CN102901813B - Detect enzyme linked immunological kit and the using method thereof of residual benzene monoethanolamine A - Google Patents

Abstract

The invention discloses the enzyme linked immunological kit and using method thereof that detect residual benzene monoethanolamine A, belong to ELISA adsorption analysis method technical field.Described kit comprises: wrap by the ELISA Plate of phenolethanolamine Staphylococal Protein A, phenolethanolamine A standard items working fluid, phenolethanolamine A antibody working fluid, phenolethanolamine A ELIAS secondary antibody working fluid, substrate solution, stop buffer, concentrated cleaning solution and concentrating sample dilution.Kit of the present invention adopts indirect competitive enzyme-linked immunosorbent adsorption analysis (ELISA) method, and in the phenolethanolamine A that standard items or sample to be tested remain and ELISA Plate, pre-coated antigen competes phenolethanolamine A antibody jointly.The method may be used for the phenolethanolamine A in direct-detection animal derived food, urine sample, animal tissue and blood serum sample, the feature such as to have easily and fast, sensitive, is applicable to batch samples and detects, this kit sensitivity 0.1ng/mL.

Description

Detect enzyme linked immunological kit and the using method thereof of residual benzene monoethanolamine A

Technical field

The invention discloses a kind of enzyme linked immunological kit and the detection method thereof that detect phenolethanolamine A in animal tissue and metabolin thereof, belong to ELISA adsorption analysis method (Enzyme-Linked Immunosorbent Assay, ELISA) technical field.The present invention relates to the enzyme linked immunological kit of the content for detecting phenolethanolamine A residual in animal-derived food, blood and urine and use the detection method of this kit.

Background technology

Phenolethanolamine A (Phenylethylamine, PEA) is also called Ke Lunba amine, and formal name used at school is 2-[4-(4-nitrobenzophenone) butyl-2-base amino]-1-methoxybenzene ethanol, is a kind of chemical substance of Prof. Du Yucang.Chemical structural formula is as follows:

Gram rumba amine is the isomers of Formoterol, is the accessory substance of ractopamine synthesis, has the effect that same clenbuterol hydrochloride is identical with Ractopamine, belong to the one of beta-2-agonists, have repartitioning function effect.

With other clenbuterol hydrochloride kinds as compared with Ractopamine etc., stimulating animal growth can be used for as adjuvant equally, strengthen protein and deposit in animal body, improve basic metabolism level, make body fat be tending towards decomposing, promote that growth speed of pigs is accelerated, the red hair of skin is bright, lean meat percentage is high.After people eats the pork containing this clenbuterol hydrochloride, there will be the toxicity symptoms such as nauseating, dizzy, weakness of limbs, hand tremor, particularly larger to heart disease, hyperpietic's harm.Long-term eating then likely causes chromosome aberration, brings out malignant tumour.

Phenolethanolamine A is classified as " forbidding the material used in feed and drinking water for animals " by the Ministry of Agriculture's No. 1519 bulletin, animal and veterinary station, Zao Shan town, Guangan District, Guangan City, Sichuan Province's in September ,-2010 is detected the earliest in the customary forbidden drug monitoring in certain pig farm in Sichuan Province, sow, piglet and growing and fattening pigs urine is detected respectively with Ractopamine test card, find that this growing and fattening pigs urine examination is positive, be confirmed to be novel additive phenolethanolamine A afterwards, Dec, the Ministry of Agriculture issued No. 1519 bulletin, prohibited the use of this chemicals.

Prior art is for the detection mainly detection method such as high performance liquid chromatography-tandem mass method, high performance liquid chromatography of phenolethanolamine A, but the testing process of these methods is loaded down with trivial details, detection time is long, the apparatus expensive of needs, complicated operation, is difficult to promote.According to a set of residue of veterinary drug monitor and detection technology that China has been formed at present, ELISA as a kind of accurately, the detection method of sensitivity, high performance-price ratio, be food safety monitoring, residue of veterinary drug monitoring field carry out the Perfected process that screens.The present invention has the phenolethanolamine A antibody of high-affinity, high specific by distinctive immune animal preparation, sets up a kind of racing ELISA detecting method that can detect phenolethanolamine A.The features such as this method has easy and simple to handle, quick, and highly sensitive, specificity is good.

Summary of the invention

Technical matters to be solved by this invention: a kind of enzyme linked immunological kit that can detect residual benzene monoethanolamine A is fast and accurately provided.Another technical matters to be solved by this invention be to provide with a kind of simple to operate, consuming time short, detection sensitivity is high and be applicable to the method for detection residual benzene monoethanolamine A that batch samples detects.

For solveing the technical problem, through lot of experiments analysis, the invention provides following technical scheme and solving described technical matters:

Detect an enzyme linked immunological kit of residual benzene monoethanolamine A, comprise ELISA Plate 96 hole, each 1 mL of phenolethanolamine A standard items working fluid, phenolethanolamine A antibody working fluid 7 mL, phenolethanolamine A ELIAS secondary antibody working fluid 12 mL, substrate solution 12 mL, stop buffer 7 mL, concentrated cleaning solution 40 mL and concentrating sample dilution 40 mL;

Wherein, described ELISA Plate is wrapped by the ELISA Plate of phenolethanolamine Staphylococal Protein A, and phenolethanolamine Staphylococal Protein A is buffered liquid (CBS) with the carbonate bag of 0.05 mol/L pH 9.6 to be diluted to 1:8000 ratio, and 100 is micro-

L/ hole, 37 DEG C, 2h, takes out 4 DEG C of placements and spends the night; Take out ELISA Plate and get rid of liquid in plate, micro-with the concentrated cleaning solution 300 after dilution

L/ hole, washes plate 3 times, 1min/ time; Then add 0.5% BSA to close, 150 is micro-

L/ hole, places 2 h for 37 DEG C; Take out ELISA Plate and get rid of liquid in plate, pat dry residual liquid in plate with after the towel parcel of cleaning; ELISA Plate after patting dry fitly is put and puts GMP workshop constant temperature (25 DEG C) and dry; Sampling observation requires between plate endoporus and hole, and the OD value coefficient of variation between plate is all less than 10%; Preserve at ELISA Plate being sealed rearmounted 4 DEG C after the assay was approved.

Wherein, the concentration of described phenolethanolamine A standard items is respectively 0 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL.

Wherein, described phenolethanolamine A antibody working fluid is diluted to 1:10000 ratio with antibody diluent;

Described phenolethanolamine A ELIAS secondary antibody working fluid becomes 1:2000 ratio by ELIAS secondary antibody diluted;

Described substrate solution comprises the NaHPO of 0.05 mol/L ₄12H ₂citric Acid Mono (the C of O, 0.025 mol/L ₆h ₈o ₇h ₂o), 3,3,5, the 5-tetramethyl benzidines (TMB) of 0.2 mmol/L, the carbamide peroxide of 0.5 mmol/L and the DMF of 5%;

Described stop buffer is the sulfuric acid (H of 2 mol/L ₂sO ₄);

Described concentrated cleaning solution is 20 times of concentrated cleaning solutions, and it comprises 0.05% Tween-20, and the PBST of 0.01 mol/L, between pH value range 7.0-7.5;

Described concentrating sample dilution is 2 times of concentration and dilution liquid, and its composition is 0.01 mol/L, pH7.4 phosphate PBS damping fluid;

The using method of the enzyme linked immunological kit of above-mentioned detection residual benzene monoethanolamine A, the method comprises the following steps:

(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and nitrogen dries up and redissolved in sample diluting liquid working fluid;

(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) and balance 30 more than min, notice that often kind of liquid reagent must shake up before using;

(3) get bag by the ELISA Plate of phenolethanolamine Staphylococal Protein A, add standard items/sample 50 μ L/ hole in the micropore of correspondence;

(4) add phenolethanolamine A antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;

(5) carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10 s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;

(6) add phenolethanolamine A ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, take out and repeat to wash plate step 5;

(7) add substrate solution 100 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;

(8) add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance (please running through data in 5min);

(9) with the absorbance of standard items test and the log concentration value drawing standard curve of standard items, the content of phenolethanolamine A in reference standard curve calculation sample.

Wherein in step (3), phenolethanolamine A standard items and the sample handled well add in corresponding micropore simultaneously.

Sample after wherein said process is through the sample of following process:

I, pig urine

Get urine sample to be checked, if when sample is more muddy, can 5000 more than r/min, centrifugal 5 min or filtration;

II, Swine serum or cow's serum

Get serum sample to be checked, if when sample is more muddy, can 5000 more than r/min, centrifugal 5 min or filtration;

III, pork flesh tissue or pork liver flesh tissue

(1) accurately take in 2.0 ± 0.05 g homogenised sample to 50 mL centrifuge tubes;

(2) first add 3% trichloroacetic acid 4 mL, shake up with adding 2mL acetonitrile again after vortex instrument whirling motion extremely evenly and fully vibrating;

Centrifugal 10 min of (3) 4000 r/min;

(4) get supernatant 3mL in another centrifuge tube, in it, add 1 mol/LNa ₂cO ₃solution 400 mL, and mix (adjust ph to 9.8 ± 0.2);

(5) add 5 mL ethyl acetate, vibrate 30 s, centrifugal 5 ~ 10 min of 4000 r/min; Get 3 mL supernatant liquids to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

(6) add the sample diluting liquid after 1 mL dilution and fully vibrate and shake up;

(7) 50 μ L are got for analyzing.

Wherein, phenolethanolamine A concentrating sample dilution (2 ×) dilutes by 1:1 volume ratio by the sample diluting liquid deionized water in step III, that is: 1 part of phenolethanolamine A concentrating sample dilution (2 ×)+1 part of deionized water.

The present invention adopts enzyme linked immunosorbent assay (ELISA) method to detect phenolethanolamine A.For detecting the measuring principle of the enzyme linked immunological kit of residual phenolethanolamine A: antigen-specific sexual competition body fixing in the phenolethanolamine A in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of phenolethanolamine A in judgement sample according to the depth of colour developing.If the phenolethanolamine A content in sample is few, colour developing is dark; Otherwise, then develop the color shallow.The features such as kit of the present invention has easily and fast, sensitive, are applicable to batch samples and detect.

Accompanying drawing explanation

Fig. 1 is the typical curve of phenolethanolamine A standard items.

Embodiment

Below adopt more specifically embodiment or embodiment to describe the present invention, but protection scope of the present invention is not intended to be limited to embodiment described herein or embodiment.

The inventive method adopts enzyme linked immunosorbent assay (ELISA) to detect phenolethanolamine A.The principle of this assay method is: based on antigen-specific sexual competition body fixing in the phenolethanolamine A in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of phenolethanolamine A in judgement sample according to the depth of colour developing.Sample light absorption value becomes negative correlation with the content of residue phenolethanolamine A contained by it, is multiplied by its corresponding extension rate more again, can draws the residual quantity of phenolethanolamine A in sample with typical curve.

A preferred embodiment of the invention, the operation steps of the inventive method is:

Get 96 orifice plates being coated with phenolethanolamine Staphylococal Protein A, add phenolethanolamine A standard items with the sample 50 μ L/ hole handled well in the micropore of correspondence; Add phenolethanolamine A antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Carefully open cover plate film, dried by liquid in hole, with wash operating solution 250 μ L/ hole, fully wash 4 ~ 5 times, every minor tick 10 s, pats dry with thieving paper; Add phenolethanolamine A ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, take out and repeat to wash plate as above; Add substrate solution 100 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance; The absorbance tested with standard items and the log concentration value drawing standard curve of standard items, the content of phenolethanolamine A in reference standard curve calculation sample.

A preferred embodiment of the invention, first testing sample is carried out pre-service by the inventive method, be fluid sample by sample preparation to be tested, or is dissolved in sample diluting liquid with Solvent Extract methods testing sample.

Kit of the present invention or detection method can detect various animal derived food or animal excrements, as animal tissue (pig, ox, chicken etc.), feed, urine, serum etc. and other animal derived food or material.Below enumerate the preprocess method of several sample:

I, pig urine

Get urine sample to be checked, if when sample is more muddy, can 5000 more than r/min, centrifugal 5 min or filtration;

II, Swine serum or cow's serum

Get serum sample to be checked, if when sample is more muddy, can 5000 more than r/min, centrifugal 5 min or filtration;

III, pork flesh tissue or pork liver flesh tissue

(1) accurately take in 2.0 ± 0.05g homogenised sample to 50 mL centrifuge tube;

(2) first add 3% trichloroacetic acid 4 mL, shake up with adding 2 mL acetonitriles again after vortex instrument whirling motion extremely evenly and fully vibrating;

Centrifugal 10 min of (3) 4000 r/min;

(4) get supernatant 3 mL in another centrifuge tube, in it, add 1 mol/LNa ₂cO ₃solution 400 mL, and mix (adjust ph to 9.8 ± 0.2);

(5) add 5 mL ethyl acetate, vibrate 30 s, centrifugal 5 ~ 10 min of 4000 r/min; Get 3 mL supernatant liquids to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

(6) add the sample diluting liquid after 1 mL dilution and fully vibrate and shake up;

(7) 50 μ L are got for analyzing.

The present invention is for detecting residual phenolethanolamine A, this enzyme linked immunological kit may be used for the phenolethanolamine A in direct-detection animal derived food, urine sample, animal tissue and blood serum sample, have easily and fast, the feature such as sensitive, be applicable to batch samples detect, this kit has high sensitivity (0.1 ng/mL).

Embodiment

Embodiment 1 prepares PEA-ELISA kit

PEA is small haptens, and only respond originality, but do not have immunogenicity, could be used for immune animal Dispersal risk after needing macromolecular substances to combine.

Phenolethanolamine A (PEA) medicine belongs to small-molecule substance, is haptens, and only respond originality, but do not have immunogenicity, need have immunogenicity with macromolecular carrier albumen connection side.Get phenolethanolamine A, the amino reactive group in its molecular structure is transformed, with the coupling of carrier protein phase, prepare the public antigen for phenolethanolamine A.This research have employed phenolethanolamine A and has prepared artificial antigen as haptens and carrier protein (BSA etc.) by carbodlimide method.

The preparation of PEA-BSA antigen:

By PEA haptens and carrier protein BSA by the combination of 11:1 than being blended in the carbonate buffer solution (CBS) of 0.05 mol/L pH 9.6, then add into carbodiimide, stir l ~ 2h, put room temperature reaction 24h, finally dialyse two days with the PBS of 0.15 mol/L pH 7.6, remove unreacted haptens, PEA-BSA bond can be obtained.

The preparation of anti-phenolethanolamine A antibody:

Select 3 monthly ages healthy White Rabbit, by three immunity, each PEA-BSA immunogene consumption is 50 μ g/0.05 mL, and each immunization interval time is 2 weeks.Initial immunity is respectively by immunizing antigen and Fei Shi adjuvant and Freund's incomplete adjuvant mixed in equal amounts, and the subcutaneous multi-point injection in strength portion, secondary, three immunity directly inject abdominal cavity.Merge first 3 days every mouse peritoneals to inject 25 μ g PEA-BSA and carry out booster immunization, after immunity, the 6th d ear vein gets blood, adopts indirect competitive ELISA method to measure and tires.Directly inject at leg muscle with haptens after obtaining more efficient valency, 8 d rear neck arteries adopt whole blood, are separated antiserum, adopt caprylic acid-ammonium antibody purification, save backup after making freeze-dried powder in-20 DEG C.

The preparation of enzyme mark PEA:

The 25% glutaraldehyde carbonate buffer solution (CBS) of 0.05 mol/L pH 9.6 is diluted to 5%, and take the glutaraldehyde that 5 mg HRP are dissolved in 0.5 mL 5%, 37 DEG C of water-baths are in conjunction with 2 h; Add 0.15 mol/L NaCl 1.0 mL, fully after mixing, put 4 DEG C of precooling 10 min; Add absolute ethyl alcohol 2.4 mL, put upside down mixing, immediately centrifugal 10 min of 1000 r/min; Discard supernatant, precipitate and wash once with 75% cold ethanol, centrifuge tube is inverted, air-dry; Add the CBS 0.5 mL dissolution precipitation thing of 0.05 mol/L pH 9.6; 5 mg PEA antibody are dissolved in 0.5 mL 0.15 mol/LNaCl, then mix with hydroformylation HRP solution; Add the CBS solution of 1.0 mol/L pH 9.6, regulate pH to 9.0 ~ 9.5, at 4 DEG C, electromagnetic agitation is in conjunction with 24 h; Add 0.1 mL 0.15 mol/L lysine, to close residual aldehyde radical, cessation reaction, 2 h at being positioned over 4 DEG C; By reactant by Sephadex G-200 gel column, use PBS wash-out, collect the first eluting peak; Or reactant is loaded bag filter, with 0.01 mol/L pH7.2 PBS, 4 DEG C of dialysed overnight are also collected; Be enzymic-labelled antibody (PEA-HRP).

Bag is by the preparation of plate:

Be buffered liquid (CBS) with bag and envelope antigen is diluted to 1:8000 concentration, 100 μ L/holes, are positioned over 37 DEG C and hatch 2h, take out 4 DEG C of placements and spend the night; Take out ELISA Plate and get rid of liquid in plate, with cleansing solution (PBST) 300 μ L/ hole, wash plate 2 times, 30 s/ time; Add 0.5%BSA, 150 μ L/ holes, place 2h for 37 DEG C; Get rid of liquid in plate, with cleansing solution (PBST) 300 μ L/ hole, wash plate 4 ~ 5 times.Vacuum is drained, and preserves at this bag is placed on 4 DEG C by plate sealing.

The preparation of reagent:

(1) 6 kind of phenolethanolamine A standard items, its phenolethanolamine A concentration is respectively 0 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL, and they carry out corresponding proportion dilution with the PBS dilution of 1.0 mol/L pH 7.4 to prepare;

(2) concentrating sample dilution (8) is 2 times of concentration and dilution liquid, and its composition is 0.01 mol/L, pH7.4 phosphate PBS damping fluid;

(3) concentrated cleaning solution is 20 times of concentrated cleaning solutions, and it comprises 0.05% Tween-20, and the PBST of 0.01 mol/L, between pH value range 7.0-7.5;

(4) substrate solution comprises the NaHPO of 0.05 mol/L ₄12H ₂citric Acid Mono (the C of O, 0.025 mol/L ₆h ₈o ₇h ₂o), 3,3,5, the 5-tetramethyl benzidines (TMB) of 0.2 mmol/L, the carbamide peroxide of 0.5 mmol/L and the DMF of 5%;

(5) stop buffer is the sulfuric acid (H of 2 mol/L ₂sO ₄);

According to one embodiment of the present invention, based on the reagent of above-mentioned preparation, the enzyme linked immunological kit for detecting residual phenolethanolamine A of the present invention comprises following material:

(1) 96 ELISA Plate × 1 piece, hole: be coated with coupled antigen;

(2) titer × 6 bottle: 1mL/ bottle, 0 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL;

(3) high standard product: 1mL/ bottle, 100 ng/mL;

(4) ELIAS secondary antibody 12 mL;

(5) antibody concentrated solution 0.8 mL;

(6) substrate solution 14 mL;

(7) stop buffer 7 mL;

(8) 20 × concentrated cleaning solution 40 mL;

(9) 2 × concentrating sample dilution 50 mL.

Use points for attention during this kit:

(1) room temperature is not got back to room temperature (20 ~ 25 DEG C) lower than 20 DEG C or reagent and sample the OD value of all standards can be caused on the low side;

(2) washing the situation if there is plate hole drying in plate process, then there will be typical curve non-linear, the phenomenon that repeatability is bad.Next step operation should be carried out immediately so wash after plate pats dry;

(3) need to be shaken up before often adding a kind of reagent;

(4) reaction terminating liquid is 2M hydrochloric acid, avoids contacting skin;

(5) kit of date of expiration was not used; Also do not use any reagent in the kit of the term of validity, doping had used the kit of the term of validity can cause the reduction of sensitivity; Do not exchange the reagent used in different lot number kit;

(6) condition of storage: preserve kit in 2 ~ 8 DEG C, not freezing, put no ELISA Plate microwell plate into valve bag and reseal.Standard substance and colourless colour former to photaesthesia, under therefore will avoiding being directly exposed to light;

(7) sign that reagent is rotten: chromogenic agents have any color to show colour former goes bad, should be abandoned it.When absorbance (450/630 nm) value of 0 standard is less than 0.5 (A450 nm<0.5), represents that reagent may go bad, please don't use;

(8) this kit optimal reaction temperature is 25 DEG C, too high or too low for temperaturely will cause detecting absorbance and sensitivity and changes.

Testing result calculates and analyzes:

With 6 kinds of phenolethanolamine A standard concentration 0 ng/mL in the kit of above-mentioned preparation, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL, measure absorbance at 450/630 nm place.

The calculating of percentage absorptance, the percentage absorptance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the percentage absorbance of standard items or sample, then is multiplied by 100%, namely

Percentage absorbance (%)=

The wherein mean absorbance values of B-standard solution or sample solution, B ₀the mean absorbance values of-0ppb standard solution.

With standard items percentage absorptance for ordinate, be horizontal ordinate drawing standard curve with the semilog of phenolethanolamine A standard concentration (ppb), obtain straight-line equation.Typical curve is shown in accompanying drawing 1.Y=－17.529X+99.32，R ²=0.9982。By the B/B of sample ₀value substitutes in typical curve, and read the concentration of corresponding sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of phenolethanolamine A in sample.

Embodiment 2 is added phenolethanolamine A and is reclaimed

The PEA mother liquor of 1 ppm is diluted to debita spissitudo, add to and be accredited as without in the residual urine of PEA or pork sample by high performance liquid chromatography (HPLC) method, the final concentration added is 0.2 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL, 4.0 ng/mL, the sample of often kind of concentration repeats 5, for adding recovery experiment.

Embodiment 3 PEA-ELISA kit detects pig urcine sample

Detect the pig urcine sample being added with 3 kinds of variable concentrations PEA such as 0.5 ng/mL, 1.0 ng/mL and 2.0 ng/mL with PEA-ELISA kit of the present invention, concrete detecting step is as follows:

First processed by sample: get urine sample to be checked, at room temperature, 5000 r/min, centrifugal 5 min, get supernatant point sample.

Get bag by the ELIAS strip of phenolethanolamine Staphylococal Protein A, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add phenolethanolamine A antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10 s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper; Add phenolethanolamine A ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, take out and repeat to wash plate step; Add substrate solution 100 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;

Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance, and reference standard curve calculates the content of phenolethanolamine A in test specimens product.In pig urine samples, the interpolation recovery experiment of 3 kinds of variable concentrations PEA the results are shown in Table 1.

Table 1. pig urcine sample adds recovery experiment result

Wherein, average recovery rate is the numerical value calculated by " (practical measurement value/theoretical add value) × 100% ", and it can show the accuracy of the inventive method; The coefficient of variation is the repeatability representing five revision tests.

Embodiment 4 PEA-ELISA kit detects pork sample

Detect the pork sample being added with 3 kinds of variable concentrations PEA such as 0.5 ng/mL, 1.0 ng/mL and 2.0 ng/mL with PEA-ELISA kit of the present invention, concrete detecting step is as follows:

First sample is processed: (1) accurately takes in 2.0 ± 0.05g homogenate pork sample to 50 mL centrifuge tube; (2) first add 3% trichloroacetic acid 4 mL, shake up with adding 2 mL acetonitriles again after vortex instrument whirling motion extremely evenly and fully vibrating; Centrifugal 10 min of (3) 4000 r/min; (4) get supernatant 3mL in another centrifuge tube, in it, add 1 mol/LNa ₂cO ₃solution 400 mL, and mix (adjust ph to 9.8 ± 0.2); (5) 5 mL ethyl acetate are added, the centrifugal 5 ~ 10min of vibration 30s, 4000 r/min; (6) get 3 mL supernatant liquids to flow down in 50 ~ 60 DEG C of nitrogen and dry up; (7) add the sample diluting liquid after 1 mL dilution and fully vibrate and shake up; (8) 50 mL are got for analyzing.

Get bag by the ELIAS strip of phenolethanolamine Staphylococal Protein A, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add phenolethanolamine A antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10 s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper; Add phenolethanolamine A ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, take out and repeat to wash plate step; Add substrate solution 100 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance, and reference standard curve calculates the content of phenolethanolamine A in test pork sample.In pork sample, the interpolation recovery experiment of 3 kinds of variable concentrations PEA the results are shown in Table 1.

Table 2. pork sample adds recovery experiment result

Embodiment 5 PEA-ELISA kit and liquid chromatography-tandem mass meet experiment

PEA-ELISA kit test method carries out according to the method for embodiment 4, liquid chromatography-tandem mass is according to Sun Zhiwen (Chinese Veterinary Journal 2011,47th volume, 4th phase) method detects: take accurately take 2 g (be accurate to 0.01 g) pork sample in 50 mL centrifuge tubes, add 0.2 mol/L ammonium acetate solution (pH value=5.2) 8.0 mL, add β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase 40 μ L again, vortex mixes, and at 37 DEG C, lucifuge water-bath is vibrated 16 h.Room temperature is placed to after enzymolysis, vortex mixes, 10000 r/min high speed centrifugation 10 min, incline and supernatant in another 50 mL centrifuge tube, add 0.1 mol/L perchloric acid solution 5 mL, vortex mixes, with perchloric acid adjust pH to 1.0 ± 0.2, after centrifugal 10 min of 10000 r/min, supernatant is transferred in another 50 mL centrifuge tube.With 10 mol/L NaOH solution adjust pH to 9.5 ± 0.2, add ethyl acetate 15 mL, vortex mixes, and centrifugal 5 min of 10 min that vibrate, 5 000 r/min, take out upper organic phase in another 50 mL centrifuge tube, merge organic phase, at 50 DEG C, nitrogen dries up, and dissolves with 2% formic acid solution 5 mL, for subsequent use.MCX solid-phase extraction column is use methyl alcohol, water, each 3 mL activation of 2% formic acid solution successively, get the whole post excessively of reserve liquid, then use each 3 mL drip washing of 2% formic acid solution, methyl alcohol successively, drain, with 3% ammoniacal liquor methanol solution 2.5 mL wash-out; Eluent dries up with nitrogen at 50 DEG C.Residue methyl alcohol-0.1% formic acid solution (10+90, v/V) 0.2 mL dissolves, and vortex mixes, 15000 r/min high speed centrifugation 10 min, gets supernatant appropriate, measures for high performance liquid chromatography one tandem mass spectrometer.The detection method of PEA-ELISA kit of the present invention and the related coefficient of HPLC method are R ²=0.9931, this show PEA-ELISA kit method and instrumental method correlativity good, reliable test result.

Comparative example 1 contrast agent box detects pig urine samples

Detect the pig urine samples being added with 3 kinds of variable concentrations PEA such as 0.5 ng/mL, 1.0 ng/mL and 2.0 ng/mL with contrast agent box, detection method is identical with embodiment 3.In pig urine samples, the interpolation recovery experiment of 3 kinds of variable concentrations PEA the results are shown in Table 3.Contrasted from table 1 and table 3, the recovery of embodiment is all more than 90%, and the comparative example recovery is between 71%-80%.This shows, in the detection of pig urine samples, embodiment has the better recovery and repeatability than comparative example, and namely enzyme linked immunological kit of the present invention has higher accuracy of detection.

Table 3. contrast agent box detects pig urine samples and adds recovery experiment result

Comparative example 2 contrast agent box detects pork sample

Detect the pork sample being added with 3 kinds of variable concentrations PEA such as 0.5 ng/mL, 1.0 ng/mL and 2.0 ng/mL with contrast agent box, detection method is identical with embodiment 4.In pork sample, the interpolation recovery experiment of 3 kinds of variable concentrations PEA the results are shown in Table 4.Contrasted from table 2 and table 4, the recovery of embodiment is all more than 90%, and the comparative example recovery is between 60% ~ 89%, and the embodiment coefficient of variation is all within 20%, and comparative example coefficient of variation when 0.5 ng/mL addition is 22.9%, does not meet testing requirement.This shows, in the detection of pork sample, embodiment has the better recovery than comparative example, and measurement result is more stable than comparative example.

Table 4. contrast agent box detects pork sample and adds recovery experiment result

Claims (1)

1. detect a method of phenolethanolamine A in animal tissue and metabolin thereof, it is characterized in that the method comprises the following steps:

The preprocess method of sample:

I, pig urine

Get pig to be checked urine, if when sample is more muddy, 5000 more than r/min, centrifugal 5 min or filtration;

II, Swine serum or cow's serum

Get Swine serum to be checked or ox blood final proof, if when sample is more muddy, 5000 more than r/min, centrifugal 5 min or filtration;

III, pork flesh tissue or pork liver flesh tissue

(1) accurately take in 2.0 ± 0.05g homogenised sample to 50 mL centrifuge tube;

(2) first add 3% trichloroacetic acid 4 mL, shake up with adding 2 mL acetonitriles again after vortex instrument whirling motion extremely evenly and fully vibrating;

Centrifugal 10 min of (3) 4000 r/min;

(4) get supernatant 3 mL in another centrifuge tube, in it, add 1 mol/LNa ₂cO ₃solution 400 mL, and mix adjust ph to 9.8 ± 0.2;

(5) add 5 mL ethyl acetate, vibrate 30 s, centrifugal 5 ~ 10 min of 4000 r/min; Get 3 mL supernatant liquids to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

(6) add the sample diluting liquid after 1 mL dilution and fully vibrate and shake up;

(7) 50 μ L are got for analyzing;

Preparation PEA-ELISA kit

The preparation of PEA-BSA antigen:

By PEA haptens and carrier protein BSA by the combination of 11:1 than being blended in the carbonate buffer solution (CBS) of 0.05 mol/L pH 9.6; then carbodiimide is added; stir l ~ 2h; put room temperature reaction 24h; finally dialyse two days with the PBS of 0.15 mol/L pH 7.6; remove unreacted haptens, PEA-BSA antigen can be obtained;

The preparation of anti-phenolethanolamine A antibody:

Select 3 monthly ages healthy White Rabbit, by three immunity, each PEA-BSA antigen consumption is 50 μ g/0.05 mL, and each immunization interval time is 2 weeks; Initial immunity respectively by immunizing antigen and Fei Shi adjuvant and Freund's incomplete adjuvant mixed in equal amounts, the subcutaneous multi-point injection in strength portion, secondary, three immunity directly inject abdominal cavity; Merge first 3 days every mouse peritoneals to inject 25 μ g PEA-BSA antigens and carry out booster immunization, after immunity, the 6th d ear vein gets blood, adopts indirect competitive ELISA method to measure and tires; Directly inject at leg muscle with PEA haptens after obtaining more efficient valency, 8 d rear neck arteries adopt whole blood, are separated antiserum, adopt caprylic acid-ammonium antibody purification, save backup after making freeze-dried powder in-20 DEG C;

The preparation of the anti-PEA of enzyme mark:

The 25% glutaraldehyde carbonate buffer solution (CBS) of 0.05 mol/L pH 9.6 is diluted to 5%, and take the glutaraldehyde that 5 mg HRP are dissolved in 0.5 mL 5%, 37 DEG C of water-baths are in conjunction with 2 h; Add 0.15 mol/L NaCl 1.0 mL, fully after mixing, put 4 DEG C of precooling 10 min; Add absolute ethyl alcohol 2.4 mL, put upside down mixing, immediately centrifugal 10 min of 1000 r/min; Discard supernatant, precipitate and wash once with 75% cold ethanol, centrifuge tube is inverted, air-dry; Add the CBS 0.5 mL dissolution precipitation thing of 0.05 mol/L pH 9.6; 5 mg PEA antibody are dissolved in 0.5 mL 0.15 mol/LNaCl, then mix with hydroformylation HRP solution; Add the CBS solution of 1.0 mol/L pH 9.6, regulate pH to 9.0 ~ 9.5, at 4 DEG C, electromagnetic agitation is in conjunction with 24 h; Add 0.1 mL 0.15 mol/L lysine, to close residual aldehyde radical, cessation reaction, 2 h at being positioned over 4 DEG C; By reactant by Sephadex G-200 gel column, use PBS wash-out, collect the first eluting peak; Or reactant is loaded bag filter, with 0.01 mol/L pH7.2 PBS, 4 DEG C of dialysed overnight are also collected; Be enzymic-labelled antibody PEA-HRP;

Bag is by the preparation of plate:

Be buffered liquid CBS with bag and envelope antigen is diluted to 1:8000 concentration, 100 μ L/holes, are positioned over 37 DEG C and hatch 2h, take out 4 DEG C of placements and spend the night; Take out ELISA Plate and get rid of liquid in plate, with cleansing solution PBST 300 μ L/ hole, wash plate 2 times, 30 s/ time; Add 0.5%BSA, 150 μ L/ holes, place 2h for 37 DEG C; Get rid of liquid in plate, with cleansing solution PBST 300 μ L/ hole, wash plate 4 ~ 5 times; Vacuum is drained, and preserves at this bag is placed on 4 DEG C by plate sealing;

The preparation of reagent:

(1) 6 kind of phenolethanolamine A standard items, its phenolethanolamine A concentration is respectively 0 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL, and they carry out corresponding proportion dilution with the PBS dilution of 1.0 mol/L pH 7.4 to prepare;

(2) concentrating sample dilution is 2 times of concentration and dilution liquid, and its composition is 0.01 mol/L, pH7.4 phosphate PBS damping fluid;

(3) concentrated cleaning solution is 20 times of concentrated cleaning solutions, and it comprises 0.05% Tween-20, and the PBST of 0.01 mol/L, between pH value range 7.0-7.5;

(4) substrate solution comprises the NaHPO of 0.05 mol/L ₄12H ₂citric Acid Mono (the C of O, 0.025 mol/L ₆h ₈o ₇h ₂o), the TMB (TMB) of 0.2 mmol/L, the carbamide peroxide of 0.5 mmol/L and the DMF of 5%;

(5) stop buffer is the sulfuric acid (H of 2 mol/L ₂sO ₄);

Based on the reagent of above-mentioned preparation, the enzyme linked immunological kit for detecting phenolethanolamine A in animal tissue and metabolin thereof comprises following material:

(1) 96 ELISA Plate × 1 piece, hole: be coated with coupled antigen;

(2) titer × 6 bottle: 1mL/ bottle, 0 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL;

(3) high standard product: 1mL/ bottle, 100 ng/mL;

(4) ELIAS secondary antibody 12 mL;

(5) antibody concentrated solution 0.8 mL;

(6) substrate solution 14 mL;

(7) stop buffer 7 mL;

(8) 20 × concentrated cleaning solution 40 mL;

(9) 2 × concentrating sample dilution 50 mL;

Get 96 orifice plates being coated with phenolethanolamine Staphylococal Protein A, add phenolethanolamine A standard items with the sample 50 μ L/ hole handled well in the micropore of correspondence; Add phenolethanolamine A antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Carefully open cover plate film, dried by liquid in hole, with wash operating solution 250 μ L/ hole, fully wash 4 ~ 5 times, every minor tick 10 s, pats dry with thieving paper; Add phenolethanolamine A ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, take out and repeat to wash plate as above; Add substrate solution 100 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance; The absorbance tested with standard items and the log concentration value drawing standard curve of standard items, the content of phenolethanolamine A in reference standard curve calculation sample.