CN103698504B - Phthalate total amount ELISA enzyme-linked immunologic detecting kit and using method thereof - Google Patents
Phthalate total amount ELISA enzyme-linked immunologic detecting kit and using method thereof Download PDFInfo
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- CN103698504B CN103698504B CN201310752662.XA CN201310752662A CN103698504B CN 103698504 B CN103698504 B CN 103698504B CN 201310752662 A CN201310752662 A CN 201310752662A CN 103698504 B CN103698504 B CN 103698504B
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G—PHYSICS
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Abstract
The invention discloses a kind of phthalate total amount ELISA enzyme-linked immunologic detecting kit and using method thereof, belong to enzyme linked immunosorbent detection technical field.Described kit comprises following component: two anti-, phthalic acid standard solution, substrate solution, substrate buffer solution, reaction terminating liquid, the concentrated cleaning solutions being coated with the ELISA Plate of phthalic acid antigen, phthalic acid antibody working fluid, horseradish peroxidase-labeled.The present invention discloses the method utilizing described kit to detect phthalate total amount in sample, sample, after pre-treatment, utilizes kit to detect, processes testing result, analyzes phthalate total amount in sample.The kit of detection phthalic acid provided by the invention adopts indirect competitive enzyme-linked immunosorbent adsorption analysis technology, highly sensitive, good stability, and cost is low, is applicable to very much the examination of a large amount of sample, has important application value.
Description
Technical field
The present invention relates to ELISA enzyme linked immunosorbent detection technical field.More specifically, a kind of phthalate total amount ELISA enzyme-linked immunologic detecting kit and using method thereof is related to.
Background technology
Phthalic ester (phthalate acid esters, PAEs) compounds is also called peptide acid esters, the general chemical constitution of phthalic ester is made up of a rigid plane aromatic hydrocarbons and two plastic non-linear aliphatic side chainses, in colorless oil thick liquid under normal temperature, be insoluble in water and not volatile, solidifying point is low, be soluble in the organic solvents such as methyl alcohol, ethanol, ether, be mainly used in plastic products, be used for improving plasticity and toughness, also use the raw materials for production of used as pesticides, dyestuff, cosmetics, spices and medical equipment.PAEs is connected with hydrogen bond or Van der Waals force with plastic molecules, not with covalent bond form strong bonded, therefore enter in room air, air, food, potable water and other materials easily via modes such as drip washing migration or evaporations, also can be discharged in environment by flue dust sedimentation in plastics-production or combustion process.Past thinks that the toxicity of PAEs is very low always, and thus milli is unrestrictedly produced.But research shows afterwards, it have kind many, be difficult to degrade, feature that bioconcentration is strong, all have larger toxicity to human body, biosome and plant.Research finds that multiple PAEs has general toxicity and specific toxicity, and part PAEs has teratogenesis and mutagenic effect to animal, and demonstrates stronger endocrine disrupting.The harm of phthalate pollutant to the mankind is mainly manifested in carcinogenicity, teratogenesis and inhibitive ability of immunity, especially to cause human body reproductive function abnormal the most noticeable.PAEs compounds enters in the environment of our existence in large quantities along with flue dust, discarded object, industry and house refuse, waste water etc., threatens the health of the mankind, thus causes the concern of more and more people.
Due to human society a large amount of production and widely use, phthalic ester has become one of environmental contaminants that the earth the most extensively exists, the environmental pollution that PAEs causes has received global concern, phthalate compound is called " second global PCB pollutant " by people, 1977 American National Environmental Protection Agency (EPA) these 6 kinds of PAEs compounds of DEHP, DOP, BBP, DBP, DEP and DMP are classified as the toxic pollutant of priority acccess control, these 3 kinds of PAEs compounds of DEP, DMP and DOP are also defined as Environment Priority control polluted articles by China.In the standard GB/T/T 21911-2008 of the mensuration of Phthalic Acid Esters in Food, regulation is limited to 1.5mg/kg containing detecting of each phthalate compound in oil sample, is not limited to 0.05mg/kg containing detecting of each phthalate compound in oil sample.In May, 2011, there is global the first plasticiser pollution case in Taiwan, detect a large amount of DEHP in some food and beverage product, find after deliberation, DEHP adds in food emulsifying agent by food additives manufacturer, to improve food uniformity coefficient.The white wine plasticiser disturbance produced in 2012 has more increased the weight of the fear of people to plasticiser.
At present, the analysis of PAEs adopts instrumental method more, liquid phase-mass spectrometry, gas phase-mass spectrometry, close bright moon (Chinese Journal of New Drugs, 2013) etc. people is with the phthalate compound in gas chromatography mass spectrometry chromatography determination gelatin hollow capsule, and the people such as Yang Rongjing (environmental chemistry, 2012) detect 17 kinds of phthalic ester plasticizers in food contact material by high performance liquid chromatography-tandem mass method.Instrument analytical method can realize multi-residue determination, and its advantage is that to detect degree of accuracy high, but hinders it to apply, normally as the confirmation method in laboratory because its instrumentation degree is high, detection time is long, process is loaded down with trivial details, testing cost is expensive etc.
Enzyme-linked immuno assay (ELISA) Fast Detection Technique is because cost is low, simple to operate, speed fast, one-time detection sample size is large, instrumentation degree is low, and being worth us to promote becomes conventional screening technique.The ultimate principle of ELISA immune analysis method is: make antigen or antibody be attached to certain surface of solid phase carriers, and keep its immunocompetence.Make antigen or antibody and certain enzyme connect into enzyme-labelled antigen or antibody, this enzyme-labelled antigen or antibody had both retained its immunocompetence, retained again the activity of enzyme.When measuring, react by the antigen of different steps and surface of solid phase carriers or antibody by inspection sample (mensuration antibody wherein or antigen) and enzyme-labelled antigen or antibody.By the method for washing, the antigen antibody complex that solid phase carrier is formed is separated with other materials, the enzyme amount be finally combined on solid phase carrier becomes certain ratio with the amount of tested substance in sample.After adding the substrate of enzyme reaction, substrate is become color products by enzymatic, and the amount of product is directly related with the amount of tested substance in sample, therefore can carry out qualitative or quantitative test according to the depth of color reaction.Because the catalysis frequency of enzyme is very high, therefore can greatly iodine effect, thus make the susceptibility that assay method reaches very high.
But immune analysis method mostly is the detection of single ester class, because phthalic ester is of a great variety, and the phenomenon that multiple compound is detected is very general simultaneously, but detecting method used is all instrumental method simultaneously, therefore, be necessary very much to set up the multi-residue determination method (multi-residues analysis) that simultaneously can detect multiple PAEs.There is not commercial phthalic ester ELISA detection kit both at home and abroad at present, about the report of phthalic ester immuno analytical method (IA) is also few, mainly concentrate on both at home and abroad at present and set up the research of specific immunity detection method for single phthalic ester, there is no the holoantigen detecting phthalate total amount and design the correlative study prepared with antibody.
In sum, the instrument detection method that current domestic and international existing phthalate adopts is complicated, loaded down with trivial details, be difficult to be applied to actual batch detection, and other immunoassay detection methods existing cannot realize multi-residue determination, have had a strong impact on the detecting & monitoring of phthalate.Therefore develop that stability is high, simple to operate, equipment requirement is low, the cheap and ELISA kit of High sensitivity, low detectability, for accurately fast, high flux detects phthalate and has very important economy and social effect.
Summary of the invention
The technical problem to be solved in the present invention is the technical deficiency overcoming existing phthalate multi-residue determination, a kind of high specific, high sensitivity, cheap, simple to operate are provided, the enzyme-linked immunologic detecting kit of phthalate total amount can be detected in enormous quantities fast.
The object of this invention is to provide a kind of phthalate total amount ELISA enzyme-linked immunologic detecting kit.
Another object of the present invention is to provide application and the using method of described kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides a kind of phthalate total amount ELISA enzyme-linked immunologic detecting kit, comprise following ingredients:
(1) ELISA Plate of phthalic acid antigen is coated with;
(2) phthalic acid antibody working fluid;
(3) two of horseradish peroxidase-labeled resist;
(4) phthalic acid standard solution;
(5) substrate solution;
(6) substrate buffer solution;
(7) reaction terminating liquid;
(8) concentrated cleaning solution.
Wherein, described phthalic acid antigen is the conjugate of haptens 4-aminophthalic acid and OVA, uses glutaraldehyde method phthalic acid and ovalbumin (OVA) covalent coupling to be synthesized and obtains;
1.69g sodium carbonate and 2.95g sodium bicarbonate are preferably dissolved in 1L distilled water by the coating buffer of phthalic acid antigen and obtain by bag, and the bag of phthalic acid antigen is 0.5mg/L by concentration; Confining liquid preferably gets that 0.1g bovine serum albumin(BSA) (BSA), 5g glycocoll are molten, 5g sucrose obtains in 100mL phosphate buffer (PBS solution).The concentration of described PBS solution is 0.01mol/L, and pH value is 7.4, and compound method is: the NaH accurately taking 2.9g
2pO
412H
2the KH of KCl, 0.2g of NaCl, 0.2g of O, 8.5g
2pO
4, be settled to 1L with water, sterilizing.
Described ELISA Plate is 96 or 40 hole ELISA Plate, and material is polystyrene, be coated with can with the phthalic acid antigen of anti-phthalic acid antibody specific binding, and the site of phthalic acid antigen is not adsorbed on closed porosity surface.
In addition, can be more as the material of fixing dibutyl phthalate antigen solid phase carrier, such as polystyrene, cellulose nitrate, tygon, polypropylene, polyacrylamide, cross-linked glucose, silicon rubber, Ago-Gel etc.The form of this carrier can be shrinkage pool, the scraps of paper, globule etc.
Described phthalic acid antibody is any one in phthalic acid monoclonal antibody, rabbit polyclonal antibody; Described phthalic acid antibody is this laboratory preparation in early stage: adopt glutaraldehyde method by phthalic acid and bovine serum albumin(BSA) (BSA) covalent coupling complex immunity SPF level Balb/c mouse gained, be preferably T liquid and dilute 8000 times during use; The formula of described T liquid is: the Tris taking 1.21g, uses 800mL deionized water dissolving, regulates solution to required pH value, be settled to 1L with deionized water with 1mol/L hydrochloric acid solution.
Two of described horseradish peroxidase-labeled resists the mouse two for horseradish peroxidase-labeled to resist or rabbit two resists.
The concentration of described phthalic acid standard solution is 1mg/mL, by PBS solution, standard solution is diluted to phthalic acid standard solution during use; Specifically with the PBS of 0.01mol/L, pH5.4, standard solution is diluted to a series of phthalic acid standard solutions that concentration is 0.0256 μ g/L, 0.128 μ g/L, 0.64 μ g/L, 3.2 μ g/L, 16 μ g/L, 80 μ g/L.
Described substrate solution is the pH5.0 phosphate citrate acid buffering solution containing 3,3,5,5-tetramethyl benzidines (TMB) or o-phenylenediamine (OPD).
Described substrate buffer solution is the pH5.0 phosphate citrate acid buffering solution containing hydrogen peroxide or urea peroxide.
Described reaction terminating liquid is 1 ~ 2mol/L sulfuric acid solution or 2mol/L sodium hydroxide solution.
Described concentrated cleaning solution is containing 0.5 ~ 1.5%(v/v) phosphate buffer of Tween-20; Described concentrated cleaning solution pH value is 7.4, and concentrated cleaning solution concentration is 0.1mol/L.With deionized water dilution 15 ~ 25 times when described concentrated cleaning solution uses.
The preparation method being coated with the ELISA Plate of phthalic acid antigen of the present invention is:
With coating buffer, phthalic acid antigen is diluted on demand, antigenic dilution is added in ELISA Plate micropore, put into 37 DEG C of environment to hatch, put into 4 DEG C of environment night incubation (good stability of the ELISA Plate obtained) again, incline coating buffer, washs with cleansing solution, then in every hole, confining liquid is added, hatch for 37 DEG C, liquid in hole of inclining, preserve with the vacuum seal of aluminium film after dry.
Present invention also offers the using method of described phthalate total amount ELISA enzyme-linked immunologic detecting kit, comprise the steps:
S1. kit is taken out from cold storage environment, be placed in equilibrium at room temperature;
S2. by standard items or add through the testing sample of pre-treatment and be coated with in the ELISA Plate hole of phthalic acid antigen, then every hole adds antibody, pats mixing, hatches;
S3. wash with cleansing solution;
S4. add horseradish peroxidase-labeled two resist, and pat mixing, hatch;
S5. wash with cleansing solution;
S6. substrate buffer solution and substrate solution equal-volume are mixed to get nitrite ion, add nitrite ion in every hole, pat mixing, lucifuge is hatched;
S7. every hole adds reaction terminating liquid, mixes, under wavelength 450nm or 492nm, is blank with air, measures each hole light absorption value;
S8. testing result treatment and analyses:
Inhibiting rate (%)=B/B
0× 100(%), in formula, B is the luminous value of variable concentrations phthalic acid standard sample wells or sample well; B
0it is 0 μ g/L concentration phthalic acid standard solution luminous value; Take inhibiting rate as ordinate, the logarithm of O-phthalic acid concentration is horizontal ordinate drawing standard curve, thus determines the content of phthalic acid in testing sample.The analysis of described testing result can also utilize computer professional software to carry out calculating and analyze.
Wherein, the equilibrium at room temperature that is placed in described in S1 refers at room temperature (20 ~ 24 DEG C) placement more than 30min, makes the temperature return of kit to room temperature.
Pre-treatment described in S2 refers to that by the phthalate Substance Transformation in testing sample be phthalic acid.
Preferably, testing sample described in step S2 is edible oil or soy sauce;
Preferably, the pre-treating method of edible oil or soy sauce sample is as follows:
S1. take sample 2.0g, be put in Backflow bottle, add the mixing of 10mL methyl alcohol, then add the KOH solution that 10mL concentration is 2mol/L, in 85 DEG C of heating water bath 2.5h, by methyl alcohol evaporate to dryness, take out and be cooled to room temperature;
S2. by volumetric concentration be 50% hydrochloric acid hydrolyzate pH is adjusted to 1 ~ 2, whirlpool concussion 3min after, ultrasonic oscillation 20min;
S3. add the normal hexane of 3 times of volumes, whirlpool concussion 3min, leaves standstill 20min, discards normal hexane layer, add the ethyl acetate of 3 times of volumes, and whirlpool concussion 3min, leaves standstill 20min, get ethyl acetate layer, add the ethyl acetate of 3 times of volumes in remaining liq again;
Three extracts are merged, are evaporated to dry to the greatest extent in rotary evaporator by S4. S3 operation in triplicate;
S5. methyl alcohol redissolves, and is directly used in detection, extension rate should be counted when result of calculation with after the PBS dilution 15 ~ 20 times of 0.01mol/L, pH5.4.
More specifically, the using method of described phthalate total amount ELISA enzyme-linked immunologic detecting kit, comprises the steps:
S1. taken out from cold storage environment by kit, be placed in room temperature (20 ~ 24 DEG C) balance more than 30min, the batten of enough standard items and testing sample usage quantity is fixed on support, and standard items and testing sample do two parallel laboratory tests, number in order;
S2. in standard sample wells, add the phthalic acid standard items of 50 μ L variable concentrations, sample well adds the testing sample of 50 μ L through pre-treatment; Then every hole adds the antibody of 50 μ L T liquid dilutions, covers the jolting mixing on micro oscillator of cover plate film, at incubated at room 30min;
S3. pour out the liquid in hole, micropore frame is upside down on thieving paper and pats (often wheel is washed plate and patted 3 times) to ensure the liquid removed completely in hole; Be filled with in hole with 300 μ L cleansing solutions, again outwell the liquid in micropore, then repetitive operation 5 times;
S4. adding 100 μ L antibody diluent PBST(formulas in every hole is NaH
2pO
412H
2o 2.9g, NaCl 8.5g, KCl 0.2g, KH
2pO
40.2g, 500 μ L Tween-20, are settled to 1L) ELIAS secondary antibody of diluting, cover the jolting mixing on micro oscillator of cover plate film, incubated at room 30min;
S5. step S3 is repeated;
S6. every hole adds 100 μ L nitrite ions (substrate buffer solution and substrate solution equal-volume are mixed to get), and pat mixing, cover cover plate film, dark at room temperature hatches 10min;
S7. 50 μ L reaction terminating liquids are added in micropore; After mixing, being blank at wavelength 450nm(with air) place measures each hole light absorption value, must read light absorption value after adding stop buffer in 60min;
S8. testing result calculates and analyzes:
Inhibiting rate (%)=B/B
0× 100(%)
In formula: B is the luminous value of variable concentrations standard sample wells (or sample well); B
0it is the mean absorbance values of 0 μ g/L standard solution.Take inhibiting rate as ordinate, the logarithm of phthalic acid standard solution concentration is horizontal ordinate drawing standard curve, thus determines the content of phthalic acid in testing sample;
According to the content of phthalic acid in testing sample, the computing formula calculating phthalate total content in testing sample is as follows:
In formula: X is the total content of PAEs in testing sample, unit is mol/g;
C is the concentration that ELISA detects the phthalic acid obtained, and unit is ng/mL;
M is the quality of testing sample, and unit is g;
N is the extension rate diluting testing sample with the PBS of 0.01mol/L, pH5.4;
V is redissolution methyl alcohol volume, and unit is mL;
166.13 is the relative molecular mass of phthalic acid, and unit is g/mol.
The preparation method of antigen of the present invention is specific as follows:
S1. haptenic synthesis: with 4-nitrophthalic acid for raw material, passes into hydrogen (H under the catalysis of palladium carbon
2) be reduced into 4-aminophthalic acid and namely obtain haptens 4-APA.
S2. the synthesis of artificial antigen: adopt glutaraldehyde method that phthalic acid haptens obtained above is prepared artificial antigen 4-APA-BSA or 4-APA-OVA with bovine serum albumin (BSA) or ovalbumin (OVA) coupling respectively.
The preparation method of phthalic acid monoclonal antibody of the present invention is specific as follows:
S1. animal immune: with artificial antigen (haptens and carrier protein couplet thing) for immunogene carries out Immunity at intervals to Balb/c mouse, indirect ELISA detects and obtains the mouse spleen containing phthalic acid specific antibody in blood.
S2. Fusion of Cells and clone: get the Balb/c mouse boosting cell that produces specific antibody and myeloma cell SP20 merges, adopts indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screens positive hole; Utilize limiting dilution assay or microclone method to carry out cloning to positive hole, obtain and set up the hybridoma cell strain producing monoclonal antibody.
S3. cell cryopreservation and recovery: get the hybridoma cryopreserving liquid being in exponential phase and make cell suspension, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal rear removal cryopreserving liquid, move into and cultivate culture in glassware.
S4. the preparation and purification of monoclonal antibody: adopt in body and induce method, by Balb/c mouse (8 week age) Intraperitoneal injection sterilizing paraffin oil, 7 ~ 14 days pneumoretroperitoneum injection hybridomas, gathered ascites after 7 ~ 10 days; Carry out ascites purifying through sad-saturated amine sulfate method or affinity chromatography, purity through SDS-PAGE electroresis appraisal, bottle packing ,-20 DEG C of preservations.
The preparation method of phthalic acid rabbit polyclonal antibody of the present invention is specific as follows:
Adopt new zealand white rabbit as immune animal, with haptens 4-aminophthalic acid and carrier protein couplet thing for immunogene carries out immunity to new zealand white rabbit, repeatedly measure serum antibody titer after immunity, Culling heart blood, obtain the polyclonal antibody of purifying through amine sulfate fractional precipitation.
The thinking that the present invention designs detection is as follows:
First by antigen coated for phthalic acid on solid phase carrier (ELISA Plate), then standard specimen or pretreated testing sample is added, add phthalic acid antibody again, after hatching a period of time, add that horseradish peroxidase (HRP) marks again two resist, and the phthalic acid in envelope antigen and testing sample competes antibody, during testing sample phthalate content height, the antibody be then combined with solid phase antigen is just few, otherwise the antibody be combined on solid phase antigen is just many.The antibody that ELIAS secondary antibody is combined with solid phase antigen combines, add substrate after reaction to carry out developing the color being measured, when antibody amount one timing, the testing sample added is more containing phthalic acid, the antibody be combined with solid phase antigen is fewer, color reaction weakens, percentage absorbance is low, otherwise, then color reaction strengthens, percentage absorbance increases, thus map according to the semilog relation between percentage absorbance and O-phthalic acid concentration and obtain typical curve, again according to the typical curve of phthalic acid and the percentage absorbance of measuring samples, the concentration of phthalic acid in testing sample can be extrapolated.
The present invention is according to the imagination that can obtain total material phthalic acid after phthalic ester plasticizer hydrolysis, design can detect the kit of phthalic acid total content, thus calculate phthalate total amount, reflect by the detection of phthalate total amount the situation whether food pollutes by plasticiser and pollute.In testing sample, the computing method of phthalic acid total amount are: substitute into the content obtaining phthalic acid in liquid to be measured in typical curve with the light absorption value of phthalic acid; By phthalate integral molar quantity in the molar weight calculation sample of phthalic acid.
In testing sample (as edible oil or soy sauce), the computing formula of the total content of phthalate is as follows:
In formula: X is the total content of PAEs in testing sample, unit is mol/g;
C is the concentration that ELISA detects the phthalic acid obtained, and unit is ng/mL;
M is the quality of testing sample, and unit is g;
N is the extension rate diluting testing sample with the PBS of 0.01mol/L, pH5.4;
V is redissolution methyl alcohol volume, and unit is mL;
166.13 is the relative molecular mass of phthalic acid, and unit is g/mol.
The present invention has following beneficial effect:
Compared with prior art, the present invention has following beneficial effect:
Kit provided by the invention adopts racing ELISA detecting method, and the antibody specificity of employing is high, affinity is high, improves the sensitivity of detection, accuracy; Utilize envelope antigen to carry out bag quilt to ELISA Plate, compared to antibody bag quilt, the antigen coated bag of the present invention is by better effects if, and the holding time is longer, thus improves precision and stability that kit detects.
In addition, simple pre-treatment is carried out to sample, after ester-type hydrolysis is phthalic acid, kit of the present invention directly can be utilized to detect the content of phthalic acid, phthalate total amount in sample can be calculated according to formula.Experimental result shows, and kit of the present invention is 0.36 ~ 5.29ng/mL to the maximum detection range of phthalate total amount, and sensitivity is 1.38ng/mL, detects and is limited to 0.17ng/mL.
Kit of the present invention have simple to operate fast, the advantage such as stability is high, pin-point accuracy is sensitive, detectability is low, and it is low for equipment requirements, with low cost, be highly suitable for trace analysis and the batch detection of phthalate total amount, be applicable to the examination of a large amount of sample, there is important application value.
Accompanying drawing explanation
Fig. 1 is phthalic acid canonical plotting.
Embodiment
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
In embodiment use the preparation method of reagent as follows:
Coating buffer: 1.69g sodium carbonate and 2.95g sodium bicarbonate are dissolved in 1L distilled water and obtain.
Confining liquid: get 0.1gBSA(bovine serum albumin(BSA)), 5g glycocoll, 5g sucrose is in 100mLPBS(0.01mol/L, pH7.4) solution obtains.
Concentrated cleaning solution is for containing 0.5 ~ 1.5%(v/v) phosphate buffer of Tween-20; Concentrated cleaning solution pH value is 7.4, and concentrated cleaning solution concentration is 0.1mol/L, with deionized water dilution 15 ~ 25 times during use.
Phthalic acid standard solution: with hplc grade methanol, phthalic acid standard solution is diluted to 1mg/mL for subsequent use, during use, standard solution is diluted to a series of phthalic acid standard solutions that concentration is 0.0256 μ g/L, 0.128 μ g/L, 0.64 μ g/L, 3.2 μ g/L, 16 μ g/L, 80 μ g/L by the PBS of 0.01mol/L, pH5.4,4 DEG C of preservations.
Substrate nitrite ion: be the pH5.0 phosphate citrate acid buffering solution containing 3,3,5,5-tetramethyl benzidine (TMB) or o-phenylenediamine (OPD).
Substrate buffer solution: be the pH5.0 phosphate citrate acid buffering solution containing hydrogen peroxide or urea peroxide.
Stop buffer: be 1 ~ 2mol/L sulfuric acid solution or 2mol/L sodium hydroxide solution.
Phthalic acid monoclonal antibody: laboratory is obtained for early stage, sees embodiment 1 step (3).
Horseradish peroxidase mark two resists: commercial reagents.
the preparation of embodiment 1 haptens, artificial antigen
1, haptenic synthesis
(1) 0.845g(4mmol is taken) 4-nitrophthalic acid and 1.8g palladium carbon is dissolved in 20mL ethanol, slowly passes into hydrogen (H
2), stir under normal temperature, reaction 2h.
(2) reaction terminates rear filtration, and supernatant rotary evaporation concentrates.
(3) again filter with after acetic acid ethyl dissolution, obtain gray solid (i.e. haptens 4-APA) 0.72g after filtrate decompression evaporate to dryness, productive rate ﹥ 99%.
2, the synthesis of artificial antigen
(1) BSA(or OVA of 0.0025mmol is got) be dissolved in 5mLPBS, more separately get 0.1mmol haptens 4-APA and be dissolved in 0.5mL dimethyl formamide (DMF), and dropwise drop in PBS, be called A liquid;
(2) by volumetric concentration be 25% glutaraldehyde PBS dilute 10 times after draw 200 μ L, under magnetic agitation, dropwise slowly add in A liquid, time for adding is 2h.12h is reacted at 4 DEG C;
(3) load bag filter, to dialyse 3d with PBS, change water every day three times.Obtain immunogene 4-APA-BSA and coating antigen 4-APA-OVA respectively.
(4) be sub-packed in 0.5mL centrifuge tube, frozen for subsequent use in-20 DEG C of refrigerators.
the preparation of embodiment 2 phthalic acid monoclonal antibody
1, animal immune
(1) the present embodiment selects 47 week ages, and healthy female SPF Balb/c mouse (purchased from Guangdong Medical Lab Animal Center), numbers respectively; The immunogene 4-APA-BSA prepared by embodiment 1 carries out immunity.
(2) first immunisation: take 4-APA-BSA as immunogene, immunizing dose be 50 μ g/ only, by immunogene normal saline dilution to 1mg/mL(in protein concentration), add isopyknic Freund's complete adjuvant fully emulsified, adopt the immunity of belly multiple spot.
(3) booster immunization: interval is got same dose immunogene and added the emulsification of equivalent incomplete Freund's adjuvant after three weeks, booster immunization once, after repeating four immunity, abdominal cavity booster immunization once, extracting spleen cell after 3 days.
(4) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 5:1 ratio and SP2/0 myeloma cell fusion, adopts indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screens positive hole.Microclone method is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
(5) cell cryopreservation and recovery: get the hybridoma cryopreserving liquid (formula is 1mL dimethyl sulfoxide (DMSO) (DMSO), 9mLRPMI-1640 nutrient culture media) being in exponential phase and make 5 × 10
6the cell suspension of individual/mL, is sub-packed in cryopreservation tube, preserves for a long time in-70 DEG C of ultra low temperature freezers.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
(6) preparation and purification of monoclonal antibody: adopt in body and induce method, by Balb/c mouse (8 week age) lumbar injection hybridoma 5 × 10
6individual/only, gather ascites after 14 days.Carry out ascites by sad-saturated amine sulfate method to purify, and use determined by ultraviolet spectrophotometry antibody concentration, bottle packing ,-20 DEG C of preservations.
the preparation of embodiment 3 enzyme-linked immunologic detecting kit component of the present invention
1, the composition of kit
(1) be coated with the ELISA Plate of phthalic acid antigen: the 96 removable ELISA Plate in hole, wrapped by phthalic acid antigen and confining liquid, phthalic acid antigen is 4-APA-OVA(phthalic acid and OVA conjugate), wrapping by concentration is 0.5mg/L.
The bag quilt of ELISA Plate microwell plate: envelope antigen coating buffer is diluted to 0.5mg/L, adds 100 μ L in every hole, 37 DEG C of overnight incubation, and liquid in hole of inclining, cleansing solution washs 2 times, pats dry.Then add 120 μ L confining liquids in every hole, 37 DEG C hatch 3h after, liquid in hole of inclining, is placed in 37 DEG C of baking ovens and dries, use aluminium foil bag vacuum seal, 4 DEG C of preservations.
(2) phthalic acid standard solution: accurately take phthalic acid standard model hplc grade methanol and be diluted to 1mg/mL; 0.0256 μ g/L, 0.128 μ g/L, 0.64 μ g/L, 3.2 μ g/L, 16 μ g/L, 80 μ g/L phthalic acid standard solutions are mixed with respectively, 4 DEG C of preservations with the PBS damping fluid of 0.01mol/L, pH5.4 during use.
(3) two of horseradish peroxidase-labeled resist, and with the PBST damping fluid of pH7.4,0.01mol/L, (formula is: the Na of the NaCl taking 8.5g, 2.9g
2hPO412H
2the KH of the KCl of O, 0.2g, 0.2g
2pO
4, 500 μ L polysorbas20s, then be settled to 1L with deionized water) dilution; Working concentration is 1:5000.
(4) phthalic acid monoclonal antibody 3mg/mL, during use, (formula is the Tris of 1.21g to T liquid, uses 800mL deionized water dissolving; Regulate solution to required pH value with 1mol/L hydrochloric acid solution; 1L is settled to deionized water) dilution, working concentration is 1:8000.
(5) substrate buffer solution: the pH5.0 phosphate citrate buffer that 30% hydrogen peroxide 30 μ L is dissolved in 19mL (is filled a prescription as the Na of 25.7mL 0.2M
2hPO
4, the citric acid of 24.3mL 0.1M, adding distil water 50mL) in, 4 DEG C of preservations.
(6) substrate solution: 3,3,5,5-tetramethyl benzidine (TMB) 80mg is dissolved in the phosphate citrate buffer of 10mL pH5.0,4 DEG C of preservations.
(7) concentrated cleaning solution is containing 0.5 ~ 1.5%(v/v) phosphate buffer (pH7.4,0.1mol/L) of Tween-20, described concentrated cleaning solution pH value is 7.4, and concentrated cleaning solution concentration is 0.1mol/L;
With deionized water dilution 15 ~ 25 times when described concentrated cleaning solution uses.
(8) reaction terminating liquid: be 1 ~ 2mol/L sulfuric acid solution or 2mol/L sodium hydroxide solution.
2, reagent packing: above-mentioned various reagent is prepared on request, measures qualified rear aseptic subpackaged.Phthalic acid monoclonal antibody working fluid 7mL/ bottle, the anti-7mL/ bottle of horseradish peroxidase mark two, phthalic acid standard solution 1mL/ bottle, substrate solution 7mL/ bottle, substrate buffer solution 7mL/ bottle, reaction terminating liquid 7mL/ bottle, concentrated cleaning solution 50mL/ bottle.Label after packing, indicate lot number and the term of validity, 4 DEG C of preservations.
3, the assembling of kit: will ELISA Plate 1 piece and phthalic acid antibody working fluid, anti-, each 1 bottle of substrate solution, substrate buffer solution, reaction terminating liquid, the concentrated cleaning solution of horseradish peroxidase mark two of phthalic acid antigen be coated with respectively, phthalic acid standard solution 6 bottles, operation instructions 1 part, be placed in assigned address in kit, kit encapsulates after the assay was approved, 4 DEG C of preservations.
the application of embodiment 4 enzyme-linked immunologic detecting kit of the present invention
1, sample pre-treatments
Soy sauce and edible oil sample process: take sample 2.0g, be put in Backflow bottle, adds the mixing of 10mL methyl alcohol, then add the KOH solution that 10mL concentration is 2mol/L, in 85 DEG C of heating water bath 2.5h, finally by methyl alcohol evaporate to dryness, takes out and be cooled to room temperature.With 1:1 hydrochloric acid, hydrolyzate PH is adjusted to 1 ~ 2, after whirlpool concussion 3min, ultrasonic oscillation 20min.Add the normal hexane of 3 times of volumes, whirlpool concussion 3min, leaves standstill 20min, discards normal hexane layer, add the ethyl acetate of 3 times of volumes, whirlpool concussion 3min, leaves standstill 20min, gets ethyl acetate layer, the ethyl acetate of 3 times of volumes is added again in remaining liq, repeat aforesaid operations, three extracts are merged, is evaporated to dry to the greatest extent in rotary evaporator.Methyl alcohol redissolves, and is directly used in detection, extension rate should be counted when result of calculation with after the PBS dilution 15 ~ 20 times of 0.01mol/L, pH5.4.
2, kit of the present invention is utilized to detect
(1) taken out from cold storage environment by kit, be placed in room temperature (20 ~ 24 DEG C) balance more than 30min, the batten of enough standard items and testing sample usage quantity is fixed on support, and standard items and testing sample do two parallel laboratory tests, number in order.
(2) add the phthalic acid standard items of 50 μ L variable concentrations in standard sample wells, sample well adds the testing sample of 50 μ L through pre-treatment.Then every hole adds the antibody of 50 μ L T liquid dilutions, covers the jolting mixing on micro oscillator of cover plate film, at incubated at room 30min.
(3) pour out the liquid in hole, micropore frame is upside down on thieving paper and pats (often wheel is washed plate and patted 3 times) to ensure the liquid removed completely in hole.Be filled with in hole with 300 μ L cleansing solutions, again outwell the liquid in micropore, then repetitive operation 5 times.
(4) add the ELIAS secondary antibody that 100 μ L PBST dilute in every hole, cover the jolting mixing on micro oscillator of cover plate film, at incubated at room 30min.
(5) step (3) is repeated.
(6) every hole adds 100 μ L nitrite ions (substrate buffer solution and substrate solution equal-volume are mixed to get), and pat mixing, cover cover plate film, dark at room temperature hatches 10min.
(7) adding 50 μ L reaction terminating liquids in micropore, after mixing, is blank at wavelength 450nm(with air) place measures each hole light absorption value, must read light absorption value after adding stop buffer in 60min.
3, testing result calculates and analyzes
Inhibiting rate computing formula is:
Inhibiting rate (%)=B/B
0× 100(%)
In formula: B is the luminous value in variable concentrations standard solution hole (or sample well); B
0it is the mean absorbance values of 0 μ g/L standard solution.Take inhibiting rate as ordinate, the logarithm of phthalic acid standard solution concentration is horizontal ordinate drawing standard curve, thus determines the content of phthalic acid in testing sample.The analysis of testing result can also utilize computer professional software to carry out calculating and analyze.
4, testing result
(1) by obtaining phthalic acid canonical plotting (as shown in Figure 1) to the Analysis of test results of standard items, indicating kit of the present invention to phthalic acid linear detection range is 0.36 ~ 5.29ng/mL, sensitivity is 1.38ng/mL, detects and is limited to 0.17ng/mL.
(2) content obtaining phthalic acid in testing sample in above-mentioned typical curve is substituted into the light absorption value of phthalic acid.
Result shows, and the phthalate content in sample soy sauce is 1.2ng/mL.Phthalate content in sample edible oil is 3.2ng/mL.
5, the calculating of phthalate total amount in testing sample
(1) computing method
The content obtaining phthalic acid in testing sample in above-mentioned typical curve is substituted into the light absorption value of phthalic acid; By phthalate integral molar quantity in the molar weight calculation sample of phthalic acid.
In testing sample (as edible oil or soy sauce), the computing formula of the total content of phthalate is as follows:
In formula: X is the total content of PAEs in testing sample, unit is mol/g;
C is the concentration that ELISA detects the phthalic acid obtained, and unit is ng/mL;
M is the quality of testing sample, and unit is g;
N is the extension rate diluting testing sample with the PBS of 0.01mol/L, pH5.4;
V is redissolution methyl alcohol volume, and unit is mL;
166.13 is the relative molecular mass of phthalic acid, and unit is g/mol.
(2) result of calculation display, the phthalate total amount in sample soy sauce is 1.44 × 10
-7mol/g.Phthalate total amount in sample edible oil is 3.85 × 10
-7mol/g.
embodiment 5 enzyme-linked immunologic detecting kit precision of the present invention and accuracy test
1, the replica test of phthalic acid standard solution
In the ELISA Plate prepared according to the method embodiment 3 from 3 batches, respectively extract 20 micropores out, measure the luminous value of 1 μ g/L phthalic acid standard solution according to the detection method of embodiment 4 kit, repeats 20 times, calculating coefficient of variation CV%, result is as shown in table 1.
Table 1 phthalic acid standard solution replica test
Result shows, the variation within batch coefficient range that kit standard product of the present invention detect is between 5.4 ~ 7.1%, and interassay coefficient of variation is 7.8%.
2, testing sample repeatability and accuracy test
Accuracy refers to the matching degree of measured value and true value, and in enzyme-linked immunoassay, accuracy often represents with the recovery, and precision often represents with the coefficient of variation.In blank sample, add phthalic acid to final concentration be 1,2,4 μ g/L, each concentration each 10 parallel, measure 3 batches.Calculating mean value, TIANZHU XINGNAO Capsul, variation within batch coefficient, interassay coefficient of variation.Result is as shown in table 2.
Table 2 sample repeatability and accuracy test result
Result shows, the TIANZHU XINGNAO Capsul of edible oil, soy sauce sample is between 81 ~ 109%, and variation within batch coefficient is between 3.8 ~ 8.9%, and interassay coefficient of variation is between 7.6 ~ 10.8%.
In sum, enzyme-linked immunologic detecting kit of the present invention has extraordinary precision and accuracy, and meets the standard of country for all kinds of index of kit.
embodiment 6 enzyme-linked immunologic detecting kit storage life test of the present invention
1, kit prepared by embodiment 3 is positioned over 2 ~ 8 DEG C, get respectively and deposit 0,2,4,6,8,9,10,11 and kit after 12 months, each parameter such as absorbance, 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient of phthalic acid standard model (1 μ g/L) is measured.
2, placed 12 days under 37 DEG C of conditions of preserving by kit, every day, each parameter such as absorbance, 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient to phthalic acid standard model (1 μ g/L) measured.
3, by kit-20 DEG C of Refrigerator stores 12 days, every day, each parameter such as absorbance, 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient to phthalic acid standard model (1 μ g/L) measured.
4, result shows, through three kinds of condition food preservation test, the absorbance of phthalic acid standard model (1 μ g/L) declines and is less than 8%, and indices all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2 ~ 8 DEG C.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (1)
1. the application of phthalate total amount ELISA enzyme-linked immunologic detecting kit in the phthalate total amount detecting soy sauce, it is characterized in that, described kit comprises following component:
(1) be coated with the ELISA Plate of phthalic acid antigen, described phthalic acid antigen is the conjugate of haptens 4-aminophthalic acid and ovalbumin; Described ELISA Plate is 96 or 40 hole ELISA Plate, and material is polystyrene, is coated with phthalic acid antigen, and the site of phthalic acid antigen is not adsorbed on closed porosity surface;
(2) phthalic acid antibody working fluid, described phthalic acid antibody is the phthalic acid monoclonal antibody that adopts glutaraldehyde method and obtained by phthalic acid and bovine serum albumin(BSA) covalent coupling complex immune SPF level Balb/c mouse or phthalic acid rabbit polyclonal antibody; 8000 times are diluted with T liquid during use; The formula of described T liquid is: the Tris taking 1.21g, uses 800mL deionized water dissolving, regulates solution to required pH value, be settled to 1L with deionized water with 1mol/L hydrochloric acid solution.
(3) two of horseradish peroxidase-labeled resist;
(4) the phthalic acid standard solution of 1mg/mL; Standard solution is diluted to phthalic acid standard solution with phosphate buffer when using by described phthalic acid standard solution;
(5) substrate solution: the pH5.0 phosphate citrate acid buffering solution containing 3,3,5,5-tetramethyl benzidine or o-phenylenediamine;
(6) substrate buffer solution: the phosphate citrate acid buffering solution of the pH5.0 containing hydrogen peroxide or urea peroxide;
(7) reaction terminating liquid: 1 ~ 2mol/L sulfuric acid solution or 2mol/L sodium hydroxide solution;
(8) concentrated cleaning solution: containing 0.5 ~ 1.5%(v/v) phosphate buffer of Tween-20, the pH value of concentrated cleaning solution is 7.4, concentration is 0.1mol/L;
The concrete grammar of described application comprises the steps:
S1. kit is taken out from cold storage environment, be placed in equilibrium at room temperature;
S2. by standard items or add through the testing sample of pre-treatment and be coated with in the ELISA Plate hole of phthalic acid antigen, then every hole adds antibody, pats mixing, hatches;
S3. wash with cleansing solution;
S4. add horseradish peroxidase-labeled two resist, and pat mixing, hatch;
S5. wash with cleansing solution;
S6. substrate buffer solution and substrate solution equal-volume are mixed to get nitrite ion, add nitrite ion in every hole, pat mixing, lucifuge is hatched;
S7. every hole adds reaction terminating liquid, mixes, under wavelength 450nm or 492nm, is blank with air, measures each hole light absorption value;
S8. testing result treatment and analyses: the content determining phthalic acid in sample, according to the total content of phthalate in the cubage sample of phthalic acid;
Wherein, testing sample described in step S2 is soy sauce; The pre-treating method of soy sauce sample is as follows:
S21. take sample 2.0g, be put in Backflow bottle, add the mixing of 10mL methyl alcohol, then add the KOH solution that 10mL concentration is 2mol/L, in 85 DEG C of heating water bath 2.5h, by methyl alcohol evaporate to dryness, take out and be cooled to room temperature;
S22. by volumetric concentration be 50% hydrochloric acid hydrolyzate pH is adjusted to 1 ~ 2, whirlpool concussion 3min after, ultrasonic oscillation 20min;
S23. add the normal hexane of 3 times of volumes, whirlpool concussion 3min, leaves standstill 20min, discards normal hexane layer, add the ethyl acetate of 3 times of volumes, and whirlpool concussion 3min, leaves standstill 20min, get ethyl acetate layer, add the ethyl acetate of 3 times of volumes in remaining liq again;
Three extracts are merged, are evaporated to dry to the greatest extent in rotary evaporator by S24. S23 operation in triplicate;
S25. methyl alcohol redissolves, and is directly used in detection, extension rate should be counted when result of calculation with after the PBS dilution 15 ~ 20 times of 0.01mol/L, pH5.4.
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| CN104130144A (en) * | 2014-07-30 | 2014-11-05 | 新疆农垦科学院 | Synthetic method and application of universal hapten and antigen for phthalate plasticizer |
| CN104530221B (en) * | 2014-12-17 | 2017-09-01 | 西南大学 | A kind of synthetic method of phthalate compound general artificial antigen |
| CN105738611A (en) * | 2015-11-23 | 2016-07-06 | 轻工业环境保护研究所 | Benzo(a)pyrene enzyme-linked immunosorbent assay (ELISA) kit and detection method thereof |
| CN109724930B (en) * | 2018-12-27 | 2021-10-26 | 北京普赞生物技术有限公司 | Detection kit and detection method for dibutyl phthalate in edible oil and grease-containing food |
| CN110530835B (en) * | 2019-09-27 | 2022-03-04 | 山西省农业科学院农产品加工研究所 | Fluorescence method detection method for phthalate plasticizer in oil substances |
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