CN101419230A - ELISA detecting method for di-isooctyl-phthalate - Google Patents

Abstract

The invention relates to an enzyme-linked immunoassay method for diisooctyl phthalate, and belongs to the technical field of immunodetection. The invention utilizes immunity of synthesized diisooctyl phthalate immunogen to obtain a polyclonal antibody, and takes the diisooctyl phthalate as standard and conjugate of diisooctyl phthalate hapten and OVA as envelope antigen, to establish indirect competitive enzyme-linked immunoassay method for the diisooctyl phthalate. The invention establishes the indirect ELISA method for the diisooctyl phthalate, and provides a quick and high-efficiency detecting method for detecting residual of the diisooctyl phthalate. The method adopts the polyclonal antibody, so the cost is lower and the stability and repetitiveness are good; the sensitivity is 0.085ng/mL, and the linear range is between 0.1 and 25ng/mL; and high specificity and compatibility of an immunoreaction make the ELISA have extremely high selectivity and sensitivity.

Description

A kind of enzyme-linked immune detection method of diisooctyl phthalate

Technical field

The present invention relates to a kind of phthalate ester residue detection method, to a kind of enzyme-linked immune detection method of diisooctyl phthalate, belong to technical field of immunoassay specifically.

Background technology

(Diethylhexyl phthalate DEHP) is a kind of of phthalate ester to diisooctyl phthalate.Phthalate ester is especially used in the processing of Polyvinylchloride (PVC) in processing of plastic is produced widely as plastifier.Owing to do not form covalent bond between phthalate ester plastifier and the plastic matrix, but be connected with Van der Waals force with hydrogen bond, maintenance chemical property independently separately each other, thereby just can stripping when contained water, grease in touching packaged food.Through discovering that phthalate ester is a kind of environmental hormone material, it disturbs biological is generation, release, transfer, metabolism, reaction and the elimination that keeps the normal hormone of homeostasis and modulated growth processes, or causes the bad health effect and the change of endocrine function in int biological offspring.Phthalic acid two (2-ethylhexyl) ester claims diisooctyl phthalate again, is a kind of of consumption maximum in the phthalic ester plasticizer, is widely used, and the content in plastics can reach 60%.At present, the detection of relevant diisooctyl phthalate both at home and abroad mainly contains high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc., but these methods not only need expensive instrument and equipment, the operating personnel of specialty, to the requirement of sample also than higher, and need further sample pre-treatment just can carry out, this can not reach modern and detect quick, convenient, requirement accurately.In recent years, carried out the research of terephalic acid ester para-immunity analytical approach both at home and abroad, but still there is not report both at home and abroad at the enzyme linked immunosorbent detection of diisooctyl phthalate, in order to remedy this blank, be necessary to set up a kind of enzyme-linked immune detection method at diisooctyl phthalate.

Summary of the invention

The object of the present invention is to provide the residual immunological detection method of a kind of fast detecting diisooctyl phthalate and higher sensitivity and specificity are arranged.

Technical scheme of the present invention is as follows: utilize that the petty official is transmitted, (a kind of preparation method of diisooctyl phthalate artificial antigen) synthetic immunogen immune such as Xu Liguang, Ma Wei obtain polyclonal antibody, as envelope antigen, set up the residual indirect competitive enzyme-linked immunosorbent method of diisooctyl phthalate with the conjugate of diisooctyl phthalate haptens and OVA.

The present invention realizes by following steps:

(1) carbonate buffer solution with 0.05M dilutes envelope antigen with 1:5000～1:8000, adds in the ELISA Plate, and every hole adds 100 μ L.4 ℃ of overnight incubation use the above-mentioned carbonate buffer solution 200 μ L/ holes that contain 0.1% gelatin as confining liquid, sealing 2h then;

(2) with 0.01,0.1,0.5,1,5,10, the diisooctyl phthalate standard items of 20ng/mL series concentration gradient or the sample of handling well are in enzyme mark hole separately, and every hole adds 50 μ L; Add antiserum simultaneously, every hole 50 μ L with antibody diluent dilution 1:6000～1:9000.Wash 3～5 times with the PBST cleansing solution behind 37 ℃ of competition 1h.

(3) the goat-anti rabbit (GAR-HRP) of horseradish peroxidase-labeled is diluted with 1:3000～1:5000 with antibody diluent, every hole adds 100 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of effect 1h.

(4) configuration colour developing liquid: A:0.933g citric acid, 3.68g Na ₂HPO ₄12H ₂O, 18 μ L30% H ₂O ₂Be settled to 100mL with ultrapure water;

B:60mg 3,3/, 5, and 5-tetramethyl benzidine (TMB) is dissolved in the 100mL ethylene glycol;

Before using A is mixed with the 5:1 volume ratio with B.

(5) every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 15min; Add stop buffer 2M sulfuric acid solution at last, every hole 100 μ L; Microplate reader 450nm surveys light absorption value A ₄₅₀, calculate the diisooctyl phthalate concentration of testing sample with the contrast of work typical curve.

More detailed step is:

Main solution preparation

1) preparation phosphate 0.01M (PBS) damping fluid:

Na ₂HPO ₄·12H ₂O 3.62g

KH ₂PO ₄ 0.2g

NaCl 0.2g

KCl 8.0g

Add ultrapure water and be diluted to 1000mL.

2) preparation (0.05M) pH9.6 carbonate (CBS) buffer solution:

Na ₂CO ₃ 1.59g

NaHCO ₃ 2.93g

Add ultrapure water and be diluted to 1000mL.

3) preparation PBST solution: the PBS solution that contains 0.05% Tween-20.

4) preparation confining liquid: the carbonate buffer solution that contains 0.1% gelatin.

5) preparation antibody diluent: the PBST solution that contains 0.1% gelatin.

6) colour developing liquid:

A liquid: 0.933g citric acid, 3.68g Na ₂HPO ₄12H ₂O, 18 μ L, 30% H ₂O ₂Be settled to 100mL with ultrapure water.

B liquid: 60mg 3,3/, 5,5/-tetramethyl benzidine (TMB) is dissolved in the 100mL ethylene glycol;

Before using A is mixed with the 5:1 volume ratio with B.

7) H of stop buffer: 2M ₂SO ₄

The step of indirect competitive ELISA experimental technique is as follows:

The methanol solution that in advance standard items of diisooctyl phthalate is mixed with 1000 μ g/mL is as the work mother liquor, and is stand-by 4 ℃ of preservations.Preparation PBST solution (0.01mol/L, pH7.4,0.15mol/L NaCl, 0.5% Tween-20) is prepared serial reaction liquid based on this, in order to dilution competition thing titer and antiserum.

A, bag quilt: with the coating antigen coated elisa plate of setting concentration, 100 μ L/ holes, 4 ℃ are spent the night.

B, washing: use PBST detersive enzyme target 3 times, each 3min, 200 μ L/ holes dry ELISA Plate.

C, sealing: contain the carbonate buffer solution of 0.1% gelatin, the 37 ℃ of sealings in 200 μ L/ holes 2h.

D, washing: same b.

E, competition: with PBST diisooctyl phthalate is diluted to 0.01,0.1,0.5,1,5,10, the 20ng/mL series concentration, other establishes a PBST blank, 50 μ L/ holes.Every then hole adds the antiserum of 8000 times of 50 μ L dilutions, in 37 ℃ of incubation 1h.

F, washing: same b.

G, add ELIAS secondary antibody (goat-anti rabbit GAR-HRP, 1:4000), 100 μ L/ holes, 37C reacts 1h.

H, washing: same b.

I, colour developing: add colour developing liquid 100 μ L/ holes, colour developing 15min.

J, termination: add stop buffer 100 μ L/ holes.

K, mensuration: the light absorption value that detects 450nm with microplate reader.

Beneficial effect of the present invention: the present invention has set up the indirect ELISA method of diisooctyl phthalate, for the diisooctyl phthalate residue detection provides a kind of detection means rapidly and efficiently, because what adopt is polyclonal antibody, expense is lower and stable and repeated better.Sensitivity is 0.085ng/mL, and the range of linearity is 0.1～25ng/mL, half amount of suppression (IC ₅₀) be 3.6ng/mL.Immunoreactive high specific and high-affinity make ELISA have high selectivity and sensitivity, and sample pre-treatment process is simple.

Description of drawings

The standard of Fig. 1 diisooctyl phthalate suppresses curve.

Specific embodiments

Further specify the present invention by the following examples.

One, instrument:

TGL-40B table-type low-speed hydro-extractor, Anting Scientific Instrument Factory, Shanghai;

The KFLOW water purification machine, Kai Folong company;

The horizontal shaking table of ZD-9556, granary science and education equipment factory;

Costar96 hole 8 * 12 removable ELISA Plate, the lucky safe bio tech ltd in Shanghai;

MuLtiska Mks microplate reader, Thermo Labsystems company;

Can debug pipettor, Thermo Labsystems company;

Turbine mixer, Shanghai Hu Xi instrumental analysis factory.

Two, reagent:

The goat-anti rabbit (GAR-HRP) of horseradish peroxidase-labeled, health becomes bio-engineering corporation;

Tetramethyl benzidine (TMB), Huamei Bio-Engrg Co.;

Other reagent are analytical reagent.

Three, step

1. immunogene and coating antigen is synthetic

Synthetic immunogen and coating antigen (diisooctyl phthalate haptens and bovine serum albumin BSA or ovalbumin OVA conjugate), with the coupling of diazotising method, concrete steps are as follows:

1. prepare A liquid: get the 0.023mmol haptens in the 25mL beaker, add the 1mol/L hydrochloric acid of 2mL dimethyl formamide, 0.2mL water and 0.2mL, form yellow solution, be cooled to 0-5 ℃.Progressively drip the 1mol/L sodium nitrite solution then, with the starch potassium iodide paper monitoring, become pewter up to test paper and stop to drip, low temperature stirs 1h down, and this liquid is A liquid.

2. prepare B liquid: the bovine serum albumin (or ovalbumin of 102mg) that takes by weighing 150mg is dissolved in the pH9.0 borate buffer solution of 6mL, and cryopreservation, this liquid are B liquid.

Borate buffer solution: 0.2mol/L boric acid: boric acid 12.37g adds water to 1000mL; 0.05mol/L borax: borax 19.07g adds water to 1000mL; Above-mentioned two solution are mixed the borate buffer solution that is pH9.0 with the ratio of volume ratio 2:8.

3. under low temperature stirs, A liquid dropwise is added drop-wise to B liquid, and regulates pH, pH is remained on stir under 9,4 ℃ of conditions and spend the night, promptly obtain the artificial antigen mixed liquor with the sodium hydroxide solution of 1mol/L.

4. the artificial antigen mixed liquor is moved in the bag filter, dialysed 4-6 days with the deionized water of 6 * 1L.Use desivac that the liquid in the bag filter is made powder at last, promptly obtain artificial antigen: diisooctyl phthalate-bovine serum albumin (or coating antigen: diisooctyl phthalate-ovalbumin).

The antibody titer determination step:

1) coating antigen is cushioned liquid with bag and makes the serial dilution bag by 96 hole ELISA Plate, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out ELISA Plate and be back to room temperature next day, and 200 μ L PBST solution are injected in every hole, and the 3min that vibrates on the shaking table firmly gets rid of cleansing solution, pats dry on thieving paper, continues washing 2 times.Following washing methods is identical.

2) after the abundant washing, with sealing damping fluid sealase target, 200 μ L/ holes, taking-up is dried stand-by behind the incubation 2h in 37 ℃ of incubation casees.

3) positive serum serial dilution correspondence is joined preceding 7 ranks of ELISA Plate, eighth row adds negative serum, and 100 μ L/ holes are hatched washing behind the 1h, patted dry for 37 ℃.

4) every hole adds 100 μ L, and the goat-anti rabbit GAR-HRP of the mark of 1:4000 dilution is hatched washing behind the 1h, patted dry for 37 ℃.

5) every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out every hole, back and adds 100 μ L stop buffers (sulfuric acid of 2mol/L), measures light absorption value A with microplate reader ₄₅₀

The antibody specificity determination step:

A, bag quilt: with the coating antigen coated elisa plate of setting concentration, 100 μ L/ holes, 4 ℃ are spent the night.

B, washing: use PBST detersive enzyme target three times, each 3min, 200 μ L/ holes dry ELISA Plate.

C, sealing: contain the carbonate buffer solution of 0.1% gelatin, 200 μ L/ holes, 37 ℃ of sealing 2h.

D, washing: same b.

E, competition: with PBST with the diisooctyl phthalate mother liquor be diluted to 0, the standard solution series concentration that contains 10% ethanol of 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, 25ng/mL, other establishes a PBST blank, 50 μ L/ holes.Every then hole adds the antiserum of 4000 times of 50 μ L dilutions, in 37 ℃ of incubation 1h.

F, washing: same b.

G, add ELIAS secondary antibody (goat-anti rabbit GAR-HRP, 1:4000), 100 μ L/ holes, 37C reacts 1h.

H, washing: same b.

I, colour developing: add colour developing liquid 100 μ L/ holes, colour developing 15min.

J, termination: add stop buffer 100 μ L/ holes.

K, mensuration: the light absorption value that detects 450nm with microplate reader.

Test findings is as follows:

1, typical curve: the range of linearity of the antibody test that this experiment obtained is 0.1～25ng/mL.

2, sensitivity: sensitivity is the concentration of the pairing standard items of gained 90% maximum light absorption value, i.e. IC ₉₀Be 0.085ng/mL.

Claims (1)

1, a kind of enzyme-linked immune detection method of diisooctyl phthalate, it is characterized in that utilizing synthetic diisooctyl phthalate immunogen immune to obtain polyclonal antibody, with the diisooctyl phthalate is standard items, as envelope antigen, set up the indirect competitive enzyme-linked immunosorbent method of the diisooctyl phthalate in the animal food with the conjugate of diisooctyl phthalate haptens and OVA; Step is as follows:

(1) carbonate buffer solution with 0.05M dilutes envelope antigen with 1:5000～1:8000, add in the ELISA Plate, every hole adds 100 μ L, 4 ℃ of overnight incubation, use the above-mentioned carbonate buffer solution 200 μ L/ holes that contain 0.1% gelatin as confining liquid then, sealing 2h;

(2) with 0.01,0.1,0.5,1,5,10, the diisooctyl phthalate standard items of 20ng/mL series concentration gradient or the sample of handling well are in enzyme mark hole separately, and every hole adds 50 μ L; Add the antiserum with antibody diluent dilution 1:6000～1:9000 simultaneously, every hole adds 50 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of competition 1h;

(3) dilute with 1:3000～1:5000 with the goat-anti rabbit GAR-HRP of antibody diluent with horseradish peroxidase-labeled, every hole adds 100 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of effect 1h;

(4) configuration colour developing liquid

A liquid: 0.933g citric acid, 3.68g Na ₂HPO ₄12H ₂O, 18 μ L 30%H ₂O ₂Be settled to 100mL with ultrapure water;

B liquid: 60mg3,3 ', 5,5 '-tetramethyl benzidine is dissolved in the 100mL ethylene glycol;

Before using A liquid is mixed with the 5:1 volume ratio with B liquid;

(5) every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 15min; Add stop buffer 2M sulfuric acid solution at last, every hole 100 μ L; Microplate reader 450nm surveys light absorption value A ₄₅₀, calculate the diisooctyl phthalate concentration of testing sample with the contrast of work typical curve.