CN101315376A - ELISA detection method for carbostyril antibiotic relict - Google Patents

Abstract

The invention relates to a method for quinoxalone antibiotics multi-residue enzyme-linked immune detection, and belongs to the technology field of the antibiotics residue detection method. In the method, the polyclonal antibodies are obtained by utilizing syntactic quinoxalone antibiotics multi-cluster antigen immunization; the coupling compound of the half antigen of the quinoxalone antibiotics and an egg-white protein OVA is taken as an envelope antigen; the quinoxalone antibiotics is taken as a standard; the indirect competitive enzyme-linked immune detection method for the quinoxalone antibiotics in animal food is established; a rapid and high efficient detection means is provided for the residues of the quinoxalone antibiotics in the animal food; because the polyclonal antibodies are adopted, the cost is low, and the stability and the repeatability are good; the detection limit (IC is smaller than 90) is 0.0126 ng/ml, the median inhibitory dose (IC is smaller than 50) is 0.52 ng/ml, and the detection range (IC is smaller than 20 to 80) is 0.04467 to 14 ng/ml. In addition, the ELISA method is also evaluated in the aspects of specificity, accuracy and sensitivity, thereby conforming to the detection requirements for the multi-residue ELISA method.

Description

ELISA detection method for carbostyril antibiotic relict

Technical field

ELISA detection method for carbostyril antibiotic relict uses enzyme linked immunological adsorption method (ELISA) to detect the residual of carbostyril antibiotic in the animal food specifically.Belong to the method for antibiotic residue detection technical field.

Background technology

Quinolone is a class synthetic antibiotic class medicine, chemically relevant with nalidixic acid, its mechanism of action is the nuclear that directly acts on bacterium, the DNA gyrase that suppresses bacterium, makes enzyme not introduce otch on the dna double chain, destroy metabolism and the breeding of bacterium, rapidly kill bacteria.Initial this class medicine is used for the treatment of urinary tract infection, has developed into therapy system now and has caught, and generally used as prevention and medicine in animal feeding.In recent years, residual caused widely of these medicines in animal tissue noted.FAO (Food and Agriculture Organization of the United Nation), the joint council of the food additives expert of the World Health Organization (WHO), European Union have all formulated the maximum residue limit(MRL) of multiple QNS in animal tissue.

The various residue analysis methods of QNS mainly comprise high-efficient liquid phase technique (HPLC) and relevant therewith HPLC-UV, HPLC-FD, HPLC-DVD, LC-MS/MS, and LC-ESI-MS/MS also has fluorescent spectrometry, capillary electrophoresis etc. in addition.Lesley Johnston etc. are residual with quinolones in HPLC-MS (Massspectrometer) method detection fish and the marine product and fluoroquinolones, and all analytes all obtain the good recovery, and detection limit reaches 1～3mg/kg.The LC-ESI-MS-MS method of using Gery van Vyncht etc. detects 11 kinds of QNSs in the pig kidney.Although these method sensitivity are higher, the pre-treatment step of sample is more, and is time-consuming relatively and testing cost is higher, particularly is not suitable for the screening of a large amount of samples.

Enzyme-linked immunosorbent assay (ELISA) provides a kind of quick, sensitive, selectivity detection method of trace Permethrin.Wang Zhanhui selects four kinds of QNSs as haptens, adopt mixed anhydride method directly with BSA coupling synthetic immunogen mutually, active ester method synthesizes coating antigen, by screening, with CIP is that haptenic monoclonal antibody is discerned multiple QNs, wherein the cross reacting rate to DIF and SAR is lower than 3%, and QNs all is higher than 29% to other 12 Chinese traditional medicines.But said method requires to use monoclonal antibody, and Monoclonal Antibody process complexity costs an arm and a leg, and does not declare universal.The detection method that also lacks at present carbostyril antibiotic relict in the practicable animal food.

Summary of the invention

The objective of the invention is to set up the how residual ELISA detection method of the carbostyril antibiotic in the animal food, for the residue detection of carbostyril antibiotic in animal food provides detection means rapidly and efficiently, because what adopt is polyclonal antibody, expense is lower and stable and repeated better.Detectability (IC ₉₀) be 0.0126ng/mL, half amount of suppression (IC ₅₀) be 0.54ng/mL and sensing range (IC ₂₀～IC ₈₀) be 0.04467～14ng/mL.In addition, also assessed this ELISA method from specificity, accuracy, the sensitivity of method.The recovery is respectively 63%～95%, and the coefficient of variation is less than 20%.Except that the cross reaction with 5 kinds of QNSs (QNs) is lower than 5%, all the other 13 kinds of QNs are respectively between 16% and 112%.Meet the requirement that how residual ELISA method detects.

Technical scheme of the present invention: a kind of ELISA detection method for carbostyril antibiotic relict, utilize synthetic carbostyrile kind antibiotic multi cluster antigen immunity to obtain polyclonal antibody (Li Yali, the petty official is transmitted, Wang Canhui: Chinese patent: 200710134500.4), with the conjugate of carbostyril antibiotic haptens and ovalbumin OVA as envelope antigen, with the carbostyril antibiotic is standard items, sets up the indirect competitive enzyme-linked immunosorbent detection method of the carbostyril antibiotic in the animal food; Step is as follows:

(1) with 9% physiological saline dilution envelope antigen 1: 100～1: 200 as coating buffer, add in the ELISA Plate, every hole adds 100 μ L, 4 ℃ of overnight incubation are according to conventional ELISA method washing sealing;

(2) with polyclonal antibody with antibody diluent by after 1: 100～1: 200 dilution proportion, add in the ELISA Plate, every hole adds 35 μ L; Simultaneously every hole adds the carbostyril antibiotic standard items of one of 0.001ng/mL, 0.01ng/mL with the standard items diluted, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, a series of concentration of 1000ng/mL respectively or adds testing sample, every hole adds 65 μ L, hatches behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;

(3) add the goat-anti rabbit GAR-HRP of horseradish peroxidase-labeled, by 1: 1000～1: 3000 dilution proportion, every hole added 100 μ L with antibody diluent, hatched behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;

(4) preparation colour developing liquid

A liquid: fill a prescription to adding 0.933g citric acid, 3.68g Na in every 100mL water ₂HPO ₄12H ₂O, 18 μ L 30%H ₂O ₂

B liquid: filling a prescription is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine;

Before using with A liquid and B liquid with 4: 1 volume mixture;

(5) every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 15min; Every hole adds stop buffer 2M sulfuric acid solution 100 μ L, and microplate reader 450nm surveys light absorption value OD.

Testing sample is that the extracting method of animal food is:

(1) carbostyril antibiotic with the level of 1ng/g sample and 5ng/g sample adds negative sample, at room temperature leaves standstill 15min at least;

(2) get the sample that 2g has blended, be added in 25～50mL centrifuge tube, add 10mL ethyl acetate-mixed liquor of 1: 1 of PBS volume ratio again, use homogenizer homogenate then, ultrasonic Extraction 30min after the vortex mixed, the centrifugal 10min of 3500rpm/min;

(3) pipette the upper strata organic layer in another new band cap test tube, in 60～70 ℃ hot bath, with nitrogen dry up that (note: salt that the aqueous phase that in lower floor be alkalescence contain high concentration will to detection reaction cause severe contamination on one side.So when pipetting the upper strata organic layer, be not drawn to lower floor's water).

(4) add the stand-by (attention: concerning some sample, precipitation or turbid liquid perhaps can occur in 1mL PBS solution dissolving back.Before application of sample, can be with its centrifugal 3～5 minutes.Or heating).

More detailed step is:

1) preparation phosphate (PBS) damping fluid:

Na ₂HPO ₄.12H ₂O?3.12g，

NaH ₂PO ₄.2H ₂O 1.76g，

Add water 100mL.

2) configuration PBST solution: the PBS solution that contains 0.05%Tween-20.

3) preparation OVA/ physiological saline confining liquid: take by weighing 0.1g OVA and be dissolved in 100mL physiological saline and be mixed with 0.1% OVA/ physiological saline confining liquid.

4) configuration antibody diluent: the PBST solution that contains 0.1%OVA.

5) synthetic envelope antigen (Norfloxacin haptens and ovalbumin OVA conjugate).

6) dilute envelope antigen 1: 100～1: 200 with 9% physiological saline, add in the ELISA Plate every hole 100 μ L.4 ℃ of overnight incubation.

7) get rid of envelope antigen liquid after, with PBST, washing 2 times, add OVA/ physiological saline confining liquid, every hole 100 μ L, 37 ℃ of incubation 2h.

8) get rid of confining liquid in the ELISA Plate, with PBST washing twice, each 2～3 minutes.

9) antiserum after 1: 100～1: 200, in every hole 35 μ L adding ELISA Plate, is added the carbostyril antibiotic standard items of a series of concentration, every hole 65 μ L with the antibody diluent dilution simultaneously.Hatch behind the 1h with PBST washing 3～5 times for 37 ℃.

10) the goat-anti rabbit (GAR-HRP) of adding horseradish peroxidase-labeled is 1: 1000～1: 3000 with the antibody diluent dilution.Every hole 100 μ L.Wash 3～5 times with PBST behind 37 ℃ of effect 1h.

11) configuration colour developing liquid:

The A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water ₂HPO ₄12H ₂O, 18 μ L30%H ₂O ₂

The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine;

Before using with A and B with 4: 1 volume mixture.

12) add the every hole 100 μ L of colour developing liquid, 37 ℃ of colour developing 15min.

13) add stop buffer 2M H2SO; , microplate reader 450nm surveys light absorption value (OD).

Beneficial effect of the present invention:

With the conjugate of Norfloxacin haptens and OVA as envelope antigen, set up the indirect ELISA method of carbostyril antibiotic in the animal food, for the residue detection of carbostyril antibiotic in animal food provides detection means rapidly and efficiently, because what adopt is polyclonal antibody, expense is lower and stable and repeated better.Detectability (IC ₉₀) be 0.0126ng/mL, half amount of suppression (IC ₅₀) be 0.54ng/mL and sensing range (IC ₂₀～IC ₈₀) be 0.04467～14ng/mL.In addition, also assessed this ELISA method from specificity, accuracy, the sensitivity of method.The recovery is respectively 63%～95%, and the coefficient of variation is less than 20%.Except that the cross reaction with 5 kinds of QNs is lower than 5%, all the other 13 kinds of QNs are respectively between 16% and 112%.Meet the requirement that how residual ELISA method detects.

Description of drawings

The standard of Fig. 1 Norfloxacin 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;

KFLO water purification machine Kai Folong company;

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

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

MuLtiska Mks microplate reader Thermo Labsystems company;

Can debug pipettor Thermo Labsystems company, even matter device Fluka company;

The Hu Xi instrumental analysis of turbine mixer Shanghai.

Two, reagent:

The goat anti-rabbit igg of horseradish peroxidase-labeled (HRP-IgG) 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 (Norfloxacin haptens and bovine serum albumin(BSA) BSA conjugate) and coating antigen (Norfloxacin haptens and ovalbumin OVA conjugate) are used the glutaraldehyde method coupling, and concrete steps are as follows:

1) the Norfloxacin haptens of getting 0.1mmol is dissolved in the phosphate buffered solution of pH 7.4 of 2mL, stirs slowly to drip glutaraldehyde solution (final concentration is 1.25%) down and become A liquid.

2) get and become B liquid in the phosphate buffered solution of the bovine serum albumin of 0.002mmol or the pH 7.4 that ovalbumin (BSA or OVA) is dissolved in 10mL.

3) under the stirring condition A liquid slowly is added drop-wise in the B liquid.Stirring is spent the night, pure water dialysis 5 days.The packing freeze-drying is stored in-20 ℃ of refrigerators.

2. immunity prepares polyclonal antibody

3.ELISA course of reaction:

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 igg of HRP mark of dilution in 1: 3000 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

4, the recovery and sample extraction method determines

1) level with 1ng/g and 5ng/g adds the QNs sample, at room temperature leaves standstill 15min at least.

2) get the sample that 2g has blended, in the 50mL centrifuge tube.Add 10mL ethyl acetate-PBS (1: 1) again, use homogenizer homogenate then.Ultrasonic Extraction is 30 minutes after the vortex mixed, the rotating speed of 3500rpm/min, centrifugal 10 minutes.

3) pipette the upper strata organic layer in another new band cap test tube, in 60～70 ℃ hot bath, with nitrogen dry up that (note: salt that the aqueous phase that in lower floor be alkalescence contain high concentration will to detection reaction cause severe contamination on one side.So when pipetting the upper strata organic layer, be not drawn to lower floor's water).

4) adding the dissolving of 1mLPBS solution (notes: concerning some sample, precipitation or turbid liquid perhaps can occur.Before application of sample, can be with its centrifugal 3～5 minutes; Or heating).

5) pipette 50 μ L sample solutions, directly application of sample carries out ELISA and measures in micropore.

6) calculating of the recovery: add the sample OD value calculating corresponding inhibition ratio of concentration according to difference, find separately concentration according to corresponding inhibition ratio from typical curve again.Detectable concentration is the recovery of corresponding concentration with the ratio of actual concentration.

Test findings is as follows:

1, typical curve: the range of linearity of the Detection of antigen that the present invention obtained is to be 0.04467～14ng/mL, specifically asks for an interview Figure of description.

2, sensitivity: sensitivity is the concentration of the pairing standard items of gained 90% maximum light absorption value, i.e. IC ₉₀Be 0.0126ng/mL.

3, cross reacting rate (CR%): utilize carbostyril antibiotic to carry out cross reaction research.

By following table as can be known, except that the cross reaction with 5 kinds of QNs is lower than 5%, all the other 13 kinds of QNs are respectively between 16% and 112%.Meet the requirement that how residual ELISA method detects.

The mensuration of table 1 cross reacting rate

Determination of recovery rates

Carry out ELISA after the mark-on sample reclaims and detect, the recovery is respectively 63%～95%, and the coefficient of variation is less than 20%.

The mensuration of table 2 recovery of standard addition (n=5)

Claims (2)

1, a kind of ELISA detection method for carbostyril antibiotic relict, it is characterized in that utilizing synthetic carbostyrile kind antibiotic multi cluster antigen immunity to obtain polyclonal antibody, with the conjugate of carbostyril antibiotic haptens and ovalbumin OVA as envelope antigen, with the carbostyril antibiotic is standard items, sets up the indirect competitive enzyme-linked immunosorbent detection method of the carbostyril antibiotic in the animal food; Step is as follows:

(1) with 9% physiological saline dilution envelope antigen 1: 100～1: 200 as coating buffer, add in the ELISA Plate, every hole adds 100 μ L, 4 ℃ of overnight incubation are according to conventional ELISA method washing sealing;

(2) with polyclonal antibody with antibody diluent by after 1: 100～1: 200 dilution proportion, add in the ELISA Plate, every hole adds 35 μ L; Simultaneously every hole adds the carbostyril antibiotic standard items of one of 0.001ng/mL, 0.01ng/mL with the standard items diluted, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, a series of concentration of 1000ng/mL respectively or adds testing sample, every hole adds 65 μ L, hatches behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;

(3) add the goat-anti rabbit GAR-HRP of horseradish peroxidase-labeled, by 1: 1000～1: 3000 dilution proportion, every hole added 100 μ L with antibody diluent, hatched behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;

(4) preparation colour developing liquid

A liquid: fill a prescription to adding 0.933g citric acid, 3.68g Na in every 100mL water ₂HPO ₄12H ₂O, 18 μ L 30%H ₂O ₂

B liquid: filling a prescription is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine;

Before using with A liquid and B liquid with 4: 1 volume mixture;

(5) every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 15min; Every hole adds stop buffer 2M sulfuric acid solution 100 μ L, and microplate reader 450nm surveys light absorption value OD.

2, ELISA detection method for carbostyril antibiotic relict according to claim 1 is characterized in that testing sample is that the extracting method of animal food is:

(1) carbostyril antibiotic with the level of 1ng/g sample and 5ng/g sample adds negative sample, at room temperature leaves standstill 15min at least;

(2) get the sample that 2g has blended, be added in 25～50mL centrifuge tube, add 10mL ethyl acetate-mixed liquor of 1: 1 of PBS volume ratio again, use homogenizer homogenate then, ultrasonic Extraction 30min after the vortex mixed, the centrifugal 10min of 3500rpm/min;

(3) pipette the upper strata organic layer in another new band cap test tube, in 60～70 ℃ hot bath, with nitrogen dry up that (note: salt that the aqueous phase that in lower floor be alkalescence contain high concentration will to detection reaction cause severe contamination on one side.So when pipetting the upper strata organic layer, be not drawn to lower floor's water).

(4) adding 1mL PBS solution dissolving back is stand-by.

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