CN104020290A - Chlorothalonil enzyme-linked immunosorbent assay method - Google Patents

Abstract

The invention relates to a chlorothalonil enzyme-linked immunosorbent assay method, belonging to the technical field of enzyme-linked immunosorbent assay (ELISA). The invention discloses a chlorothalonil monoclonal antibody enzyme-linked immunosorbent assay method. A BALB/c mice is immunized by using a synthesized chlorothalonil immunogen to obtain a monoclonal antibody, and an indirect ELISA method of chlorothalonil is established by using the chlorothalonil as a standard substance and a conjugate of chlorothalonil haptin and OVA as a coating antigen, and thus a quick and efficient detection method is provided for detecting residue of the chlorothalonil, the lowest detectable limit is 0.06ng/mL, and the half maximal inhibitory concentration (IC50) is 0.3ng/mL. High sensitivity and compatibility of an immunoreaction ensures that the ELISA has extremely high sensitivity and sensitivity when used for detecting the chlorothalonil.

Description

A kind of Bravo enzyme-linked immune detection method

Technical field

A kind of Bravo enzyme-linked immune detection method, relates to a kind of Detecting Pesticide method, says more specifically and uses Enzyme-linked Immunosorbent Assay method (ELISA) to detect the residual of Bravo.Belong to enzyme linked immunosorbent detection (ELISA) technical field.

Background technology

Bravo is a kind of wide spectrum organochlorine germifuge, and the fungal disease of various crops and various greening lawns etc. is had to prevention effect.Bravo does not have interior suction conduction, once but after being sprayed onto on plant, just can there is good tackness at its body surface, be difficult for being fallen by rain drop erosion, therefore the drug effect phase longer, add it and be widely used, therefore, Bravo and metabolic product thereof have larger residual at Plants and Soils, even detect in Arctic.The method that detects at present Bravo is mainly the instrumental method such as vapor-phase chromatography (GC) and liquid phase chromatography (HPLC), but there is complex operation in these methods, consuming time, the shortcomings such as expense is somewhat expensive, can not realize the fast detecting of a large amount of samples, therefore set up a kind of fast and convenient Bravo detection method significant.Euzymelinked immunosorbent assay (ELISA) (ELISA) is that one is very efficient, responsive, detection method fast, but the monoclonal monomer that obtains high-affinity and high specific is the prerequisite of immunology detection, and wherein the synthetic of artificial antigen is a wherein important step.

Summary of the invention

The object of the invention is to have set up the residual ELISA detection method of Bravo, for Bravo residue detection provides detection means rapidly and efficiently.Lowest detection is limited to 0.06ng/mL, half amount of suppression (IC50) is 0.3ng/mL.Immunoreactive high specific and compatibility make ELISA detection Bravo have high selectivity and sensitivity.

Technical scheme of the present invention, first react and obtain haptens by the former medicine of 6-aminocaprolc acid and Bravo, obtain comlete antigen with EDC method and BSA coupling again, immunity BALB/c mouse obtains monoclonal antibody, again haptens and OVA reaction are prepared to coating antigen, taking Bravo as standard items, set up the residual indirect competitive enzyme-linked immunosorbent detection method of Bravo.

The present invention realizes by following steps:

(1) with the carbonate buffer solution dilution coating antigen of 0.05M, since 2 μ g/mL doubling dilutions, dilute 4 concentration, after dilution, add in ELISA Plate every hole 100 μ L; 4 DEG C of overnight incubation, sealing, washing;

(2) the Bravo standard items of 4 concentration gradients are added in ELISA Plate to every hole 50 μ L; Add the monoclonal antibody taking antibody diluent dilution as 1: 1000～1: 8000, every hole 50 μ L simultaneously; 37 DEG C of competition 30min, then with the phosphate buffer PBST washing containing tween 3 times, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/holes, 37 DEG C of effect 0.5h, then wash 3 times with PBST;

Described PBST solution: containing the 0.01M PBS solution of 0.05% Tween-20;

Described confining liquid: containing pH 9.6, the 0.05M carbonate buffer solution of 0.1% gelatin;

Described antibody diluent: containing the PBST solution of 0.1% gelatin;

(3) configuration nitrite ion

A liquid: fill a prescription as adding 0.933g citric acid in every 100mL ultrapure water, 3.68g Na ₂hPO ₄12H ₂o, 18 μ L 30%H ₂o ₂;

B liquid: fill a prescription and be dissolved in 100mL ethylene glycol for 60mg tetramethyl benzidine; Before using, A liquid is mixed with 5: 1 volume ratios with B liquid;

(4) add nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Add stop buffer 2M sulfuric acid solution 100 μ L/ holes, microplate reader 450nm surveys light absorption value OD, draws Bravo concentration-light absorption value typical curve according to the light absorption value of variable concentrations Bravo titer;

(5) sample detection: testing sample is operated by step (1)～(4), and gained microplate reader 450nm surveys light absorption value OD and contrasts with the institute typical curve of doing the Bravo content calculating in testing sample.

Main solution preparation

1) preparation phosphate 0.01M PBS damping fluid:

Na ₂ HPO ₄ ·12H ₂O 3.62g

KH ₂ PO ₄ 0.2g

NaCl 0.2g

KCl 8.0g

Add ultrapure water and be diluted to 1000mL.

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

Na ₂ CO ₃ 1.59g

NaHCO ₃ 2.93g

Add ultrapure water and be diluted to 1000mL.

3) preparation PBST solution: containing the PBS solution of 0.05% Tween-20.

4) preparation confining liquid: containing pH 9.6, the 0.05M carbonate buffer solution of 0.1% gelatin.

5) preparation antibody diluent: containing the PBST solution of 0.1% gelatin.

6) nitrite ion:

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 100mL ethylene glycol; Before using, A liquid is mixed with 5: 1 volume ratios with B liquid.

7) H of stop buffer: 2M ₂sO ₄.

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

Bravo standard items mother liquor is 100 μ g/mL, and matrix is normal hexane mother liquor, stand-by 4 DEG C of preservations.Mark product dilution is 15% methyl alcohol-PBS.

A, coated: with the coated enzyme-linked reaction plate of the coating antigen of setting concentration, 100 μ L/ holes, 4 DEG C are spent the night;

B, washing: use PBST washing reaction plate three times, each 3min, 200 μ L/ holes, then dry reaction plate;

C, sealing: containing the CBS of 0.1% gelatin, 200 μ L/ holes, 37 DEG C of sealing 2h;

D, washing: same to b;

E, competition: Bravo mother liquor is diluted to 0.0185,0.039 with 15% methyl alcohol-PBS, 0.156,0.3125,0.625,1.25,2.5 ng/mL series concentration, separately establish 15% methyl alcohol-PBS blank, 50 μ L/ holes; Then every hole adds the antiserum of 4000 times of 50 μ L dilutions, in 37 DEG C of incubation 0.5h;

F, washing: same to b;

G, add ELIAS secondary antibody (HRP-sheep anti-mouse igg, 1: 3000), 100 μ L/ holes, 37 DEG C of reaction 0.5h;

H, washing: same to b;

I, colour developing: add the nitrite ion 100 μ L/ holes of TMB preparation, colour developing 15min;

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

K, mensuration: detect OD by microplate reader _450nm.

Beneficial effect of the present invention: using the conjugate of Bravo haptens and OVA as coating antigen, set up the indirect ELISA method of Bravo, for the residue detection of Bravo provides detection means rapidly and efficiently.Lowest detection is limited to 0.06ng/mL, half amount of suppression (IC50) is 0.3ng/mL.Immunoreactive high specific and high-affinity make ELISA have high selectivity and sensitivity, thereby sample pretreatment process is simple.

Brief description of the drawings

The standard of Fig. 1 Bravo suppresses curve.

Embodiment

Further illustrate by the following examples the present invention.

One, instrument:

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

KFLOW water purification machine Kai Folong company

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

Costar 96 Ji Tai bio tech ltd, removable ELISA Plate Shanghai, hole 8 × 12

MuLtiska Mks microplate reader Thermo Labsystems company

Adjustable pipettor Thermo Labsystems company

The Hu Xi instrumental analysis of turbine mixer Shanghai.

Two, reagent:

Sheep anti-mouse igg (HRP-IgG) the Kang Cheng bio-engineering corporation of horseradish peroxidase-labeled

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

Other reagent are analytical reagent.

Three, step

1, immunogene and coating antigen is synthetic

Immunogene obtains Bravo haptens and bovine serum albumin(BSA) BSA coupling with carbodlimide method, and by mixed anhydride method, by Bravo haptens and the synthetic coating antigen of ovalbumin OVA coupling, concrete steps are as follows:

Immunogenic preparation

Take Bravo haptens 35.8mg, EDC 56.9mg, NHS 35.6mg, dissolves (being called A liquid) by 1mL dry DMF, and stirring at room temperature is reacted 8h.Take in the 0.01mol/L PBS that BSA 112mg is dissolved in 20mL, pH 7.2 (being called B liquid), at room temperature condition, dropwise A liquid is joined in B liquid, room temperature reaction spends the night, and obtains immunogene mixed liquor.

Bag filter pre-treatment: get the bag filter of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;

Conjugate immunogene mixed liquor is put into bag filter and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day.

Coating antigen preparation

Take 40mg haptens, be dissolved in 1mL dry DMF, successively drip the isobutyl chlorocarbonate (being called A liquid) of 32 μ L tri-n-butylamines and 17.5 μ L, 4 DEG C of stirring reaction 1h.Take in the PBS that OVA 166mg is dissolved in 30mL (being called B liquid).At 4 DEG C, dropwise A liquid is joined in B liquid, 4 DEG C of reaction 5h, obtain coating antigen mixed liquor.

Bag filter pre-treatment: get the bag filter of 10cm, boil 5min in boiling water, then use the deionized water rinsing 3min of 60 DEG C, be kept in 4 DEG C of deionized waters for subsequent use;

Coating antigen mixed liquor is put into bag filter and dialyse 3 days in the PBS of 0.01mol/L, change liquid three every day.

2, the preparation of antiserum (monoclonal antibody)

1) animal used as test: select 12 BALB/c mouse, every 4 is an experimental group;

2) antigen configuration: by immunogene physiological saline solution, be made into the solution of 2mg/mL;

3) emulsification: by above-mentioned solution and equivalent completely or incomplete Freund's adjuvant by mix and blend method by its emulsification, directly an emulsion is splashed in water, do not scatter and float on the water surface;

4) immunization method: initial immunity is by reagent good emulsification in mouse back multi-point injection, and 1mL/ only; Immunity after initial immunity is called booster immunization, booster immunization incomplete Freund's adjuvant emulsification, booster immunization is in hypodermic injection, 3 weeks booster immunizations once, the half of dosage and initial immunity;

5) blood sampling: from tail blood sampling, adopt Dot-ELISA to measure antiserum titre and inhibition after 4 booster immunizations, wait tiring and inhibition reaches after requirement, merge;

6) merge: by polyglycol (PEG4000) method, mouse boosting cell and murine myeloma cell are merged, by HAT medium culture, utilize indirect ELISA to detect positive cell hole, and further utilize indirect competitive ELISA method to measure the inhibition in positive cell hole, by limiting dilution assay, to there being the positive cell hole preferably suppressing to carry out subclone three times, final screening obtains hybridoma cell strain;

7) preparation of antibody: get 8-10 BALB/c mouse in age in week, every mouse peritoneal injection paraffin oil 1 mL; Every mouse peritoneal injection 1 × 10 after 7 days ⁶hybridoma, collected ascites since the 7th day, by ascites, by caprylic acid-ammonium purifying, the monoclonal antibody of acquisition is placed in-20 DEG C of preservations.

3, ELISA course of reaction

(1) coated: by coating antigen CTNH-OVA with 0.05M pH9.6 carbonate buffer solution since 2 μ g/mL doubling dilutions, 100 μ L/holes, 37 DEG C are reacted 2h;

(2) washing: solution in plate is inclined, dry, and with cleansing solution washing 3 times, each 3min;

(3) sealing: after patting dry, add 200 μ L/hole confining liquids, 37 DEG C of reaction 2h, washing post-drying is for subsequent use;

(4) application of sample: antiserum is started to doubling dilution from 1:1000, and join in each dilution coated hole, 100 μ L/holes, 37 DEG C of reaction 1h; Fully, after washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/holes, 37 DEG C of reaction 1h.

(5) colour developing: ELISA Plate is taken out, and fully, after washing, every hole adds the TMB nitrite ion of 100 μ L, 37 DEG C of lucifuge reaction 15min.

(6) stop and measure: every hole adds 50 μ L stop buffers with cessation reaction, then measures the OD in each hole by microplate reader ₄₅₀value.

4, determining of the recovery and sample extraction method

The cucumber sample that takes 1g pulverizing is inserted in 100mL triangular flask, adds respectively 10 μ g, 10 μ g and 2.5 μ g Bravos.Add after 10 mL acetone concussions evenly ultrasonic extraction 15 min, clean postpone supernatant N to sample ₂dry up, then with methyl alcohol dissolving, adopt indirect competitive ELISA to add recovery test.

The calculating of the recovery: the sample OD value of adding concentration according to difference is calculated corresponding inhibiting rate, then find concentration separately from typical curve according to corresponding inhibiting rate, the recovery that detectable concentration and the ratio of actual concentration are corresponding concentration.

Test findings is as follows:

Typical curve: half inhibiting rate (IC50) of the antibody that this experiment obtains is 0.3ng/mL, specifically asks for an interview Fig. 1.

Sensitivity: sensitivity is the concentration of the corresponding standard items of the maximum light absorption value of gained 90%, and IC 90 is 0.06ng/mL.

Cross reacting rate (CR%): utilize Bravo analogue and some agricultural chemicals to carry out cross reaction research.CR=IC50(cross-reacting material)/IC50(Bravo), from following table, this antibody specificity is fine, meets the requirement that how residual ELISA method detects.

The mensuration of table 1 cross reacting rate

Table 2 determination of recovery rates

Claims (1)

1. a Bravo enzyme-linked immune detection method, it is characterized in that utilizing synthetic Bravo immunogen immune BALB/c mouse to obtain monoclonal antibody, taking Bravo as standard items, using the conjugate of Bravo haptens and OVA as coating antigen, the indirect competitive enzyme-linked immunosorbent detection method of setting up Bravo, step is as follows:

(1) with the carbonate buffer solution dilution coating antigen of 0.05M, since 2 μ g/mL doubling dilutions, dilute 4 concentration, after dilution, add in ELISA Plate every hole 100 μ L; 4 DEG C of overnight incubation, sealing, washing;

(2) the Bravo standard items of 4 concentration gradients are added in ELISA Plate to every hole 50 μ L; Add the monoclonal antibody taking antibody diluent dilution as 1 ︰ 1000～1 ︰ 8000, every hole 50 μ L simultaneously; 37 DEG C of competition 30min, then with the phosphate buffer PBST washing containing tween 3 times, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/holes, 37 DEG C of effect 0.5h, then wash 3 times with PBST;

Described PBST solution: containing the 0.01M PBS solution of 0.05% Tween-20;

Described confining liquid: containing pH 9.6, the 0.05M carbonate buffer solution of 0.1% gelatin;

Described antibody diluent: containing the PBST solution of 0.1% gelatin;

(3) configuration nitrite ion

A liquid: fill a prescription as adding 0.933g citric acid in every 100mL ultrapure water, 3.68g Na ₂hPO ₄12H ₂o, 18 μ L 30%H ₂o ₂;

B liquid: fill a prescription and be dissolved in 100mL ethylene glycol for 60mg tetramethyl benzidine; Before using, A liquid is mixed with 5 ︰ 1 volume ratios with B liquid;

(4) add nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Add stop buffer 2M sulfuric acid solution 100 μ L/ holes, microplate reader 450nm surveys light absorption value OD, draws Bravo concentration-light absorption value typical curve according to the light absorption value of variable concentrations Bravo titer;

(5) sample detection: testing sample is operated by step (1)～(4), and gained microplate reader 450nm surveys light absorption value OD and contrasts with the institute typical curve of doing the Bravo content calculating in testing sample.