the nape of the neck subcutaneous injection 6~8 week old Balb/c female mices are immunized, are mixed in equal volume with immunogene and incomplete Freund's adjuvant within the 7th, 14,28 day after initial immunity Even, each supplementary immunization is primary.
2) antiserum screens
7~10 days, the 200 μ L of venous blood collection under mouse orbit after the 4th is immune, after 4 DEG C of standing 2h of blood of acquisition, 37 DEG C of incubation 8h, blood solidify completely, and serum is precipitated.Coating antigen dilution and sero-fast is screened using indirect elisa method Dilution, then its specificity is measured using indirect competitive ELISA method.Using square matrix titration determine coating antigen, antibody it is most suitable Working concentration, the specific steps are as follows:
(1) it is coated with: coating antigen being diluted to a series of concentration with coating buffer and adds to ELISA Plate, 100 holes μ L/, 4 DEG C were incubated for Night；Triclosan coating antigen is prepared according to the above-mentioned identical method of triclosan artificial antigen for preparing, only replacing carrier protein is OVA；
(2) wash: liquid in hole of inclining is washed one time with cleaning solution, and 280 holes μ L/ pat dry on blotting paper；
(3) it closes: 150 hole μ L/ of confining liquid is added, 37 DEG C of incubation 1h are directly patted dry after liquid in hole of inclining；
(4) it is loaded: a series of serum to be checked for being diluted to concentration, 100 holes μ L/, 37 DEG C of incubation 30min is added；
(5) wash: liquid in hole of inclining is washed 4 times with washing lotion, and 280 holes μ L/ pat dry on blotting paper；
(6) enzyme: HRP- sheep anti-mouse igg (1:5000 times dilutes) 100 hole μ L/, 37 DEG C of incubation 30min are added；Board-washing is same (5)；
(7) it develops the color: the TMB solution of fresh configuration is added, 100 holes μ L/ are protected from light 37 DEG C of colour developing 15min；
(8) it terminates: 2mol/L H is added2SO4, 50 holes μ L/；
(9) it measures: reading each hole OD450nm with microplate reader (dual wavelength, 630nm are with reference to optical filter wavelength).
Indirect competitive ELISA method is as follows:
(1) it is coated with: adding to ELISA Plate, 100 holes μ L/, 4 DEG C of overnight incubations after suitably being diluted coating antigen with coating buffer；
(2) wash: liquid in hole of inclining is washed one time with cleaning solution, and 280 holes μ L/ pat dry on blotting paper；
(3) it closes: 150 hole μ L/ of confining liquid is added, 37 DEG C of incubation 1h are directly patted dry after liquid in hole of inclining；
(4) it is loaded: the triclosan standard items of various concentration and the antiserum of corresponding dilution is added, 50 holes μ L/, 37 DEG C incubate Educate 30min；
(5) wash: liquid in hole of inclining is washed 4 times with washing lotion, and 280 holes μ L/ pat dry on blotting paper；
(6) enzyme: HRP- sheep anti-mouse igg (1:5000 times dilutes) 100 hole μ L/, 37 DEG C of incubation 30min are added；Board-washing is same (5)；
(7) it develops the color: the TMB solution of fresh configuration is added, 100 holes μ L/ are protected from light 37 DEG C of colour developing 15min；
(8) it terminates: 2mol/L H is added2SO4, 50 holes μ L/；
(9) it measures: each hole OD450nm (dual wavelength, 630nm are with reference to optical filter wavelength) is read with microplate reader, with OD value For ordinate, the logarithm of standard concentration is abscissa, draws standard curve with software Origin8.5, obtains IC50 (half suppression Concentration processed) value.According to the result of indirect ELISA and indirect competitive ELISA.It chooses and inhibits preferably, the high mouse of serum titer is used Make preparation monoclonal antibody.
3) recovery and culture of myeloma cell
Myeloma cell is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, after melting completely, by cell suspension It moves in the centrifuge tube of 15mL, is slowly added to cell culture fluid, the mixing of side edged, 1000rpm is centrifuged 4min.Liquid is discarded supernatant, Sedimentation cell is moved in cell bottle, DMEM complete culture solution is added, is placed in CO2It is cultivated in incubator, due to containing in frozen stock solution There is DMSO, still there are remnants in culture solution after recovery, liquid should be changed in time within second day after recovery.Myeloma cell after recovery is with containing There is the complete culture solution culture of 20% calf serum, when cell is in logarithmic growth phase, the good cell of form makes for fusion With.Cell is blown down with pipette from bottle wall when fusion, is collected in 50mL centrifuge tube, 1000rpm is centrifuged 4min, cannots be used up Full culture medium cleans cell 2 times, and it is spare to cannot be used up full culture medium resuspension.
4) preparation of splenic lymphocytes
Before cell fusion 3 days it is primary to positive mice booster immunization, be injected intraperitoneally 25 μ g antigens, adjuvant is not added, with sterilizing Normal saline dilution antigen to 0.8mL.Before fusion, mouse is plucked into eyeball bloodletting, separates serum, is saved as -20 DEG C, as Antibody positive control.
(1) positive mice cervical dislocation is put to death, is soaked in 75% alcohol, after about 3min, it is super to be put into immigration in plate Net platform；
(2) abdominal cavity is opened, sterile taking-up spleen is put into cleaning in the plate for filling the endless full nutrient solution of 10mL, removes spleen Fat and connective tissue around dirty；
(3) spleen is moved on 200 mesh filter screens in the plate that another fills the endless full nutrient solution of 10mL, spleen is cut It is broken, spleen is ground on strainer with autoclaved glass syringe inner core, then cannot be used up full nutrient solution flushing filtering net, until filter Only it is left white connective tissue on the net；
(4) splenocyte suspension is transferred in 50mL centrifuge tube, adds endless full nutrient solution to 35mL, mixes.1000rpm from Heart 10min abandons supernatant, cannots be used up full nutrient solution and cleans cell 1-2 times, further removes the connective tissue in cell suspension；
(5) cell is resuspended in the endless full nutrient solution of 10mL, and piping and druming mixes, spare after cell count.
5) cell fusion
(1) cell suspension of 2 × 107 myeloma cells, mixing are drawn containing 1 × 108 splenic lymphocytes and contained respectively In 50mL centrifuge tube, endless full nutrient solution is added to 35mL.1000rpm is centrifuged 6min, abandons supernatant.(2) plus not exclusively it cultivates Liquid makes cell suspend, and adds endless full nutrient solution to 35mL, 1000rpm and is centrifuged 6min, abandons supernatant.Centrifuge tube is inverted, with suction Water paper blots residual liquid.(3) flick centrifuge tube tube bottom, makes sedimentation cell in uniform loose's shape, is placed in 37 DEG C of water Preheating；(4) 50%PEG mono- for taking 37 DEG C dispensed to preheat is managed, left hand uniform rotation centrifuge tube, and the right hand holds the suction of 1mL pipette 50%PEG 0.8mL is taken, is slowly added into along tube wall, is added in 1min, 1.5min is stood；
(5) the endless full nutrient solution in 3min by 5mL preheating is added in fusion pipe, terminates reaction；(6) 800rpm is centrifuged 7min abandons supernatant, 5mLHAT culture solution is added, gently pressure-vaccum sedimentation cell, makes it suspend and mix, then adds HAT culture solution It is about 70mL to total volume；(7) cell suspension addition has been covered in 96 porocyte culture plates of feeder cells, 100 holes μ L/, Microscopy is set in 37 DEG C of cell incubators containing 5%CO2 and is cultivated；(8) cell growth status is observed and recorded daily, the 5th after fusion It changes liquid with HAT culture solution.
6) screening of hybridoma
Latter week is merged, antibody test is carried out to the culture supernatant in all clonal growth holes, is sieved first using indirect ELISA Select the positive cell that potency is high.The PBS to sterilize to the every Kong Zhongjia for being coated the ELISA Plate closed, 50 holes μ L/ add Hybridoma supernatant, 50 holes μ L/, is then operated according to the step of indirect ELISA, selects the cell of OD450nm > 1 Whether hole has specificity to triclosan using the antibody that indirect competitive ELISA detects the cell secretion of this some holes.
7) cloning of positive hybridoma cell
Carry out cell clone using limiting dilution assay, concrete operations are as follows: (1) the previous day preparation raising of cloning is thin Born of the same parents；(2) cell in positive hole to be cloned is suspended, is drawn in suspension to the aseptic bottle containing 1mLHT culture solution；(3) Cell suspension Trypan Blue counts；According to count results, it is diluted with HT culture medium；(4) tissue culture plate is placed in 37 DEG C 5%CO2Incubator culture 7-10 days, as the long 1/4-1/3 to hole floor space of clone cell, to its supernatant according to 6) Method and step is detected；(5) it takes positive colony to expand culture, freeze, while continuing to be cloned into positive rate according to the method described above 100%；(6) by positive monoclonal cell expansion culture to 24 orifice plates, expand after covering with to cell bottle, a part freezes；A part Continue to cultivate, for producing ascites, prepares monoclonal antibody.
8) hybridoma freezes:
(1) cell that preparation freezes is cannotd be used up full culture medium to wash twice, is then blown down from bottle wall, move into centrifuge tube In, 1000rpm is centrifuged 4min, discards supernatant；(2) according to cell concentration number, be added 1.5-2mL frozen stock solution suspension cell, so After be transferred in cryopreservation tube；(3) cryopreservation tube marked is placed in 4 DEG C of refrigerators after 0.5-1h, is moved into -80 refrigerators for 24 hours, It then moves into liquid nitrogen container and saves.
9) recovery of hybridoma:
(1) cell cryopreservation tube for preparing recovery is taken out from liquid nitrogen pipe, is put into rapidly in 37 DEG C of water-baths, is constantly stirred, It dissolves it in 1min, is transferred in centrifuge tube in super-clean bench, room temperature, 1000rpm is centrifuged 4min；(2) it discards supernatant, is added Complete culture solution, gently piping and druming makes cell suspend, and is transferred in Tissue Culture Flask；(3) it marks, cell bottle is placed in 37 DEG C, 5%CO2Constant incubator in cultivate, overnight after change liquid, continue to cultivate spare.
10) production of monoclonal antibody prepares monoclonal antibody using ascites method is induced in vivo:
(1) the Balb/c female mice for selecting health, 1-2 weeks before being inoculated with hybridoma, first to note in mouse peritoneal Penetrate sterile paraffin oil 0.5mL.It can be used in pretreated mouse 2-3 months；(2) by well-grown hybridoma, 1000rpm is centrifuged 4min, is resuspended, and adjustment cell density is 1 × 106-2× 106/mL, every mouse peritoneal injection 0.5mL is thin Born of the same parents' suspension；(3) after inoculating cell 10 days, it is seen that mouse web portion expands, and extracts ascites from abdominal cavity, raises mouse head, sterilizes abdomen Portion's skin is pierced into abdominal cavity with 5mL syringe, can generally extract 5-10mL every time.Hereafter it was taken every 1-3 days primary；(4) 3000rpm is centrifuged 10min, draws the faint yellow ascites in upper layer, packing, -20 DEG C spare.
11) ascites slightly the purifying of monoclonal antibody: is mentioned using octanoic acid-ammonium sulfate precipitation method；Specific step is as follows:
(1) ascites 5mL is taken, 10mL 0.06mol/L is added, the acetate buffer of pH 5.0 is adjusted with 0.1mol/LHCl PH to 4.8；(2) under the conditions of being stirred at room temperature, 160 μ L octanoic acids are gradually added into, 4 DEG C of standings 2h, 8000rpm are centrifuged 10min, and it is heavy to abandon It forms sediment；(3) 0.2mL 0.1mol/L PBS is added in supernatant, adjusts pH to 7.4 with 1mol/L NaOH；(4) it is added dropwise appropriate full Make solution in 45% saturation degree with ammonium sulfate, stands 1h, 4 DEG C of 8000rpm are centrifuged 30min, abandon supernatant；(5) precipitating is dissolved in 5mL's In PBS, dialyse 2 days in PBS.
The results show that the triclosan monoclonal antibody IC50 being prepared is 0.855ng/mL, the number of standard curve is drawn According to being shown in Table 1, suppression curve is shown in that attached drawing 4, Fig. 4 show that related coefficient is 0.8977, and inflection point number is 2.Illustrate that the present invention is prepared into The potency that the triclosan monoclonal antibody of mouse preparation is immunized in the triclosan artificial antigen arrived is higher, IC50 value is lower.
1 standard curve data of table
Embodiment 2The preparation of triclocarban monoclonal antibody (TCC mAb)
1, the preparation of triclocarban haptens (Formula II)
The isocyanic acid 3,4- Dichlorfop and 4- aminophenyl methyl butyrate for weighing the amount of equal substance are in 50mL round-bottomed flask In.10mL methylene chloride is added, is reacted under nitrogen protection.Chromatographic sheet monitoring reaction, after determining fully reacting, by reaction solution It is dry to be put into 40 DEG C of water-bath revolvings, after Weighed product, is dissolved with 7mL methanol, 1mol/L NaOH solution is added by the amount of 2 times of substances, It is heated at 40 DEG C, chromatographic sheet monitoring hydrolysis situation.After hydrolysis completely, pH to 2 is adjusted with the HCl of 6mol/L.By reaction solution It is extracted twice with 50mL ethyl acetate.Respectively by the water phase of extraction and organic phase TLC contact plate, determine in water phase without target product Afterwards, anhydrous sodium sulfate (anhydrous sodium sulfate is added by the amount of 10% organic phase volume) is added into the organic phase of collection, after addition Magnetic agitation 1h.Filtering, is spin-dried for, obtains triclocarban haptens.
LC-MS/MS and NMR is respectively adopted to identify the triclocarban haptens of synthesis, identified, triclocarban half Antigen is successfully prepared.LC-MS/MS qualification figure is shown in attached drawing 5, and 1H-NMR qualification figure is shown in attached drawing 6.
2, the preparation of triclocarban artificial antigen (formula IV)
With the coupling ratio of triclocarban haptens (Formula II) and carrier protein BSA for 6.8:1, trichlorine card shown in formula IV is carried out The preparation of class artificial antigen, the i.e. preparation of triclocarban immunogene.Specific step is as follows:
The triclocarban haptens (Formula II) of 60 carrier protein BSA equivalents is taken to be dissolved in 1mL n,N-Dimethylformamide, It is separately added into 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxyl of 180 carrier protein BSA equivalents Succinimide stirs 2h under room temperature, and reaction solution I is made；A certain amount of carrier protein BSA 50mg is taken to be completely dissolved in 5mL Reaction solution II is made in the PBS of pH7.4, reaction solution I is slowly added to dropwise in reaction solution II, and is stirred at room temperature for 24 hours.It will Reacting final product is packed into bag filter, is dialysed 3 days with the PBS buffer solution of 0.01mol/L, and it is not anti-to remove to change 3 dialyzates daily The small-molecule substance answered；It is centrifuged 10min with 10000rpm, is saved backup after collecting supernatant packing in -20 DEG C.Use MALDI- TOF is identified that qualification result is shown, triclocarban artificial antigen synthesizes successfully, after BSA and triclocarban hapten conjugation The MALDI-TOF qualification result of triclocarban artificial antigen is shown in attached drawing 7.
3, the preparation of triclocarban monoclonal antibody (TCC mAb)
TCC mAb is prepared according to the identical method of the above-mentioned preparation TCS mAb of height, difference is using triclocarban immunogene (formula IV) replaces triclosan immunogene (formula III).Triclocarban monoclonal antibody (TCC mAb) IC obtained50For 1.307ng/ ML, the data for drawing standard curve are shown in Table 2, and suppression curve is shown in attached drawing 8, wherein related coefficient 0.9751, inflection point number are 2.
2 standard curve data of table
Embodiment 3Detect the specificity of TCC mAb and TCS mAb and analyte (TCC and/or TCS)
Using the IC50 of enzyme-linked immunosorbent assay structure analogue compounds, TCC mAb and the TCS mAb of preparation are evaluated Cross reacting rate (CR).
1) experimental procedure
It is coated with buffer (13mM Na2CO3With 35mM NaHCO3, pH 9.6) and diluted coating antigen (every 100 μ L of hole), add Enter 96 orifice plates, 4 DEG C of overnight incubations.After cleaning buffer solution (0.01M PBS, pH 7.4,0.05% polysorbas20) cleans 3 times, 150 μ L Block buffer (0.01M phosphate buffer, pH 7.4 include 0.5%BSA, 0.05% polysorbas20) free work of closing Property ingredient 1h, 37 DEG C.Cleaning buffer solution cleans 3 times, then 37 DEG C of dry 1h.Sequentially add 50 μ LTCC, TCS or structure phase Antibody after being diluted like the standard items of object and 50 μ L with PBS is into hole, subsequent 37 DEG C of incubations 30min.Cleaning buffer solution cleans 5 times Later, 100 μ L goat anti-rabbit immunoglobulin peroxidase labels, subsequent 37 DEG C of incubations 30min are added in hole.Cleaning is slow Fliud flushing is cleaned 5 times, and 100 μ L tmb substrate solution, subsequent 37 DEG C of incubations 10min are added into hole.Finally, 50 μ L are added into hole Terminate liquid (2M H2SO4) enzymatic reaction is terminated, also, microwell plate microplate reader detects 450nm absorbance.
2) result
Cross reacting rate the results are shown in Table 3 and table 4, wherein and cross reacting rate (CR)=(antibody is to target compound IC50 of the IC50/ antibody to detection compound) × 100%.Table 3 is the obtained TCC mAb of the present invention to analyte TCC and its similar The cross reacting rate test result of object, table 4 are obtained cross reaction of the TCS mAb to analyte TCS and the like of the present invention Rate test result.
Cross reacting rate of the 3 TCC mAb of table to TCC and its structure homologue
Cross reacting rate of the 4 TCS mAb of table to TCC and its structure homologue
Embodiment 4The preparation of immune affinity column
1, prepared by matrix
(1) column material is mixed up and down, the Sepharose for taking 4mL NHS to activate with 1mL liquid-transfering gun (blue electron gun head mouth is slightly cut slightly) 4B is added in the empty reaction column tube of 6mL, and volume is 3mL bed volume after natural subsidence.(2) gravity column effect outflow saves The isopropanol of column material washes off the organic solvent in column bed with 10mL pre-cooling 1mM HCl.(3) add 2mL coupling buffer to column material In, liquid is siphoned away with syringe back suction rapidly.
2, triclosan and/or the coupling of triclocarban IgG monoclonal antibody and Sepharose 6B
(1) column tube bottom end is sealed, by 0.3mg triclosan monoclonal antibody, 0.3mg triclocarban monoclonal antibody or Mixed solution (the 0.2M NaHCO of 0.3mg triclosan monoclonal antibody, 0.3mg triclocarban monoclonal antibody3In buffer) It is added separately in the column material matrix prepared, covers upper cover, mix column material and solution up and down.(note: bed volume is molten with antibody The optimum volume ratio of liquid is 1:1.5)；
(2) above-mentioned mixture reaction 2h is mixed well under room temperature condition (20~25 DEG C) by the way of turning upside down；
(3) after making column liquid in pipe natural subsidence, efflux is collected, whether there is or not IgG in OD280 survey with ultraviolet specrophotometer Flow out column tube.If OD280 is without peak, it is believed that antibody is all coupled on column material, if OD280 has peak, according to concentration conversion column material The amount of antibody of upper coupling；
(4) after coupling buffer 2mL washes column material to dripless outflow, add about 1.5 times of bed volumes of Block buffer, room temperature Above-mentioned mixture reaction 2h is mixed well under condition (20~25 DEG C) by the way of turning upside down；
(5) after reaction until trickle to dripless outflow.It is washed with 3mL 0.1M Tris-HCl pH 8.0, 3mL 0.1mol/L acetic acid/sodium acetate (NaCl containing 0.5mol/L) is changed again, and pH 4.0 is washed.So soda acid of recycling is washed After column, washed with 5mL 20mM PBS；
(6) plus containing 0.05%NaN3PBS buffer solution in 4 DEG C of preservation column material, it is spare.
3, column is filled
It is resuspended again with 10mL PBS after Sepharose 6B after crosslinking is washed with 10mL 20mM PBS pH 7.4, dress Enter in empty affinity purification column cylinder.
4, column capacity is measured
It is determined by the way that the solution of excessive TCC (800ng) and TCS (800ng) are added in methanol/water (1:9, v/v). 5, measure 0.3mg triclosan monoclonal antibody, 0.3mg triclocarban monoclonal antibody or 0.3mg triclosan monoclonal antibody, 3 kinds of fixed forms of mixed solution of 0.3mg triclocarban monoclonal antibody to the adsorption capacity of column and the influence situation of Conjugate ratio, Wherein bisphenol A monoclonal antibody conduct control, Conjugate ratio=(total amount-outflow mAb amount that mAb is added)/plus the total amount of mAb × 100%, it the results are shown in Table 5.
5 fixed form of table is to the adsorption capacity of column and the influence of Conjugate ratio
As shown in Table 5, the Conjugate ratio of immune affinity column is approximate in three kinds of fixed forms, average out to 98.3%, and three kinds solid It is essentially identical to the adsorption capacity of TCC and TCS to determine TCC, TCS monoclonal antibody in mode.Using same in subsequent embodiment detection When be coupled TCC and TCS monoclonal antibody immune affinity column.
Embodiment 5To the trace detection of TCC in sample and/or TCS
One, sample pre-treatments
1, solid sample (vegetables, egg, chicken) pre-treatment
(1) weigh 1g sample it is homogeneous after mixed with 5mL acetonitrile；(2) 30s, ultrasonic 20min, 10000rpm centrifugation are vibrated Supernatant is taken after 10min；(3) 5mL acetonitrile repeats to extract primary, combined extract；(4) 40 DEG C of nitrogen are blown to volume less than 1mL, are added 10mL PBS is mixed.
2, fluid sample (beverage, beer) pre-treatment
(1) sample ultrasonic is deaerated 30min；(2) taking 10mL sample and PBS, 1:1 dilutes by volume；(3) 1M NaOH is used Solution adjusts pH to 9.
Two, detecting step
1, immune affinity column purification condition selects
1) select 15% methanol as sample solution
Preventing antibody activity to be destroyed selects methanol/PBS as sample solution, to determine that methanol is best dense in sample solution Degree adds the TCC and TCS of the PBS solution (10mL) containing 0%, 5%, 10%, 15%, 20% (volume content) methanol and 5ng It is downloaded in immune affinity column, the results are shown in Figure 10.When methanol content is 15vol%, the rate of recovery does not increase further, table Bright methanol content appropriate can reduce non-specific adsorption, therefore select the PBS solution of 15vol% methanol content as load liquid Carry out follow-up test.
2) select pure methanol as eluent
Suitable elution requirement helps to separate the interaction of analyte and antibody, by analyte return flowing phase.First The aqueous solution of alcohol and methanol is commonly used in immune affinity column eluent.The present embodiment compared the concentration pair of methanol in eluent The influence of TCC, TCS rate of recovery, it is determined that the optium concentration of methanol in eluent.As a result as shown in figure 11, with methanol concentration Increase, the TCC rate of recovery is increased to 75.6%, the TCS rate of recovery from 58.5% and is increased to 106.9% from 53.2%.Therefore it selects Pure methanol carries out follow-up test as eluent.And to ensure complete elution analysis object, verifying 1-5mL effluent volume is to returning The influence (referring to Figure 12) of yield, it is final to determine 3mL as best effluent volume.
2, immune affinity column purifying step
(1) it installs: taking out immune affinity column prepared by embodiment 2, restore to room temperature, top plug is taken out and is cut, Again it covers, is connect with the syringe on pump stream crosshead, open lower end plug.(2) it activates: successively by 10mL water, 10mL PBS flows through immune affinity column, and 2-3 drops/sec of flow velocity.(3) it loading: by the good sample liquid flow of pre-treatment through immune affinity column, keeps 1-2 drops/sec of flow velocity.(4) it elutes: successively affinity column being eluted with 10mL PBS and 10mL water, is blotted in column as far as possible Residual liquid.(5) it elutes: the elution of 3mL hplc grade methanol is added, 1-2 drops/sec, is collected in teat glass.
Three, testing result compares
By purifying (affinity column prepared by embodiment 4) or unpurified blank food samples in immunoaffinity chromatography It is middle that a series of analyte normal concentration is added, to make matrix to the minimum interference of testing result.Directly use UPLC-MS/ MS measurement analyte standard items (wherein, the chromatogram of 5ng/mL triclosan and triclocarban hybrid standard product is shown in Fig. 9, Fig. 9 a and 9b is respectively the chromatogram of TCC standard items ion pair 313 > 160 and ion pair 313 > 126, and Fig. 9 c and 9d are respectively TCS standard items The chromatogram of ion pair 288.8 > 35 and ion pair 286.8 > 35), establish standard curve.The method for detecting TCC in different samples Verification result is as shown in table 6, and detecting the method validation of TCS in different samples, the results are shown in Table 7.WhereinaBeer and beverages list Position is μ g/L,bWith the sample of affine in immunity column purification,cNot purified sample.Related coefficient (the R of standard curve2) be all larger than 0.99, it is linear good.
Table 6 detects the method validation result of TCC in different samples
Table 7 detects the method validation result of TCS in different samples
" ME " is Matrix effect, i.e. matrix effect, refers to the co-elute remnants matrix component of mixing sample to mesh Mark the influence of analyte ionization.Since strong ME may cause analyte sensitivity decline/increase, so as to cause UPLC-MS/MS points Analyse the inaccurate and inaccurate of result.Therefore, the influence of matrix effect should be reduced, as far as possible to ensure the accurate analysis of analyte. The common method for overcoming ME is using effective sample purification method and direct dilution method.However, direct dilution method can the side of loss The sensitivity of method.Therefore, selecting effective purification method is that reduction ME absolute value is essential.By comparing matrix matching and The slope of solvent standard curve evaluates ME.Wherein ME=[(matrix matching slope of standard curve/solvent slope of standard curve)- 1] × 100%.If value is positive, for matrix enhancement effect, signal enhancing is detected；If value is negative, for substrate inhibition effect It answers, detection signal is suppressed.If ME absolute value in 20% range, can ignore ME.Therefore, the purification of immune affinity column Effect can be evaluated by calculating the absolute value of ME.
As shown in table 6,7, through the purified ME absolute value of immunoaffinity chromatography in negligible range.On the contrary, not having There is the ME absolute value of any purifying step to show very strong substrate inhibition effect.In conclusion demonstrating immunoaffinity chromatography The advantage in detection TCC and TCS is purified, matrix matching calibration had not both been needed, and had not also needed using in expensive stable isotope Mark.
TCC's that the LOD and LOQ of detection method can be detected when being respectively signal-to-noise ratio 3:1 and 10:1 and TCS is minimum dense Degree.Table 6,7 shows that the LOD value of TCC is 0.3ng/L in drink sample, is 3-7ng/kg in food.TCS in drink sample LOD is 0.01 μ g/L, is 0.06-0.1 μ g/kg in food.The LOQ value of TCC is 1ng/L in drink sample, is 0.01- in food 0.02μg/kg.The LOQ value of TCS is 0.03 μ g/L in drink sample, is 0.2-0.3 μ g/kg in food.
By the data of table 5-7 it has been confirmed that TCC haptens provided by the invention (Formulas I) and TCS haptens (Formula II) obtain The monoclonal antibody (TCC mAb and TCS mAb) arrived is in immunoaffinity chromatography detection, TCC and TCS and its corresponding monoclonal The combination of antibody is specific.Experiment is repeated five times, the accuracy for evaluating detection method of the present invention (passes through the rate of recovery Indicate) and accuracy (being indicated by RSD).The results show that the average recovery rate of TCC and TCS be respectively 70.1-92.8% and 76.6-102.5%, RSD are lower than 14.5%, show that the accuracy of this method is good, have very high reproducibility.
Four, it is compared with other purification methods
The purification that blank drink sample is carried out using three kinds of common solid phase extraction adsorbents C18, GCB and HLB, by adding Add certain density TCC (0.5ng/mL) and TCS (5ng/mL) correct mixture, compares the purification of they and immunoaffinity chromatography Effect, wherein the operating condition of each solid phase extraction adsorbents is shown in Table 8.The results of comparison of purifying property is shown in Figure 14, drink sample point Not Yong C18, GCB and HLB purification when, the signal-to-noise ratio (signal to noise ratio, S/N) of TCC is respectively 391,458 and 412, TCS S/N is respectively 335,663 and 595.For sample after IAC is purified, the S/N of TCC and TCS are respectively 700 and 1033, Compared with its excess-three kind purification method, S/N is significantly raised, and the sensitivity of method is made to get a promotion.In addition, with IAC to sample into Row purification, before target compound chromatographic peak, in 1.51min, GCB purification in 1.83min when not occurring C18 purification Impurity Interference Peaks with occurring when HLB purification in 1.69min, illustrate that IAC can effectively remove the impurity in sample, to obtain More good clean-up effect.Illustrate that affine in immunity column chromatography method (IAC) provided by the invention can remove in food samples Impurity reduces the baseline noise of LC-MS/MS detection, the signal-to-noise ratio of improvement method.
Table 8 uses the operating condition of C18, GCB, HLB Solid Phase Extraction column purification
Embodiment 6The detection of triclosan and triclocarban in food
The purification of sample is carried out according to the optimum condition of sample-pretreating method described in embodiment 5 and immune affinity column, Purified eluted product is detected with ultra performance liquid chromatography tandem mass spectrum.
1, the filter used before UPLC-MS/MS detection is determined
Some solid granules in sample enter blocking pipeline or chromatographic column in UPLC-MS/MS system in order to prevent, lead to It often needs to filter sample with syringe filter disk before UPLC-MS/MS sample introduction.Select two different syringe filter disks into Mixed standard solution (5ng/mL) mistake of row test, nylon (0.22 μm) and glass fibre (0.22 μm) filter to TCC and TCS After filter, filtrate is collected, is analyzed with UPLC-MS/MS, wherein the ratio (retention rate) of retention analysis object in filtrate=(filtered Mixed standard solution peak area/mixed standard solution peak area) × 100%.The result is shown in Figure 13, Figure 13, which is shown, uses nylon filter When device, the loss of TCC is about 36.7%, TCS substantially lossless.After being filtered using glass fibre, do not have on the glass fibers Observe target analytes, and the loss of TCC and TCS can be ignored substantially, thus have finally chosen glass fiber filter into After row filtering, then carry out UPLC-MS/MS analysis.
Therefore, according to above-mentioned experiment, it is 15-20% that the operating condition for confirming preferred immune affinity column chromatography, which is sample solution, The PBS solution of methanol content；And/or eluent is pure methanol；And/or when sample is solid, the volume of eluent and sample Mass ratio is 3-5:1 (mL/g)；When sample is liquid, the volume ratio of eluent and sample is 3-5:10；And/or eluent makes It is filtered with glass fiber filter.
2, ultra performance liquid chromatography and mass spectrographic testing conditions are as follows:
(1) ultra performance liquid chromatography (UPLC) condition
Chromatographic column: Waters ACQUITY UPLCTMBEH C18 column (100mm × 2.1mm, 1.7 μm).Mobile phase: A, first Alcohol；B, water.Eluent gradient elution program: 9 are shown in Table.Column temperature: 40 DEG C.Sample room temperature: 4 DEG C.Sample volume: 10 μ L.
9 triclosan of table and triclocarban liquid chromatogram gradient elution program
(2) mass spectrum (MS) reference conditions:
Ionization source: ESI (-).Capillary voltage: 2.5kV.Ion source temperature: 150 DEG C.Desolvation temperature: 400 DEG C.It is de- Solvent stream amount: 900L/h.Triclosan and triclocarban qualitative and quantitative ion are shown in Table 10 to, collision energy.
The mass spectrometry parameters of 10 target compound of table
Note: * quota ion
3, result
The volume containing the sample and the rate of recovery of triclosan and triclocarban are investigated, using the optimal conditions of complex immunity affinity column, knot Fruit shows that the maximum adsorption capacity of triclosan and triclocarban is respectively 332ng and 128ng.
1) vegetables, in drink sample TCC and TCS detection
The rate of recovery of triclosan and triclocarban in vegetables and beverage is shown in Table 11.
The rate of recovery and relative standard deviation of table 11 TCC and TCS in vegetables and beverage
2) detection of TCC and TCS is shown in Table 12 in chicken egg sample.
The concentration of TCC and TCS in 12 actual sample of table
Testing result in table 12 by dispersive solid-phase extraction-high performance liquid chromatography detection identification (specific method referring to " point Dissipate triclosan and triclocarban in Solid Phase Extraction-high Performance Liquid Chromatography fruit ", Wang Li etc., " food safety quality testing Journal ", 2015 (10): 4205-4211.), as a result with it is almost the same in table 12.Detected in chicken and egg sample TCC and TCS, wherein the concentration range that TCC is detected is from 0.08-0.1 μ g/kg, and the concentration range that TCS is detected is from lower than LOQ to 6.7 μg/kg.TCC and TCS in egg and chicken may be to be contained by contaminated water or feed, and biological accumulation causes.Another party Face, it is also possible to which food surface institute is contaminated to cause because being cleaned using sponge or food wet tissue.It is edible to contain TCC and TCS Chicken derived food to human health constitute potential threat.This also reflects the important of TCC and TCS detection accuracy from side Property.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this A little simple variants all belong to the scope of protection of the present invention.

Claims (10)

1. a kind of triclosan monoclonal antibody and/or triclocarban monoclonal antibody are in detection triclosan and/or triclocarban Purposes, which is characterized in that the triclosan monoclonal antibody is prepared by triclosan haptens shown in Formulas I, institute Stating triclocarban monoclonal antibody is prepared by triclocarban haptens shown in Formula II,
2. purposes as described in claim 1, which is characterized in that prepare trichlorine stranger used in the triclosan monoclonal antibody Work antigen is that triclosan haptens shown in Formulas I and carrier protein are combined and obtained as shown in formula III；Prepare the triclocarban list Triclocarban artificial antigen used in clonal antibody is that triclosan haptens shown in Formula II and carrier protein combine as shown in formula IV It obtains,
3. purposes as claimed in claim 1 or 2, which is characterized in that the method for the detection includes immunoassay method, biology Sensor, Surface enhanced Raman scattering；The immunoassay method includes immunoaffinity chromatography, ELISA, colloid Golden immunochromatography, chemiluminescence immune assay or suspension array.
4. purposes according to claim 3, which is characterized in that the method for the detection further includes and ultra high efficiency liquid phase color Spectrum, mass spectrum or ultra performance liquid chromatography and mass spectrometry.
5. a kind of triclosan hapten compound, structure are shown in formula I:
6. a kind of triclosan artificial antigen, structure is as shown in formula III, wherein the coupling ratio of triclosan haptens and carrier protein For 0.5-1:1, preferably 0.7:1.
7. it is a kind of detection triclosan and/or triclocarban method, comprising the following steps: using triclosan monoclonal antibody and/ Or triclocarban monoclonal antibody prepares immune affinity column, is selected using immune affinity column triclosan and/or triclocarban After selecting property is combined and is enriched with, then detect triclosan and/or triclocarban, which is characterized in that prepare the triclosan monoclonal Triclosan haptens used in antibody is shown in formula I, prepares triclocarban used in the triclocarban monoclonal antibody half Antigen as shown in Formula II,
8. it is a kind of detection triclosan and/or triclocarban method, comprising the following steps: using triclosan monoclonal antibody and/ Or triclocarban monoclonal antibody prepares immune affinity column, is selected using immune affinity column triclosan and/or triclocarban After the enrichment of selecting property and purification, then detect triclosan and/or triclocarban, which is characterized in that the triclosan monoclonal antibody Triclosan artificial antigen used prepares triclocarban people used in the triclocarban monoclonal antibody as shown in formula III Work antigen as shown in formula IV,
9. method as claimed in claim 7 or 8, which comprises the following steps:
1) sample is splined on immune affinity column, elution；
2) triclosan and/or triclocarban in eluted product are detected.
10. method as claimed in claim 9, which is characterized in that the filler mesostroma and Dan Ke of immune affinity column in step 1) The volume ratio of grand antibody is 1:1~1:1.5；And/or
The method that detection triclosan and/or triclocarban use ultra performance liquid chromatography and mass spectrometry in step 2)；
The sample is selected from food, body fluid, sewage, weaving or daily chemical product；And/or the detection includes sample pre-treatments, The solid sample pre-treatment includes mixing solid sample with acetonitrile, being ultrasonic, and centrifuging and taking supernatant, nitrogen is blown；The liquid Sample pre-treatments include by fluid sample ultrasonic degassing；It is 15- that the operating condition of preferred immune affinity column chromatography, which is sample solution, The PBS solution of 20vol% methanol content；And/or eluent is pure methanol；And/or when sample is solid, eluent and sample Volume mass ratio be 3-5:1 (mL/g)；When sample is liquid, the volume ratio of eluent and sample is 3-5:10；And/or it washes De- liquid is filtered using glass fiber filter.
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