CN104558182A - Monoclonal antibody for detecting dibutyl phthalate and butyl benzyl phthalate, and application of monoclonal antibody - Google Patents

Abstract

The invention discloses a specificity monoclonal antibody capable of distinguishing dibutyl phthalate and butyl benzyl phthalate, and further discloses an enzyme linked immunosorbent assay method and kit for detecting the dibutyl phthalate and the butyl benzyl phthalate. According to the invention, the monoclonal antibody is secreted by a hybridoma cell strain DBP3B2 of which the preservation number is CCTCC NO: C201497. Compared with the prior art, the monoclonal antibody disclosed by the invention can distinguish the dibutyl phthalate and the butyl benzyl phthalate at the same time, the lowest detectable limit (IC50) to DBP is only 44.33 microgram/L, the lowest detectable limit to BBP is only 62.59 microgram/L, and the monoclonal antibody has a higher cross reaction rate to the BBP; therefore, the monoclonal antibody has higher detection sensitivity and specificity for two phthalate plasticizers of the DBP and BBP.

Description

For detecting monoclonal antibody and the application thereof of dibutyl phthalate and phthalic acid tolyl butyl ester

Technical field

The invention belongs to wild animal resources and immunological technique field, be specifically related to a kind of monoclonal antibody and the application thereof that can identify dibutyl phthalate and phthalic acid tolyl butyl ester.

Background technology

Phthalate (phthalate esters, PAEs) is commonly called as fluidizer or phthalate, is the derivative of phthalic acid.Phthalate basic structure is made up of a phenyl ring and two side chains, two side-chain radicals may be the same or different, kind more than 20 is had according to the difference of side chain, common are dimethyl phthalate (dimethyl phthalate, DMP), diethyl phthalate (diethyl phthalate, DEP), dibutyl phthalate (dibutyl phtalate, DBP), dipropyl phthalate (dipropyl phthalate, DPrP), phthalic acid two (2-ethylhexyl) ester (di (2-ethylhexyl) phthalate, DEHP) etc.Phthalate Chang Zuowei softening agent is used for industrial production, and this kind of material adds in plastics, improving the snappiness of plastics, because being that a kind of physical property is combined with plastics, As time goes on, migrating to external environment.Because fluidizer can replace plam oil, produce thickening effectiveness, illegal businessman adds cheaply poisonous fluidizer to jelly, beverage, in the food such as Yoghourt, enters human body by food, produces class female hormone effect, can cause endocrine disturbance etc.After within 2011, detecting fluidizer first in Taiwan Food, within 2012, Jiu Gui Jiu also detects fluidizer, makes fluidizer contaminated food products become popular focus, more and more receives the concern of human consumer.

European Union issued 2007/19/EC instruction in 2007, regulation DEHP, DBP, BBP, DIDP, DINP, DnOP, DAP Special migration (SML) is respectively 1.5mg/kg, 0.3mg/kg, 30mg/kg, 9mg/kg, 9mg/kg, 5mg/kg, is less than 0.01mg/kg, and every day, maximum intake was 0.3mg/kg.Environmental Protection Agency specifies can not more than 0.1% in the toy that DBP, DEHP and BBP content in child-bearing article and toy can not use at child-bearing article and 3 years old children more than 0.1%, DINP, DIDP and DNOP.China GB 9685-2008 " food product containers, wrapping material additive use hygienic standard " also clear stipulaties DEHP, DBP, DINP maximum residue limit in food and foodstuff additive is respectively 1.5mg/kg, 0.3mg/kg, 9.0mg/kg, and GB/T 21911-2008 " mensuration of Phthalic Acid Esters in Food " specifies: containing oil sample with do not contain the detection limit of each phthalate compound of oil sample and be respectively 1.5mg/kg, 0.05mg/kg.Therefore, preparation can identify the monoclonal antibody of phthalate and to research and develop efficient enzyme-linked immunoassay method very necessary.

CN 103257115 A for haptens, has prepared the monoclonal antibody that can identify DBP with 4-aminophthalic acid dibutylester; CN 104004719 A is using 4-aminophthalic acid dibutylester as haptens, prepare the monoclonal antibody that can identify DBP, BBP, DiBP, DNP, DMP, the compound that Tetra hydro Phthalic anhydride and propyl carbinol, 4-Aminobutanoicacid are obtained by reacting by CN 103869070 A, as haptens, has prepared the monoclonal antibody that can identify DBP, DiBP; CN 103204925 A is with phthalic acid and 6-(fluorenes methoxy carbonyl sulphonyl-amino)-1-hexanol generation esterification; slough fluorenes methoxy carbonyl acyl protecting groups again; prepared a kind of phthalate universal hapten, the sensitivity of antibody to DBP, DEHP and DINP of preparation is low.Visible, the enzyme-linked immune detection method of current phthalate mainly concentrates on haptenic research, the monoclonal antibody adopting existing haptens to prepare or sensitivity and poor specificity, the phthalic ester kind that can identify is single, does not still have the monoclonal antibody that simultaneously can identify DBP and BBP in prior art.

Summary of the invention

First object of the present invention is to provide a kind of highly sensitive and can the monoclonal antibody of specific recognition dibutyl phthalate and phthalic acid tolyl butyl ester.

Second object of the present invention utilizes this monoclonal antibody, prepares a kind of enzyme linked immunological kit detected for dibutyl phthalate and phthalic acid tolyl butyl ester.

3rd object of the present invention utilizes this test kit, sets up a kind of enzyme-linked immunoassay method that can be used for dibutyl phthalate and phthalic acid tolyl butyl ester residue detection in food and drink.

The present invention is achieved through the following technical solutions:

Can identify a monoclonal antibody for dibutyl phthalate and phthalic acid tolyl butyl ester, it is by preserving number secreted by the hybridoma cell strain DBP3B2 of CCTCC NO:C201497.

Above-mentioned hybridoma cell strain DBP3B2, is deposited in China typical culture collection center (CCTCC), and its preserving number is CCTCC NO:C201497.

Monoclonal antibody described in preparation immunogen used reacts the haptens 4-hydroxyl phthalic dibutylester generated, with the conjugate of human serum albumin (HSA) by 4-hydroxyl phthalic and propyl carbinol.

Further, the invention provides and a kind ofly detect the enzyme-linked immunoassay method that in beverage, dibutyl phthalate and phthalic acid tolyl butyl ester are residual, the method comprises the following steps:

(1) by 4-hydroxyl phthalic and propyl carbinol be haptens 4-hydroxyl phthalic dibutylester (4-DBHP) prepared by raw material, N, N-carbonyl dimidazoles (CDI) method of employing and ovoserum albumin (OVA) coupling obtain coating antigen (4-DBHP-CDI-OVA);

(2) monoclonal antibody is prepared with the hybridoma cell strain DBP3B2 that preserving number is CCTCC NO:C201497;

(3) use the coating antigen bag of step (1) by solid phase carrier;

(4) enzyme linked immunosorbent detection is carried out after being extracted by testing sample.

Preferably, the extracting method of described testing sample is: beverage is centrifugal, gets supernatant, gets 2mL, and adding volume ratio is PBS damping fluid: ethanol=8: the solution 8mL of 2 dilutes.

The present invention using said monoclonal antibody and coating antigen as core reagent with other conventional agent combination, make the enzyme-linked immunologic detecting kit that can detect dibutyl phthalate and phthalic acid tolyl butyl ester, in conjunction with above-mentioned enzyme-linked immunoassay method, achieve the enzyme linked immunosorbent detection to dibutyl phthalate and phthalic acid tolyl butyl ester.

Major advantage of the present invention is:

1) the present invention is when prepared by antibody, adopt 4-DBHP as haptens, fully expose antigenic determinant, the monoclonal antibody prepared by this antigen can identify dibutyl phthalate and phthalic acid tolyl butyl ester simultaneously, and existing monoclonal antibody cannot identify this two kinds of medicines simultaneously.

2) the present invention set up ELISA method and test kit to the lowest detectable limit (IC of DBP ₅₀) be only 44.33 μ g/L, 62.59 μ g/L are only to the lowest detectable limit of BBP, and to BBP, there is higher cross reacting rate, illustrate have higher detection sensitivity and specificity to these two kinds of phthalic ester fluidizers of DBP and BBP.

3) ELISA method that the present invention sets up can detect dibutyl phthalate in beverage and phthalic acid tolyl butyl ester simultaneously, method accuracy is high, detection efficiency is high, compared with the existing methods, detection kind there is obvious advantage, can save time and cost, have good marketable value.The sample treatment related in method is simple, and easy to operate, organic reagent used is little to operator's body harm.

Accompanying drawing explanation

Fig. 1 is the UV scanning collection of illustrative plates of haptens used in the present invention, human serum albumin (HSA) and immunogen (4-DBHP-CDI-HSA).

Fig. 2 is the UV scanning collection of illustrative plates of haptens used in the present invention, ovoserum albumin (OVA) and coating antigen (4-DBHP-CDI-OVA).

Fig. 3 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and dibutyl phthalate standard substance, X-axis is dibutyl phthalate concentration of standard solution logarithmic value, and Y-axis is that the optical density value of dibutyl phthalate standard solution is divided by " zero " hole optical density value (B/B ₀).

Embodiment

Below by embodiment, the invention will be further described, but do not limit the present invention.

The preparation of embodiment 1 immunogen and coating antigen

1.1 immunogen 4-DBHP-CDI-HSA preparations

Take 4-hydroxyl phthalic 3g and be dissolved in propyl carbinol 4mL, under agitation condition, add dense H ₂sO ₄0.3mL catalysis, after 100 DEG C of stirring and refluxing 4h, revolves steaming, product 10%Na ₂cO ₃washing, to water layer colourless (pH value is 8), is got organic layer and is revolved steaming, be i.e. haptens 4-hydroxyl phthalic dibutylester (4-DBHP).Take 28mg 4-DBHP and be dissolved in 200 μ L1, in 4-dioxane, add 50mg CDI (N simultaneously, N-carbonyl dimidazoles) and 60mgDMAP (DMAP), the activation of mixture room temperature is spent the night, and the reaction solution of activation is added drop-wise to HSA40mg, 4 DEG C of reactions are spent the night, centrifugally remove supernatant, dialysis 3d, changes 2 dialyzates every day, dialyzate is pH7.4 phosphate buffered saline buffer, centrifugal removing precipitation after dialysis, lyophilized, obtains coating antigen 4-DBHP-CDI-HSA.Synthetic route is as follows:

The preparation of 1.2 coating antigen 4-DBHP-CDI-OVA

Take 28mg 4-DBHP and be dissolved in 200 μ L1, in 4-dioxane, add 50mg CDI and 60mgDMAP simultaneously, the activation of mixture room temperature is spent the night, the reaction solution of activation is added drop-wise to OVA44mg, and 4 DEG C of reactions are spent the night, and centrifugally remove supernatant, dialysis 3d, change 2 dialyzates every day, dialyzate is pH7.4 phosphate buffered saline buffer, centrifugal removing precipitation after dialysis, lyophilized, both obtained coating antigen 4-DBHP-CDI-OVA.Synthetic route is as follows:

The preparation of embodiment 2 monoclonal antibody

The preparation of hybridoma: with reference to Yang Hanchun " animal immunology ", immunogen 4-DBHP-CDI-HSA conjugate immunity Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov) prepared with embodiment 1.Immune programme for children is: fundamental immunity, by the neck dorsal sc being injected in mouse containing the immunogenic protein emulsion of 50 μ g of immunogen and isopyknic Freund's complete adjuvant emulsification, carried out booster immunization with Freund's incomplete adjuvant emulsification containing the immunogenic protein emulsion of 100 μ g at interval of 15 days later.From immunity three times.Within after each immunity the 8th day, adopt tail blood, separation of serum, indirect elisa method detects serum antibody titer.The mouse of immuno-competent (height of tiring, sensitivity good) stops immunity in order to merging.

During fusion, the Balb/C mouse of last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, isolates splenocyte, with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of fresh preparation by 1 ~ 2 × 10 ⁷individual SP2/0 and 10 ⁸the ratio of individual immune spleen cell (1:10 ~ 1:15) is in 50mL centrifuge tube, and with 15mLRPMI-1640 basal liquid re-suspended cell, the centrifugal 5min of 1500r/min, washes cell 1 time.The substratum that temperature is bathed by centrifugal gap, the water of temperature bath, super clean bench put into by the polyoxyethylene glycol (PEG) etc. of temperature bath.Then take out the thieving paper of sterilizing, by the centrifuge tube that myeloma cell and immune spleen cell be housed emptying to the greatest extent, tipping upside down on and thieving paper controls solid carbon dioxide dripping, rapping at the bottom of pipe and cell is loosened.Open timing register, draw 0.8mLPEG, the hand-held centrifuge tube that cell mixing is housed with 1mL suction pipe, place it in a moment in water-bath, be added drop-wise to slowly on cell mixing by PEG, limit edged stirs gently, adds in 1min, Keep agitation 30s.Get 10mL basal liquid with suction pipe, be slowly added on fused cell along tube wall, limit edged shakes gently (can not blow and beat), adds 1mL respectively in 5min, 2mL, 3mL, 4mL, finally add basal liquid to 40mL, after adding, cover lid, puts upside down several times repeatedly, and cell is mixed.800r/min 5min is centrifugal, abandons supernatant.Draw the HAT substratum containing feeder cell, stirred gently by the fused cell in centrifuge tube, be dropwise added dropwise in the serum bottle containing feeder cell, stirring and evenly mixing near liquid level with suction pipe, action wants light, is stirred gently by cell, must not blow and beat.Put upside down mixing.Then be seeded on Tissue Culture Plate by cell, two, every hole, is placed in incubator and cultivates.Single cell fusion can inoculate 4 ~ 6 piece of 96 orifice plate.Also can plant less as required, the cell count of generally pressing SP2/0 calculates, and every hole inoculum size is about containing 10 ⁴a left and right SP2/0 cell.In 37 DEG C, 5%CO ₂cultivate in incubator.

The same day of merging counts 0d, and front 3d tries not kinetocyte plate, keeps incubator homeostasis.3d adds in every hole 1 HAT perfect medium; 5d every hole sucking-off l/2 culture supernatant (100 μ L), then add 1 HT perfect medium; Suck l/2 ~ 3/4 culture supernatant every the same method of 2d later, after 7d, change to HT perfect medium.

Cell colony to be fused grows to culture hole 1/10 ~ 1/5, screens with the indirect ELISA method set up simultaneously.Compared with zero medicine hole, medicine hole OD value repressedly can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, select the cell hole only having 1-2 single colony of 2 ~ 6 strong positives, adopt limited dilution method to carry out cloning.

Through 3 ~ 4 time clonings, finishing screen selects the monoclonal hybridoma strain of secreting anti-dibutyl phthalate antibody, applicant is by its called after DBP3B2, and China typical culture collection center (CCTCC) preservation being positioned at Wuhan City, Hubei Province Wuhan University is sent on May 20th, 2014, deposit number is CCTCC NO:C201497.Chromosome counting has been carried out to this clone, result shows, and the chromosomal mean number of SP2/0 is 58, and splenocyte karyomit(e) is 40, and the chromosome number of hybridoma is 102.2, the cell of SP2/0 really of fused cell and the hybrid product of splenocyte are described.By this cell strain through abdominal injection Balb/C mouse, manufacture order clonal antibody.Adopt and carry out hypotype qualification purchased from the mouse monoclonal antibody Rapid ELISA homotype detection kit of Thermo Scientific company to the monoclonal antibody that the present invention obtains, result is mouse IgG ₁hypotype.

The foundation of embodiment 3 racing ELISA detecting method

The preparation (reagent that the present embodiment uses all adopts following methods preparation except another indicating) of 3.1 reagent

Phosphate buffered saline buffer: NaCl 8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o 2.9g, KCl 0.2g, adds distilled water to 1000mL, regulates pH to 7.4;

Coating buffer: get Na ₂cO ₃1.59g, NaHCO ₃2.93g, adds tri-distilled water to 1000mL, adjust ph to 9.6;

Washings: NaCl 8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o 2.9g, KCl 0.2g, Tween 20 0.5mL, adds distilled water to 1000mL, regulates pH to 7.4;

Confining liquid: ovalbumin 1g is dissolved in 100mL phosphate buffered saline buffer;

Substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

Substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% Urea Peroxide 6.4mL, adds distilled water to 1000mL;

Substrate cocktail: by A liquid and B liquid by volume 1:1 mix and get final product, now with the current;

Stop buffer: 2mol/L sulphuric acid soln.

Tentatively determining of 3.2 coating antigen concentration and antibody working concentration

Square formation volumetry: select the 4-DBHP-CDI-OVA of above-mentioned synthesis as coating antigen, 8 concentration such as 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L are diluted to coating buffer, at 96 hole enzyme plates, from first to the 8th leu adds, and 4 DEG C are spent the night; Wash 3 times, pat dry, add confining liquid 200 μ L, 37 DEG C of closed 60min; Wash 3 times, pat dry, the first row to the 8th row of enzyme plate add successively 100 μ L phosphate buffered saline buffers dilution extension rate be 500,1000,2000,4000,8000,16000,32000,64000 monoclonal antibody, hatch 30min for 37 DEG C, wash 3 times, pat dry; The sheep anti-mouse igg antibody that each hole adds the HRP mark of 1:5000 times of phosphate buffered saline buffer dilution (is called for short two to resist, the anti-sheep anti-mouse igg antibody being HRP mark of following indication two, purchased from Wuhan Fei Yi Bioisystech Co., Ltd) 100 μ L, hatch 30min for 37 DEG C, wash 5 times, pat dry; Each hole adds 100 μ L Substrate cocktail, and lucifuge colour developing 15min, adds 50 μ L stop buffers, measure optical density value (OD value) by automatic microplate reader at 450nm wavelength place.The results are shown in Table 1, tentatively determine that bag is 4 μ g/mL by concentration, antibody working concentration is 1:8000.

Table 1 wraps tentatively determining by concentration and antibody dilution

The determination of 3.3 best coating antigen concentration and antibody working concentration

With the bag determined at first by concentration coated elisa plate, dibutyl phthalate phosphate buffered saline buffer is diluted to 0,160,80,40,20,10 μ g/L, 6 concentration, antibody take 1:8000 as intermediate concentration, designs two concentration gradients by equal difference, its " 0 " hole and IC ₅₀value is in table 2.Select OD value at about 2.0, IC ₅₀the lower corresponding antibody dilution of value is best anti-extent of dilution.Result shows, selects 1:8000 to be optimum antibody extent of dilution.

Table 2 optimum antibody extent of dilution is optimized

Antibody dilution multiple (1:X)	" 0 " hole OD value	IC 50(μg/L)
			7500	2.143	50.85
8000	2.092	48.43
			8500	2.018	49.96

The foundation of 3.4 typical curves

Dibutyl phthalate phosphate buffered saline is become 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L, 6 concentration gradients, each concentration repeats 5 holes, measures, replication 5 times according to indirect competitive ELISA method.With the logarithmic value of dibutyl phthalate strength of solution for X-coordinate, B/B ₀for ordinate zou drawing standard curve, obtain IC ₅₀.This test kit IC ₅₀value is 44.33 ± 1.42 μ g/L.

3.5 cross reaction tests

By phthalate: dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), dipropyl phthalate (DPrP), diallyl phthalate (DAP), diamyl phthalate (DPP), dimethyl phthalate (DMP), diethyl phthalate (DEP), diisopropyl phthalate (DiPrP), diisobutyl phthalate (DiBP), dihexyl phthalate (DHP), dicyclohexyl phthalate (DCHP), dinoctyl phthalate (DOP), dimixo-octyl phthalate (DiOP), 2-nonyl-phthalate ester (DNNP), phthalic acid (2-ethylhexyl) ester (DEHP), the different certain herbaceous plants with big flowers ester (DIDP) of phthalic acid two, diisononyl phthalate (DINP), dinoctyl phthalate (DnOP), becomes proper concn with phosphate buffered saline, measures the IC of each medicine by the ELISA method set up ₅₀value, the multiple hole of each medicine 3, with monoclonal antibody to the cross reacting rate of dibutyl phthalate for 100%, utilize formula 1 to calculate the cross reacting rate of monoclonal antibody to each medicine.The results are shown in Table 3, this antibody has certain recognition capability to BBP, DPrP, DAP, DPP, and its cross reacting rate is respectively 71.40%, 12.83%, 2.99%, 1.75%, is less than 1% to the cross reacting rate of other phthalate; Meanwhile, this antibody is to the lowest detectable limit (IC of DBP and BBP ₅₀) be respectively 44.33 μ g/L and 62.59 μ g/L, significantly lower than other phthalate.Therefore this antibody can be used for the detection of DBP and BBP.

Table 3 test kit of the present invention is to the cross reacting rate of phthalate

Medicine	IC 50(μg/L)	Cross reacting rate (%)	Regression equation	Correlation coefficient r
					DBP	44.33	100	y＝－0.581x+1.4584	0.9969
BBP	62.59	71.40	y＝－0.7234x+1.7996	0.9984
					DPrP	348.24	12.83	y＝－0.3427x+1.3711	0.9916
DAP	1493	2.99	y＝－0.3649x+1.6583	0.9924
					DPP	2548.58	1.75	y＝－0.3874x+1.8196	0.9925
DMP	>4469	<1
					DEP	>4469	<1
DiPrP	>4469	<1
					DiBP	>4469	<1
DHP	>4469	<1
					DCHP	>4469	<1
DOP	>4469	<1
					DiOP	>4469	<1
DNNP	>4469	<1
					DEHP	>4469	<1
DIDP	>4469	<1
					DINP	>4469	<1
DnOP	>4469	<1

The assembling of embodiment 4ELISA test kit

4.1 ELISA kit of the present invention are made up of following part:

1) solid phase carrier (enzyme plate) of coating antigen 4-DBHP-CDI-OVA is coated with;

2) 6 bottles, dibutyl phthalate standardized solution, concentration is respectively 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L;

3) preserving number is the monoclonal antibody of the hybridoma cell strain DBP3B2 secretion of CCTCC NO:C201497;

4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);

5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄12H ₂o 29.0g, KCl 2.0g, adds distilled water to 1000mL;

6) concentrated cleaning solution: NaCl 80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄12H ₂o 29.0g, KCl 2.0g, Tween20 5mL, add distilled water to 1000mL;

7) substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

8) substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, adds distilled water to 1000mL, regulates pH to 5.0 ~ 5.4;

9) stop buffer: 2mol/L sulphuric acid soln.

The preparation of 4.2 enzyme plates

With coating buffer, 4-DBHP-CDI-OVA is diluted to 4mg/L, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L washingss and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 120min for 37 DEG C, incline liquid in hole, washings washs 3 times, pats dry, and preserves with masking foil vacuum-sealing.

The mensuration program of embodiment 5 enzyme linked immunological kit

The preparation of 5.1 reagent

1) sample diluting liquid: use after the concentrated phosphoric acid salt buffer tri-distilled water provided in test kit is diluted 10 times.

2) washings: use after the washings tri-distilled water provided in test kit is diluted 10 times.

3) Substrate cocktail: according to each institute expense, by the substrate solution A of preparation and substrate solution B by volume 1:1 mixing, now with the current.

5.2 sample pre-treatments: beverage is centrifugal, get supernatant, get 2mL, and adding volume ratio is PBS damping fluid: the solution 8mL of ethanol=8:2 dilutes.

5.3 determination step

1) application of sample: add dibutyl phthalate series concentration standardized solution or sample solution 50 μ L in enzyme plate micropore, then add monoclonal antibody working fluid 50 μ L, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

2) wash: pour out the liquid in hole, add washings 250 μ L in every hole and wash 3 times and pat dry;

3) the sheep anti-mouse igg antibody working fluid that horseradish peroxidase (HRP) marks is added: in every hole, add the sheep anti-mouse igg antibody working fluid 100 μ L that horseradish peroxidase (HRP) marks, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

4) wash: pour out the liquid in hole, in every hole, add washings 250 μ L, wash 3 times and pat dry;

5) substrate is added: add Substrate cocktail 100 μ L in every hole, be placed in wet box, 37 DEG C of constant-temperature incubation 15min;

6) stop buffer is added: in every hole, add stop buffer 50 μ L;

7) measure: the optical density value (OD value) measuring every hole by microplate reader at 450nm place.

5.4 results judge

Typical curve:

With measured standard substance OD value divided by " zero " hole OD value (B/B ₀) be ordinate zou, the logarithmic value of dibutyl phthalate concentration is that X-coordinate makes typical curve, and line linearity of going forward side by side returns, and provides regression equation.

In beverage, dibutyl phthalate concentration calculates:

The inhibiting rate (the OD value of the sample obtained is divided by " zero " hole OD value) of calculation sample, substitute in the regression equation of typical curve, and be multiplied by dilution factor 5, calculate dibutyl phthalate concentration in beverage (μ g/kg) and be converted to phthalic acid tolyl butyl ester concentration (extension rate 5, μ g/kg) according to formula 2.

The sensitivity of embodiment 6 test kit of the present invention, precision, accuracy, replica test

The sensitivity test of 6.1 test kits of the present invention

With the IC of typical curve ₅₀value and organize lowest detectable limit (LOD) as the sensitivity index of detection kit of the present invention.The dilution of dibutyl phthalate standard substance is become 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L, 6 concentration, the multiple hole of each concentration 5, according to indirect competitive ELISA method replication 5 times, get the IC measured for 5 times ₅₀mean value.LOD is determined by following steps, measures the OD value of 20 parts of blank drink samples, goes out corresponding dibutyl phthalate concentration, then calculate the mean value of dibutyl phthalate concentration according to the regression equation calculation of typical curve with standard deviation (SD), according to formula calculate the lowest detectable limit in tissue, according to formula calculate the minimum quantitative limit in beverage.The IC of dibutyl phthalate of the present invention ₅₀value is 48 μ g/L, LOD value 9.4 μ g/L, and LOQ value is 15 μ g/L.The IC of phthalic acid tolyl butyl ester ₅₀value is 72 μ g/L, LOD value 14.8 μ g/L, and LOQ value is 20.8 μ g/L.

The precision test of 6.2 test kits of the present invention

Dibutyl phthalate standard substance are diluted to 160 μ g/L, 80 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L, 6 concentration, the multiple hole of every concentration 5, according to indirect competitive ELISA method replication 5 times, the regression equation calculation of application standard curve goes out the measured value of each concentration dibutyl phthalate standardized solution, the variation coefficient in computing board and between plate, the results are shown in Table 4.

Error in the plate of table 4 typical curve and between plate

The accuracy of 6.3 test kits of the present invention, replica test

Add in drink sample by dibutyl phthalate and phthalic acid tolyl butyl ester, its TIANZHU XINGNAO Capsul, between 77.3% ~ 90%, criticizes interior and interassay coefficient of variation≤8.6%; Concrete measurement result is in table 5.

TIANZHU XINGNAO Capsul in table 5 beverage

Claims (8)

1. can identify a monoclonal antibody for dibutyl phthalate and phthalic acid tolyl butyl ester, it is by preserving number secreted by the hybridoma cell strain DBP3B2 of CCTCC NO:C201497.

2. a strain of hybridoma strain DBP3B2, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201497, the monoclonal anti physical efficiency identification dibutyl phthalate of described hybridoma cell strain secretion and phthalic acid tolyl butyl ester.

3. monoclonal antibody according to claim 1 is preparing the application in the enzyme linked immunological kit detecting dibutyl phthalate and phthalic acid tolyl butyl ester.

4. comprise the test kit of monoclonal antibody described in claim 1.

5. test kit according to claim 4, this test kit is the enzyme linked immunological kit for detecting dibutyl phthalate and phthalic acid tolyl butyl ester.

6. the test kit described in claim 4 or 5 is detecting the application that in food, dibutyl phthalate and phthalic acid tolyl butyl ester remain.

7., for detecting the enzyme-linked immunoassay method that in beverage, dibutyl phthalate and phthalic acid tolyl butyl ester remain, comprise the following steps:

(1) with 4-hydroxyl phthalic dibutylester for haptens, obtain coating antigen with the coupling of ovoserum albumin;

(2) monoclonal antibody is prepared with the hybridoma cell strain DBP3B2 that preserving number is CCTCC NO:C201497;

(3) use the coating antigen bag of step (1) by solid phase carrier;

(4) enzyme linked immunosorbent detection is carried out after being extracted by testing sample.

8. according to claim 7 for detecting the enzyme-linked immunoassay method that in beverage, dibutyl phthalate and phthalic acid tolyl butyl ester remain, it is characterized in that: the extracting method of described testing sample is: beverage is centrifugal, get supernatant, get 2mL, adding volume ratio is PBS damping fluid: ethanol=8: the solution 8mL of 2 dilutes.