CN105067805B - Irgacure 907 detection kit and preparation and application thereof - Google Patents

Irgacure 907 detection kit and preparation and application thereof Download PDF

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CN105067805B
CN105067805B CN201510452064.XA CN201510452064A CN105067805B CN 105067805 B CN105067805 B CN 105067805B CN 201510452064 A CN201510452064 A CN 201510452064A CN 105067805 B CN105067805 B CN 105067805B
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irgacure
specific antigen
concentration
mouse
sulfydryl
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CN105067805A (en
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唐穗平
刘付建
王娜
陈纪文
陈满英
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Guangdong Testing Institute of Product Quality Supervision
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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Abstract

The present invention relates to a kind of Irgacure 907 detection kit and preparation and application thereof, belong to additive detection technique field.This kit includes the ELISA Plate that each micropore endoperidium concentration is 0.5 3.0 μ g/mL Irgacure 907 specific antigens, concentration is the Irgacure 907 specific antibody working solution of 0.5 3.0 μ g/mL, and the consumption of described Irgacure 907 specific antigen and Irgacure 907 specific antibody is 1:1.The kit of the present invention can quickly detect the Irgacure 907 content in papery packing articles, food liquid analogies, dry food analogies and beverage, there is specific height, result is accurate and pre-treatment simple, and instrument and equipment is required the feature low, testing cost is low.

Description

Irgacure 907 detection kit and preparation and application thereof
Technical field
The present invention relates to a kind of additive detection technique, particularly relate to a kind of Irgacure 907 detection kit and Preparation and application.
Background technology
Irgacure 907, formal name used at school is 2-methyl isophthalic acid-(4-methyl mercapto) phenyl 2-morpholinyl-1-acetone (2-Methyl- 4'-(methylthio)-2-morpholinopropiophenone, No. cas is 71868-10-5), it is a kind of light trigger (Photoinitiators, PIs).
Along with popularizing of UV typography, PIs is widely used in papery bag as the main component of UV curable ink Dress printing, contributes to causing or be catalyzed corresponding polymerisation, improves the solidification drying efficiency of UV printing ink.But there is research table Bright, after UV printing-ink has solidified, the PIs wherein remained can occur chemical transport under certain condition, it is possible to through bag Package material or the food in being packed by surface contact stain, thus the health of human body is caused potentially hazardous.Therefore, auspicious The country such as scholar, European Union and tissue have promulgated that regulation and permission log are to limit the use of PIs in food contact material, wherein in succession The Special migration (SML) of regulation Irgacure 907 is 0.01mg/kg.
Therefore, in food paper packing material, food simulants and paper wrapper food, the detection research of PIs has become research Focus, but current detection method is based on chromatographic mass spectrometry instrumental method, these methods have the longest, be difficult to scene The defect of detection, so needing a kind of method the most quickly detecting Irgacure 907 badly.
Summary of the invention
Based on this, it is necessary to for the problems referred to above, it is provided that a kind of Irgacure 907 detection kit, this detection kit Based on enzyme linked immunoassay, can quickly detect the content of Irgacure 907.
A kind of Irgacure 907 detection kit, specifically includes that
1) ELISA Plate of Irgacure 907 specific antigen it is coated: Irgacure in each micropore of described ELISA Plate The concentration of 907 specific antigens is 0.5-3.0 μ g/mL；
2) Irgacure 907 specific antibody working solution: the concentration of this Irgacure 907 specific antibody is 0.5- 3.0μg/mL；
Described Irgacure 907 specific antigen is 1:1 with the amount ratio of Irgacure 907 specific antibody.
The Cleaning Principle of above-mentioned Irgacure 907 detection kit is: be adsorbed in by Irgacure 907 specific antigen On solid phase carrier ELISA Plate, add testing sample detection liquid or Irgacure 907 standard liquid and Irgacure 907 is special Property antibody working solution, makes coated Irgacure 907 on the Irgacure 907 in testing sample and solid phase carrier specifically resist Former competition binding Irgacure 907 specific antibody, after hatching, adds ELIAS secondary antibody and carries out the amplification of enzymatic activity, survey after colour developing The absorbance of random sample product, this value is negative correlation with the amount of Irgacure 907 in sample, compares with calibration curve and can draw Irgacure 907 concentration range.Thus this kit can quickly detect the content of Irgacure 907.
Wherein in an embodiment, this kit also includes ELIAS secondary antibody, described ELIAS secondary antibody be concentration by 1: The sheep anti mouse two of the horseradish peroxidase-labeled of the dilution proportion of 8000 resists.Above-mentioned ELIAS secondary antibody has optimal enzymatic activity and puts Big effect.
Wherein in an embodiment, described Irgacure 907 specific antigen is Irgacure 907 and carrier protein Conjugate, the concentration of this Irgacure 907 specific antigen is 2 μ g/mL；Described Irgacure 907 specific antibody is Mouse resource monoclonal antibody, the concentration of this Irgacure 907 specific antibody is 2 μ g/mL.Select the anti-of mentioned kind and concentration Original antibody, has optimum response ratio, and enzyme linked immunoassay is the most thorough, and the immune complex precipitation of formation is most, maximum.
Wherein in an embodiment, this kit also includes Irgacure 907 standard solution, substrate developer, washes Wash liquid, stop buffer, confining liquid and concentrating sample dilution.Testing staff is made to have when using this kit the most excellent Point.
Wherein in an embodiment, described carrier protein is albumin rabbit serum, bovine serum albumin(BSA), the white egg of egg white In vain, the one in thyroglobulin；The concentration of described Irgacure 907 standard solution is respectively 1 × 103μg/L、1× 102μg/L、1×101μg/L、1×100μg/L、0.1μg/L；Described substrate developer is made up of developer A and developer B, institute Stating developer A is hydrogen peroxide or urea peroxide, and described developer B is o-phenylenediamine or tetramethyl benzidine；Described cleaning solution For the 0.01M containing 0.05%-0.5% Tween-20, the phosphate buffer of pH7.5；Described stop buffer is the sulphur of 1-2mol/L Acid solution；Described confining liquid is the 0.01M containing 5% skimmed milk power, the phosphate buffer of pH7.5；Described concentrating sample dilutes Liquid is the phosphate buffer of 0.01M, pH7.5；The material of described ELISA Plate is in polystyrene, polyethylene, polypropylene Kind.
The invention also discloses the preparation method of a kind of above-mentioned Irgacure 907 detection kit, mainly include following Step:
1) Irgacure 907 specific antigen is prepared:
Being dissolved in dichloromethane by Irgacure 907, temperature of reaction system controls at-10 DEG C to-1 DEG C, drips tribromo Change and recover after boron dichloromethane to room temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtain the 2-methyl with phenol sulfydryl- 4-sulfydryl-2-morpholine propiophenone；Above-mentioned 2-methyl-4-sulfydryl-2-morpholine propiophenone and potassium carbonate are scattered in dimethyl formyl In amine, add 4-bromobutyrate, be heated to reflux, make the phenol sulfydryl of 2-methyl-4-sulfydryl-2-morpholine propiophenone by ethyl butyrate Replaced, obtained intermediate product；Subsequently this intermediate product is added in trifluoroacetic acid, back flow reaction, slough the protection of ethyl, naked Exposing carboxyl, preparing can be with the haptens of the Irgacure 907 of protein molecule；Again by its with N-hydroxy-succinamide and React after carbodiimide mixing, prepare Irgacure 907 specific antigen with carrier protein couplet；Described carrier protein For the one in albumin rabbit serum, bovine serum albumin(BSA), oralbumin, thyroglobulin；
2) coated elisa plate:
Above-mentioned Irgacure 907 specific antigen is coated in ELISA Plate, in each micropore of described ELISA Plate The concentration of Irgacure 907 specific antigen is 0.5-3.0 μ g/mL；
3) Irgacure 907 specific antibody is prepared:
Using step 1) in Irgacure 907 specific antigen that obtains as immunogene, mouse is carried out immunity；Immunity After, take the mouse that serum titer is high, take its splenocyte and carry out cell fusion with myeloma cell SP2/0；Use limiting dilution assay Screening hybridoma, obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain；And take not exclusively Adjuvant lumbar injection carries out the mouse after sensitization, is expelled in mouse peritoneal by hybridoma suspension, collects ascites, through pungent Acid-ammonium sulfate precipitation method carries out ascites purifying, obtains Irgacure 907 monoclonal antibody purified；Preparation concentration is 0.5- The Irgacure 907 monoclonal antibody working solution of 3.0 μ g/mL.
Use Irgacure 907 specific antigen and specific antibody that said method prepares, have the best excellent Point.
Wherein in an embodiment, step 1) in, prepare Irgacure 907 specific antigen method particularly includes:
Being dissolved in 20mL anhydrous methylene chloride by the Irgacure 907 of 1mmol, temperature of reaction system controls at-5 DEG C, Being slowly added dropwise 10mL Boron tribromide dichloromethane solution, recover to room temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtains 2-methyl-4-sulfydryl-2-morpholine propiophenone with phenol sulfydryl；By 0.8mmol2-methyl-4-sulfydryl-2-morpholine propiophenone and Potassium carbonate is scattered in anhydrous dimethyl formamide, adds 1.52g 4-bromobutyrate, be heated to reflux 3h, make under stirring at normal temperature The phenol sulfydryl of 2-methyl-4-sulfydryl-2-morpholine propiophenone is replaced by ethyl butyrate, obtains intermediate product；Subsequently is produced from centre Thing adds in excess trifluoroacetic acid, return stirring reaction 1h, sloughs the protection of ethyl, exposes carboxyl, and preparing can be with albumen The haptens of the Irgacure 907 of matter coupling；React after again it being mixed with N-hydroxy-succinamide and carbodiimide, with ox Seralbumin coupling prepares Irgacure 907 specific antigen.
Wherein in an embodiment, step 3) in, prepare Irgacure 907 specific antibody method particularly includes:
Using step 1) in Irgacure 907 specific antigen that obtains as the immunogene Balb/c mouse to 7-8 week old Carrying out immunity, first immunisation uses complete Freund's adjuvant and Irgacure 907 specific antigen emulsifying soln, and antigen concentration is 1mg/mL, dosage is 200 μ g/, and booster immunization uses incomplete Freund's adjuvant specific with Irgacure 907 the most every time Antigenic solution emulsifies, the same initial immunity of dosage；After initial immunity two weeks, at interval of 1 week booster immunization once, immunity 5-7 time altogether, When antibody titer no longer raises, carry out last immunity；It is not added with immunologic adjuvant for the last time directly with Irgacure 907 Specific antigen aqueous solution lumbar injection, the same initial immunity of dosage；After afterbody takes blood examination survey serum titer, take serum titer high Mouse, aseptically, takes its splenocyte and carries out cell fusion in the ratio of 10:1 with myeloma cell SP2/0；Employing has Limit dilution method screening hybridoma, obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain；And Using Freund's incomplete adjuvant lumbar injection Balb/C mouse to carry out sensitization before Yu Yizhou, dosage is 0.5mL/；By cell concentration it is The hybridoma suspension of 1.2 ten thousand/mL is expelled in mouse peritoneal, and dosage is 0.5mL/；Inoculation hybridoma 7-10 After it, collect ascites, repeatedly collect for several times；It is stored in 4 DEG C of Refrigerator stores；Ascites purifying is carried out through octanoic acid-ammonium sulfate precipitation method, To Irgacure 907 monoclonal antibody purified.
The invention also discloses the using method of a kind of above-mentioned Irgacure 907 detection kit, mainly include following Step:
1) sample pre-treatments
Accurately measure testing sample, be quantitatively adding organic solvent, quantitatively draw after extraction or extraction supernatant, add after drying up Enter organic solvent miscible with water to redissolve, and dilute constant volume with water, obtain sample detection liquid；
2) detection
Use above-mentioned kit to detect, add in the ELISA Plate being coated with Irgacure 907 specific antigen Standard items or sample detection liquid, add Irgacure 907 specific antibody working solution, washes plate after incubation, adds ELIAS secondary antibody Carry out the amplification of enzymatic activity, again wash plate, add substrate developer, stop buffer, then measure absorbance (OD) value with ELIASA；
3) interpretation of result
With inhibiting rate I% as ordinate, with the logarithm lg [Irgacure 907] of Irgacure 907 concentration as abscissa, Draw Irgacure 907 Competitive assays curve；The inhibiting rate of sample is substituted into calibration curve regression equation, from calibration curve Reading the concentration corresponding to sample, the extension rate being multiplied by its correspondence is the actual content of Irgacure 907 in sample；
Described inhibiting rate computing formula is as follows:
I = B - B N B 0 - B N × 100 %
Wherein: I inhibiting rate
The mean absorbance values of B sample solution
B0The mean absorbance values of 0 μ g/L standard liquid
BNReference blank comparison mean absorbance values.
In above-mentioned using method, by sample pre-treatments, the Irgacure 907 in determinand preferably can be carried Take out, and be enriched with as required, then by the integrated enzyme reaction kit measurement concentration of high sensitivity high specific, have Detection limit is low, the advantage that accuracy is high.
Above-mentioned steps 1) in sample pre-treatments, for different types of testing sample, pre-treating method has following difference:
For papery packing articles, accurately cut testing sample, be quantitatively adding organic solvent, and sample is completely soaked in In this organic solvent, quantitative Aspirate supernatant after extraction, add organic solvent after drying up and redissolve, and dilute constant volume with water, obtain Sample detection liquid；
For food liquid, accurately weigh testing sample, be quantitatively adding organic solvent, mixing, add NaCl, inhale after extraction Take supernatant, add organic solvent after drying up and redissolve, and dilute constant volume with water, obtain sample detection liquid；
For dry food, accurately weigh testing sample, be quantitatively adding organic solvent, mixing, quantitatively draw after extraction Clear liquid, adds organic solvent and redissolves, and dilute constant volume with water, obtain sample detection liquid after drying up.
Wherein in an embodiment, step 1) in, described organic solvent is acetonitrile；It is high and easy that acetonitrile has extraction efficiency Volatilize tractable feature.
Step 2) in, detection method particularly includes: be first coated 96 with Irgacure 907 specific antigen of 2 μ g/mL Hole ELISA Plate, 100 μ L/ holes, it is positioned over 4 DEG C of refrigerators and is coated overnight, wash with cleaning solution after closing with 250 μ L/ hole confining liquids, and Pat dry；Being subsequently adding Irgacure 907 standard solution or sample detection liquid 50 μ L, adding concentration is 2 μ g/mL Irgacure 907 specific antibody working solution 50 μ L, mixes, 37 DEG C of incubation 1h；Washing subsequently pats dry, and adds with dilution Liquid presses the anti-working solution of sheep anti mouse two of the horseradish peroxidase-labeled of 1:8000 dilution proportion, 100 μ L/ holes, 37 DEG C of incubation 1h； Finally washing pats dry, and every hole adds 50 μ L developer B, 10 μ L developer A, and develop the color 15min, and adding concentration is the sulfuric acid of 2mol/L Solution, 50 μ L/ holes, terminate reaction, setting ELIASA, at 450nm, measures absorbance.
Compared with prior art, the method have the advantages that
A kind of Irgacure 907 detection kit of the present invention, utilizes competitive enzyme-linked immune determination method to detect Irgacure 907, there is specific height, result is accurate, and instrument and equipment is required the feature low, testing cost is low.
Meanwhile, the reagent in this kit is to facilitate the working solution form of use to provide, simple to operate, quick, for using Person saves the time and reduces the error caused because of step complexity.It is suitable for detecting in batch samples quickly and accurately Irgacure 907。
The preparation method of Irgacure 907 detection kit of the present invention, is prepared by specific method Irgacure907 specific antigen and specific antibody, make the kit finally given have high specific, highly sensitive excellent Point.
The using method of Irgacure 907 detection kit of the present invention, has sample pre-treatments easy, and detection limit is low, The advantage that accuracy is high.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail, but present disclosure does not cause any limit System.
Embodiment 1
A kind of Irgacure 907 detection kit, mainly comprises composition as follows:
1) Irgacure 907 specific antigen working solution.
Prepare by the following method: the Irgacure 907 of 0.279g (i.e. 1mmol) is dissolved in the anhydrous dichloromethane of 20mL In alkane, temperature of reaction system controls at-5 DEG C, is slowly added dropwise 10mL Boron tribromide dichloromethane solution, then slowly recovers to room Temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtains solid, is the 2-methyl-4-sulfydryl-2-morpholine with phenol sulfydryl Propiophenone.
0.236g (i.e. 0.8mmol) 2-methyl-4-sulfydryl-2-morpholine propiophenone and potassium carbonate are scattered in anhydrous dimethyl base In formamide (DMF), stirring at normal temperature, add 1.52g 4-bromobutyrate, be heated to reflux 3h, 2-methyl-4-sulfydryl-2-morpholine The phenol sulfydryl of propiophenone is replaced by ethyl butyrate, obtains intermediate product.
Subsequently intermediate product is added in the trifluoroacetic acid of 30 times, return stirring 1h, sloughs the protection of ethyl, exposes carboxylic Base, preparing can be with the haptens of the Irgacure 907 of protein molecule, and concrete course of reaction is as follows:
Again above-mentioned haptens is mixed with N-hydroxy-succinamide (NHS) and carbodiimide (EDC), and and bovine serum albumin White mixing, is stirred at room temperature reaction 1h, and coupling prepares Irgacure 907 specific antigen, and by concentrating sample diluted extremely Concentration is 2 μ g/mL.
2) Irgacure 907 specific antibody working solution.
A, the acquisition of monoclonal antibody:
Irgacure 907 specific antigen above-mentioned synthesis obtained is as the immunogene Balb/c mouse to 7-8 week old Carry out immunity.First immunisation uses the phosphoric acid of complete Freund's adjuvant and the 0.01M, pH7.5 of Irgacure 907 specific antigen Salt buffer emulsifies, and antigen concentration is 1mg/mL, and dosage is 200 μ g/, and booster immunization uses incomplete Freund assistant the most every time Agent emulsifies with the phosphate buffer of the 0.01M, pH7.5 of Irgacure 907 specific antigen, the same initial immunity of dosage.For the first time After immunity two weeks, at interval of 1 week booster immunization once, immunity 5-7 time, when antibody titer no longer raises, carries out last altogether Secondary immunity.It is not added with immunologic adjuvant for the last time directly with the phosphate of the 0.01M, pH7.5 of Irgacure 907 specific antigen Buffer solution lumbar injection, the same initial immunity of dosage.
Then take blood examination at afterbody and survey serum titer.Take the mouse that serum titer is high, aseptically, take its splenocyte Cell fusion is carried out with myeloma cell SP2/0 in the ratio of 10:1.Use limiting dilution assay screening hybridoma, obtained The monoclonal antibody of full homogeneity and stable monoclonal hybridoma strain.
B, the purifying of monoclonal antibody:
Taking Balb/C mouse, use Freund's incomplete adjuvant lumbar injection to carry out sensitization before Yu Yizhou, dosage is 0.5mL/.Will Cell concentration is that the hybridoma suspension of 1.2 ten thousand/mL is expelled in mouse peritoneal, and dosage is 0.5mL/.Inoculation hybridization After oncocyte 7-10 days, collect ascites, repeatedly collect for several times.It is stored in 4 DEG C of Refrigerator stores.Carry out through octanoic acid-ammonium sulfate precipitation method Ascites purifies.
Method particularly includes: every 1 part of ascites adds 3 parts of sodium acetate buffers (concentration 0.05mol/L, pH4.0), uses The NaOH of 0.1mmol/L adjusts pH to 4.5, stirs 30min at 4 DEG C, and period calculates 40 μ L/mL's by ascites volume before dilution Amount is slowly added to octanoic acid；Stand 3h, centrifugal (11000r/min, 30min) at 4 DEG C, take supernatant, be maintained in 4 DEG C of environment, (NH is added in 30min4)2SO4Make final concentration of 0.277g/mL, stand 1h, 4 DEG C centrifugal (11000r/min, 30min), abandon Clear liquid, obtains monoclonal antibody precipitation, and is 2 μ g/mL by concentrating sample diluted to concentration.
3) sheep anti mouse two of horseradish peroxidase-labeled resists.
The sheep anti mouse two of the horseradish peroxidase-labeled provided by commercial company resists.
4) Irgacure 907 standard solution 6 bottles.
Concentration is respectively as follows: 1 × 103μg/L、1×102μg/L、1×101μg/L、1×100μg/L、0.1μg/L、0μg/L。
5) ELISA Plate.
96 hole polystyrene ELISA Plates, are provided by commercial company.
6) substrate developer.
Be made up of developer A and developer B, developer A be concentration be the aqueous hydrogen peroxide solution of 30%, developer B is Concentration is the DMSO solution of the tetramethyl benzidine of 10mg/mL.
7) cleaning solution.
For containing the 0.01M that weight ratio is 0.05%-0.5% Tween-20, the phosphate buffer of pH7.5.
8) stop buffer.
Sulfuric acid solution for 2mol/L.
9) confining liquid.
For the 0.01M containing 5% skimmed milk power, the phosphate buffer of pH7.5.
10) concentrating sample dilution.
For the phosphate buffer of 0.01M, pH7.5, (i.e. phosphate concentration is the phosphate-buffered of 0.01M/L, pH7.5 Liquid).
11) valve bag.
Thered is provided by commercial company.
Embodiment 2
The foundation of indirect competitive ELISA method.
Using the Competitive assays rate of indirect competitive ELISA method detection Irgacure 907 monoclonal antibody, method is as follows:
1) it is coated 96 hole ELISA Plates, 100 μ L/ holes with Irgacure 907 specific antigen of 2 μ g/mL, is positioned over 4 DEG C of ice Case and bag, by overnight, wash with cleaning solution after closing with 250 μ L/ hole confining liquids (i.e. 5% skimmed milk power), and pat dry.
2) (concentration is respectively as follows: 1 × 10 to add Irgacure 907 standard solution3μg/L、1×102μg/L、1×101μ g/L、1×100μ g/L, 0.1 μ g/L, 0 μ g/L) or sample solution 50 μ L, add the Irgacure 907 that concentration is 2 μ g/mL Specific antibody working solution 50 μ L, mixes, 37 DEG C of incubation 1h.
3), after washing pats dry, the sheep anti mouse of the horseradish peroxidase-labeled of 1:8000 dilution proportion is pressed in addition with dilution Two anti-working solutions, 100 μ L/ holes, 37 DEG C of incubation 1h.
4) washing pats dry, and every hole adds 50 μ L nitrite ion B liquid, 10 μ L nitrite ion A liquid, and develop the color 15min, adds concentration and is The sulfuric acid solution of 2mol/L, 50 μ L/ holes, terminate reaction, set ELIASA and (preferably examine with dual wavelength 450/630nm at 450nm Survey, in 5min, run through data), measure OD (absorbance) value.
With inhibiting rate I% as ordinate, with the logarithm lg [Irgacure 907 (ng/mL)] of Irgacure 907 concentration it is Abscissa, draws Irgacure 907 Competitive assays curve.The inhibiting rate of sample is substituted into calibration curve regression equation, from standard Read concentration corresponding to sample on curve, be multiplied by the extension rate of its correspondence and be the reality of Irgacure 907 in sample and contain Amount.
Inhibiting rate computing formula is as follows:
I = B - B N B 0 - B N × 100 %
Wherein: I inhibiting rate
The mean absorbance values of B sample solution
B0The mean absorbance values of 0 μ g/L standard liquid
BNReference blank comparison mean absorbance values.
Embodiment 3
The sensitivity of kit, specific, degree of accuracy experiment.
1. sensitivity determination
The indirect competitive ELISA method utilizing embodiment 2 sets up the standard working curve of Irgacure 907 detection, and result is such as Under:
Irgacure 907 is at 0.1 μ g/L～1 × 103Have good linear in the range of μ g/L, IC50=26.7ng/mL, Low detection is limited to 0.32ng/mL, and detection range (between suppression 20%～80%) is 0.32ng/mL～873.5ng/mL.
Taking separate sources, the blank sample without Irgacure 907 of unlike material, by gradient mark-on measuring It detects limit, and result is: in Food Contact paper-packed products, the detection of Irgacure 907 is limited to 0.008 μ g/dm2；Liquid is eaten In product analogies, the detection of Irgacure 907 is limited to 0.2 μ g/kg, the detection limit of Irgacure 907 in dry food analogies It is 0.08 μ g/kg；In beverage, the detection of Irgacure 907 is limited to 0.2 μ g/kg.
2. specific assay
Use embodiment 2 indirect competitive ELISA method measure Irgacure 907 analogue (Irgacure184, Irgacure 369, Irgacure 379, Irgacure 1173, Irgacure 631) and the cross reaction of monoclonal antibody mixture. By series concentration (1 × 104μg/L、1×103μg/L、1×102μg/L、1×101μg/L、1×100μ g/L, 0.1 μ g/L) upper Stating material to be added simultaneously to antibody respectively be coated in the ELISA Plate closed, concrete steps, with embodiment 2, calculate each respectively The inhibiting rate of analog.Utilize the monoclonal antibody IC to Irgacure 90750Value and monoclonal antibody are to each analog IC50The ratio of value is worth to cross reacting rate (CR%), and formula is as follows:
Result shows Irgacure 907 monoclonal antibody and Irgacure 184, Irgacure 369, Irgacure 379, Irgacure 1173, the cross reacting rate of Irgacure 631 are respectively less than 0.1%, meet the requirements.
3. accuracy determination
0.1 μ g/dm is added respectively in Food Contact paper-packed products2With 5 μ g/dm2Irgacure 907；To 3 kinds of foods Product analogies, drink sample add 0.5 μ g/kg and the Irgacure 907 of 5 μ g/kg respectively, in triplicate, does three every time Parallel, use the indirect competitive ELISA method of embodiment 2 to measure inhibiting rate, then by inhibiting rate (three parallel mean value) generation Enter calibration curve regression equation, calculate content, and calculate the rate of recovery.
Rate of recovery formula is as follows:
Result shows: the rate of recovery of Food Contact paper-packed products is 93.2%～107%；The rate of recovery of food simulants It is 88.1%～103%；The rate of recovery of beverage is 82.1%～97.5%.
Embodiment 4
Irgacure 907 content in detection Food Contact paper-packed products.
1. the pre-treatment of sample
Accurately cut 0.5dm2Printing paper packing articles sample, shreds the size to about 5mm × 5mm, is placed in 50mL plastics In centrifuge tube, adding 50mL acetonitrile, be completely soaked in acetonitrile by sample, ultrasonic 30min, 5000r/min are centrifuged 5min, take 5mL supernatant is placed in nitrogen in 40 DEG C of water-baths and is blown to do, and adds 0.1mL acetonitrile vortex 30s, is then diluted with water to 1.0mL, vortex Mixing, takes 50 μ L and is analyzed.
2. Irgacure 907 content in indirect competitive ELISA method detection sample
The method using embodiment 2 detects, and according to measurement result, calculates inhibiting rate, substitutes into calibration curve equation Obtain the content of Irgacure 907, see table 1.
The content of Irgacure 907 in table 1 Food Contact printing paper packing articles
Embodiment 5
Irgacure 907 content in detection food simulants.
1. the pre-treatment of sample
According to GB/T 23296.1-2009 " in food contact material plastics in restricted substances plastics material to food and food The guide that the test of product analogies specific transfer and content assaying method and food simulants exposure condition select " and EN " the paper and Board improvement polyphenylene oxide intending Food Contact does what analogies mensuration migrated from paper and cardboard to 14338-2003 Condition ", in conjunction with papery and the actually used situation of paper printed apcksging material, select water (analogies A) and 3% (mass concentration) Acetic acid aqueous solution (analogies B), as food liquid analogies, selects Noryl (analogies C) to simulate as dry food Thing, carries out migrating experiment, it is thus achieved that 3 kinds of corresponding food simulants samples.
Water and 3% (mass concentration) acetic acid aqueous solution: accurately weigh 5.0g food liquid analogies sample in 50mL plastics In centrifuge tube, adding 5mL acetonitrile, vortex 2min, be subsequently adding 3g NaCl, vortex 2min, 4000r/min are centrifuged 3min, draw Supernatant liquor, in nitrogen blowpipe, repeats, with 5mL acetonitrile extraction 1 time, to merge supernatant liquor, is placed in nitrogen in 40 DEG C of water-baths and blows and be concentrated into Dry, add 0.2mL acetonitrile vortex 30s, be then diluted with water to 2.0mL, vortex mixes, and takes 50 μ L and is analyzed.
Noryl: be transferred in 10mL colorimetric cylinder by the dry food analogies of 1.0g Noryl, adds 10mL acetonitrile, vortex 2min, 3000r/min be centrifuged 3min, and in Aspirate supernatant to nitrogen blowpipe, in 40 DEG C of water-baths, nitrogen is blown to Dry, add 50 μ L acetonitrile vortex 30s, then add water 150 μ L, and vortex mixes, and takes 50 μ L and is analyzed.
2. Irgacure 907 content in indirect competitive ELISA method detection sample
The method using embodiment 2 detects, and according to measurement result, calculates inhibiting rate, substitutes into calibration curve equation Obtain the content of Irgacure 907, see table 2.
Irgacure 907 content in table 2 food simulants
Embodiment 6
Irgacure 907 content in detection soft beverage packing.
1. the pre-treatment of sample
Accurately weigh 5.0g drink sample in 50mL plastic centrifuge tube, add 5mL acetonitrile, vortex 2min, be subsequently adding 3gNaCl, vortex 2min, 4000r/min be centrifuged 3min, draws supernatant liquor in nitrogen blowpipe, repeats with 5mL acetonitrile extraction 1 Secondary, merge supernatant liquor, be placed in nitrogen in 40 DEG C of water-baths and blow concentration less than 2mL, be settled to 2.0mL with acetonitrile, proceed to added with In the 2mL polytetrafluoroethylene (PTFE) centrifuge tube of 50mg N-propyl group ethylenediamine (PSA) and 25mg octadecyl silane (C18) filler, Vortex oscillation 1min, 10000r/min is centrifuged 3min, takes supernatant 1.0mL, and in 40 DEG C of water-baths, nitrogen blows and is concentrated to dryness, and adds 0.1mL acetonitrile vortex 30s, is then diluted with water to 1.0mL, and vortex mixes, and takes 50 μ L and is analyzed.
2. Irgacure 907 content in indirect competitive ELISA method detection sample
The method using embodiment 2 detects, and according to measurement result, calculates inhibiting rate, substitutes into calibration curve equation Obtain the content of Irgacure 907, see table 3.
The content of Irgacure 907 in table 3 beverage
Each technical characteristic of embodiment described above can combine arbitrarily, for making description succinct, not to above-mentioned reality The all possible combination of each technical characteristic executed in example is all described, but, as long as the combination of these technical characteristics is not deposited In contradiction, all it is considered to be the scope that this specification is recorded.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also Can not therefore be construed as limiting the scope of the patent.It should be pointed out that, come for those of ordinary skill in the art Saying, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the protection of the present invention Scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (10)

1. Irgacure 907 detection kit, it is characterised in that specifically include that
1) ELISA Plate of Irgacure 907 specific antigen it is coated: in each micropore of described ELISA Plate, Irgacure 907 is special The concentration of Specific Antigen is 0.5-3.0 μ g/mL；
2) Irgacure 907 specific antibody working solution: the concentration of this Irgacure 907 specific antibody is 0.5-3.0 μ g/ mL；
Described Irgacure 907 specific antigen is 1:1 with the amount ratio of Irgacure 907 specific antibody；
The preparation method of described Irgacure 907 specific antigen is:
Being dissolved in dichloromethane by Irgacure 907, temperature of reaction system controls at-10 DEG C to-1 DEG C, drips Boron tribromide Recovering after dichloromethane to room temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtains the 2-methyl-4-mercapto with phenol sulfydryl Base-2-morpholine propiophenone；Above-mentioned 2-methyl-4-sulfydryl-2-morpholine propiophenone and potassium carbonate are scattered in dimethylformamide, Add 4-bromobutyrate, be heated to reflux, make the phenol sulfydryl of 2-methyl-4-sulfydryl-2-morpholine propiophenone be taken by ethyl butyrate In generation, obtain intermediate product；Subsequently this intermediate product is added in trifluoroacetic acid, back flow reaction, slough the protection of ethyl, expose Carboxyl, preparing can be with the haptens of the Irgacure 907 of protein molecule；Again by itself and N-hydroxy-succinamide and carbon two React after imines mixing, prepare Irgacure 907 specific antigen with carrier protein couplet；Described carrier protein is rabbit One in seralbumin, bovine serum albumin(BSA), oralbumin, thyroglobulin；
The preparation method of described Irgacure 907 specific antibody is:
As immunogene, mouse is carried out immunity using Irgacure 907 specific antigen obtained above；After immunity, take serum The mouse that titer is high, takes its splenocyte and carries out cell fusion with myeloma cell SP2/0；Use limiting dilution assay screening hybridoma Cell, obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain；And take Freund's incomplete adjuvant abdominal cavity note Inject the mouse after row sensitization, hybridoma suspension is expelled in mouse peritoneal, collect ascites, through octanoic acid-ammonium sulfate The precipitation method carry out ascites purifying, obtain Irgacure 907 monoclonal antibody purified.
Irgacure 907 detection kit the most according to claim 1, it is characterised in that also include ELIAS secondary antibody, Described ELIAS secondary antibody is that concentration is resisted by the sheep anti mouse two of the horseradish peroxidase-labeled of the dilution proportion of 1:8000.
Irgacure 907 detection kit the most according to claim 2, it is characterised in that described Irgacure 907 is special Specific Antigen is the conjugate of Irgacure 907 and carrier protein, and the concentration of this Irgacure 907 specific antigen is 2 μ g/ mL；Described Irgacure 907 specific antibody is mouse resource monoclonal antibody, the concentration of this Irgacure 907 specific antibody It is 2 μ g/mL.
Irgacure 907 detection kit the most according to claim 3, it is characterised in that also include Irgacure 907 Standard solution, substrate developer, cleaning solution, stop buffer, confining liquid and concentrating sample dilution.
Irgacure 907 detection kit the most according to claim 4, it is characterised in that described carrier protein is rabbit blood One in pure albumen, bovine serum albumin(BSA), oralbumin, thyroglobulin；Described Irgacure 907 standard items The concentration of solution is respectively 1 × 103μg/L、1×102μg/L、1×101μg/L、1×100μg/L、0.1μg/L；Described substrate shows Toner is made up of developer A and developer B, and described developer A is hydrogen peroxide or urea peroxide, and described developer B is adjacent benzene Diamines or tetramethyl benzidine；Described cleaning solution is the 0.01M containing 0.05%-0.5% Tween-20, and the phosphate of pH7.5 delays Rush liquid；Described stop buffer is the sulfuric acid solution of 1-2mol/L；Described confining liquid is the 0.01M containing 5% skimmed milk power, pH7.5's Phosphate buffer；Described concentrating sample dilution is the phosphate buffer of 0.01M, pH7.5；The material of described ELISA Plate is One in polystyrene, polyethylene, polypropylene.
6. the preparation method of Irgacure 907 detection kit described in any one of claim 1-5, it is characterised in that main Comprise the following steps:
1) Irgacure 907 specific antigen is prepared:
Being dissolved in dichloromethane by Irgacure 907, temperature of reaction system controls at-10 DEG C to-1 DEG C, drips Boron tribromide Recovering after dichloromethane to room temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtains the 2-methyl-4-mercapto with phenol sulfydryl Base-2-morpholine propiophenone；Above-mentioned 2-methyl-4-sulfydryl-2-morpholine propiophenone and potassium carbonate are scattered in dimethylformamide, Add 4-bromobutyrate, be heated to reflux, make the phenol sulfydryl of 2-methyl-4-sulfydryl-2-morpholine propiophenone be taken by ethyl butyrate In generation, obtain intermediate product；Subsequently this intermediate product is added in trifluoroacetic acid, back flow reaction, slough the protection of ethyl, expose Carboxyl, preparing can be with the haptens of the Irgacure 907 of protein molecule；Again by itself and N-hydroxy-succinamide and carbon two React after imines mixing, prepare Irgacure 907 specific antigen with carrier protein couplet；Described carrier protein is rabbit One in seralbumin, bovine serum albumin(BSA), oralbumin, thyroglobulin；
2) coated elisa plate:
Above-mentioned Irgacure 907 specific antigen is coated in ELISA Plate；
3) Irgacure 907 specific antibody is prepared:
Using step 1) in Irgacure 907 specific antigen that obtains as immunogene, mouse is carried out immunity；After immunity, take The mouse that serum titer is high, takes its splenocyte and carries out cell fusion with myeloma cell SP2/0；Use limiting dilution assay screening miscellaneous Hand over oncocyte, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain；And take Freund's incomplete adjuvant abdomen Chamber injection carries out the mouse after sensitization, is expelled in mouse peritoneal by hybridoma suspension, collects ascites, through octanoic acid-sulphur The acid ammonium precipitation method carry out ascites purifying, obtain Irgacure 907 monoclonal antibody purified.
The preparation method of Irgacure 907 detection kit the most according to claim 6, it is characterised in that step 1) In, prepare Irgacure 907 specific antigen method particularly includes:
Being dissolved in 20mL anhydrous methylene chloride by the Irgacure 907 of 1mmol, temperature of reaction system controls at-5 DEG C, slowly Dropping 10mL Boron tribromide dichloromethane solution, recovers to room temperature, 0 DEG C of frozen water cancellation after reaction completely, suction filtration obtain with 2-methyl-4-sulfydryl-2-morpholine the propiophenone of phenol sulfydryl；By 0.8mmol2-methyl-4-sulfydryl-2-morpholine propiophenone and carbonic acid Potassium is scattered in anhydrous dimethyl formamide, adds 1.52g 4-bromobutyrate, be heated to reflux 3h, make 2-first under stirring at normal temperature The phenol sulfydryl of base-4-sulfydryl-2-morpholine propiophenone is replaced by ethyl butyrate, obtains intermediate product；Subsequently intermediate product is added Enter in excess trifluoroacetic acid, return stirring reaction 1h, slough the protection of ethyl, expose carboxyl, preparing can be even with protein The haptens of the Irgacure 907 of connection；React after again it being mixed with N-hydroxy-succinamide and carbodiimide, with cow's serum Albumin coupling prepares Irgacure 907 specific antigen.
The preparation method of Irgacure 907 detection kit the most according to claim 6, it is characterised in that step 3) In, prepare Irgacure 907 specific antibody method particularly includes:
Using step 1) in Irgacure 907 specific antigen that obtains as immunogene, the Balb/c mouse of 7-8 week old is carried out Immunity, first immunisation uses complete Freund's adjuvant and Irgacure 907 specific antigen emulsifying soln, and antigen concentration is 1mg/ ML, dosage is 200 μ g/, and booster immunization uses incomplete Freund's adjuvant and Irgacure 907 specific antigen the most every time Emulsifying soln, the same initial immunity of dosage；After initial immunity two weeks, at interval of 1 week booster immunization once, immunity 5-7 time altogether, when anti- When body titer no longer raises, carry out last immunity；It is not added with immunologic adjuvant for the last time directly special with Irgacure 907 Property antigen aqueous solution lumbar injection, the same initial immunity of dosage；Afterbody takes blood examination and surveys after serum titer, takes high little of serum titer Mouse, aseptically, takes its splenocyte and carries out cell fusion in the ratio of 10:1 with myeloma cell SP2/0；Use limited Dilution method screening hybridoma, obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain；And in Using Freund's incomplete adjuvant lumbar injection Balb/C mouse to carry out sensitization before one week, dosage is 0.5mL/；It is 1.2 by cell concentration The hybridoma suspension of ten thousand/mL is expelled in mouse peritoneal, and dosage is 0.5mL/；Inoculation hybridoma 7-10 days After, collect ascites, repeatedly collect for several times；It is stored in 4 DEG C of Refrigerator stores；Carry out ascites purifying through octanoic acid-ammonium sulfate precipitation method, obtain Irgacure 907 monoclonal antibody purified.
9. the using method of Irgacure 907 detection kit described in any one of claim 1-5, it is characterised in that main Comprise the following steps:
1) sample pre-treatments
Accurately measure testing sample, be quantitatively adding organic solvent, quantitatively draw after extraction or extraction supernatant, add after drying up with The organic solvent that water dissolves each other redissolves, and dilutes constant volume with water, obtains sample detection liquid；
2) detection
The kit described in any one of claim 1-5 is used to detect, to being coated with Irgacure 907 specific antigen ELISA Plate in add standard items or sample detection liquid, add Irgacure 907 specific antibody working solution, wash after incubation Plate, adds ELIAS secondary antibody and carries out the amplification of enzymatic activity, again wash plate, adds substrate developer, stop buffer, then surveys with ELIASA Determine absorbance；
3) interpretation of result
With inhibiting rate I% as ordinate, with the logarithm lg [Irgacure 907] of Irgacure 907 concentration as abscissa, draw Irgacure 907 Competitive assays curve；The inhibiting rate of sample is substituted into calibration curve regression equation, reads from calibration curve Concentration corresponding to sample, the extension rate being multiplied by its correspondence is the actual content of Irgacure907 in sample；
Described inhibiting rate computing formula is as follows:
I = B - B N B 0 - B N × 100 %
Wherein: I inhibiting rate
The mean absorbance values of B sample solution
B0The mean absorbance values of 0 μ g/L standard liquid
BNReference blank comparison mean absorbance values.
The using method of Irgacure 907 detection kit the most according to claim 9, it is characterised in that step 1) in, institute Stating organic solvent miscible with water is acetonitrile；
Step 2) in, use the kit described in claim 5, detection method particularly includes: first with 2 μ g/mL's Irgacure 907 specific antigen is coated 96 hole ELISA Plates, 100 μ L/ holes, is positioned over 4 DEG C of refrigerators and is coated overnight, with 250 μ L/ Hole confining liquid washs with cleaning solution after closing, and pats dry；It is subsequently adding Irgacure 907 standard solution or sample detection liquid 50 μ L, add the Irgacure 907 specific antibody working solution 50 μ L that concentration is 2 μ g/mL, mix, 37 DEG C of incubations 1h；Washing subsequently pats dry, and addition is pressed the sheep anti mouse two of the horseradish peroxidase-labeled of 1:8000 dilution proportion and resisted with dilution Working solution, 100 μ L/ holes, 37 DEG C of incubation 1h；Finally washing pats dry, and every hole adds 50 μ L developer B, 10 μ L developer A, colour developing 15min, adding concentration is the sulfuric acid solution of 2mol/L, 50 μ L/ holes, terminates reaction, and setting ELIASA, at 450nm, measures and inhales Shading value.
CN201510452064.XA 2015-07-28 2015-07-28 Irgacure 907 detection kit and preparation and application thereof Expired - Fee Related CN105067805B (en)

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