CN103265440A - Method of detecting content of phenylethanolamine A and detection kit - Google Patents
Method of detecting content of phenylethanolamine A and detection kit Download PDFInfo
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Abstract
The invention discloses a method of detecting a content of phenylethanolamine A and a detection kit. According to the method, immunogens and coating antigens are prepared by synthesizing hapten derivatives of the phenylethanolamine A and crosslinking the derivatives with carrier proteins. Four kinds of polyclonal antibodies of rabbit anti-phenylethanolamine A through immunizing animals are obtained and all the four antibodies can be used for immunoassay of the phenylethanolamine A. The invention further provides an ELISA detection kit containing the antigens and/or antibodies. The ELISA method built by the invention is high in sensitivity and strong in specificity. Four kinds of samples: pig kidney, pig liver, pork and pig forage, are added with different amounts of the phenylethanolamine A for an addition and reclamation test, and the test shows that the detecting method provided by the invention is accurate, and has relatively good correlation with an LC-MS-MS method.
Description
Technical field
The invention belongs to the research field of food safety supervision or food analysis, relate to a kind of ELISA adsorption analysis method (ELISA) that detects Phenylethanolamine A content, the invention still further relates to the correlation detection test kit.
Background technology
Receptor, agonist (abbreviation beta-2-agonists) is the phenylethanol derivative that a class formation and physiological function are similar to suprarenin and norepinephrine, because can be combined with the receptor, on most of cell membranes in tissue in the animal body and gain the name.Beta-2-agonists can significantly improve trunk lean ratio, increase weight and improve food conversion ratio.Therefore, beta-2-agonists is used for the herding productions by a large amount of illegal, and wherein the most frequently used have clenbuterol, salbutamol, a Ractopamine hydrochloride etc.Have many at home and abroad because of the edible report that contains the residual animal liver of beta-2-agonists or lung tissue generation poisoning, symptoms such as headache, tachycardia, elevation of blood pressure, manic uneasiness occur.Therefore, national governments all forbid to use beta-2-agonists in animal rearing.
Clenbuterol hydrochloride poisoning in recent years happens occasionally.A kind of novel clenbuterol hydrochloride has appearred again recently: Phenylethanolamine A.China's food safety alarm bell is beaten in the appearance of this novel clenbuterol hydrochloride again.Phenylethanolamine A is called Ke Lunba amine again, and chemical structural formula is the 2-[4-(4-nitrophenyl) butyl-2-amino]-1-anisole ethanol, English name: Phenylethanolamine A(PA), be a kind of chemical substance of synthetic.Ke Lunba amine is the by product of the synthetic Ractopamine hydrochloride of Lilly Co., Eli., has with clenbuterol hydrochloride Ractopamine hydrochloride identical effect and effect, belongs to a kind of of beta-2-agonists, has nutrition reallocation effect.Classified as the material of forbidding use in feed and animal drinking-water the end of the year 2010 by No. 1519 bulletin of the Ministry of Agriculture.
At present, Phenylethanolamine A detection method mostly is red, orange, green, blue, yellow (ROGBY).As: the high performance liquid chromatography-tandem mass method among No. 1486 bulletin-1-2010 of the Ministry of Agriculture is measured the Phenylethanolamine A in the feed.Although chromatogram analysis method is more accurate, expensive, the complicated operation of liquid chromatography-tandem mass spectrometry instrument, sample pretreatment complexity, time-consuming detects the cost height, is not suitable for the rapid screening of a large amount of samples.
That ELISA adsorption analysis method (ELISA) has is highly sensitive, high specificity, analyze advantage fast, and oneself is widely used in analysis fields such as clinical, medicine, food and environment.Thereby, set up a kind of easy high-throughout elisa assay method that is applied to Phenylethanolamine A in quantitatively conventional/screening food and the feed and have very important practical application meaning.Yet the ELISA detection method that up to the present, still lacks the Phenylethanolamine A of highly sensitive and high specific.
Summary of the invention
The objective of the invention is provides a kind of residual ELISA adsorption analysis method of Phenylethanolamine A that detects at the deficiencies in the prior art.
The present invention also aims to provide detection reagent and the detection kit of Phenylethanolamine A residue detection.
The present invention discovers, Phenylethanolamine A is carried out specific modification, obtains haptens, and coupling carrier albumen obtains antigen again, and obtains antibody as the immunogen immune animal, and the elisa assay method of setting up thus can significantly improve detection sensitivity and specificity.
Accordingly, for realizing purpose of the present invention, at first a kind of Phenylethanolamine A of the present invention haptens, it has the structural formula shown in the formula (I):
This haptens is that Phenylethanolamine A is dissolved in the dehydrated alcohol, adds hydrazine hydrate, palladium carbon after being warming up to 80 ℃, and the following 80 ℃ of back flow reaction of nitrogen protection condition obtain.Its reaction formula is as follows:
And then the invention provides Phenylethanolamine A antigen, it is above-mentioned haptens and the coupled complex of carrier proteins.
The preparation method of this antigen is dissolved in the water above-mentioned Phenylethanolamine A haptens, slowly drips NaNO
2Solution, and be 1.5 with the hydrochloric acid adjust pH, 4 ℃ of reactions in dark place 1～10 hour, after rough determination diazotization is finished, slowly drip the urea soln termination reaction, above-mentioned diazotization solution is dropwise joined in 0.01～0.02% the carrier proteins solution, regulating pH is 6～9,4 ℃ were slowly stirred 1～10 hour, dialyse 1～5 day namely.
Further the invention provides the antibody of Phenylethanolamine A, it makes as the immunogen immune animal for above-mentioned antigens.Can make polyclonal antibody or monoclonal antibody according to ordinary method.
Based on above-mentioned antigen and antibody, the invention provides Phenylethanolamine A detection kit, it comprises above-mentioned antigens and/or above-mentioned antibody.
Particularly, test kit of the present invention comprises: wrap by the enzyme plate of above-mentioned antigen above-mentioned antibody; And be selected from one or more of following reagent:
A) ELIAS secondary antibody;
B) substrate solution;
C) stop buffer;
D) standard solution;
E) confining liquid;
F) washings.
Described antigen is the coupled complex of described haptens and bovine serum albumin or oralbumin; Described antibody is that the coupled complex of described haptens and bovine serum albumin or oralbumin is the polyclonal antibody that immunogen makes; Described ELIAS secondary antibody is the horseradish peroxidase-labeled goat anti-rabbit igg; Described substrate solution is TMB solution; Described stop buffer is H
2SO
4Solution; Described standard solution is Phenylethanolamine A solution; Described confining liquid is casein solution; Described washings is the PBST damping fluid.
Above-mentioned detection kit can be for detection of Phenylethanolamine A, the especially detection of Phenylethanolamine A in food and the feed.
The present invention also provides the method that detects Phenylethanolamine A content, and this method comprises the steps:
(a). add above-mentioned antigens in enzyme plate, the 50-200ng/ hole, every hole 200 μ L, refrigerator spends the night for 4 ℃;
(b). use the PBST damping fluid, with 350 μ L/ holes, wash plate three times;
(c). add casein and blockade, every hole 300 μ L, the room temperature heat and moisture preserving was placed 1 hour;
(d). wash plate three times with the PBST damping fluid;
(e). add the antibody of 100 μ L Phenylethanolamine A standard solutions and 100 μ L1:10000 to 1:50000 dilution in every hole of enzyme plate successively respectively, the room temperature heat and moisture preserving was placed 1 hour;
(f). wash plate three times with the PBST damping fluid;
(g). add the horseradish peroxidase-labeled goat anti-rabbit igg, every hole 200 μ L, the room temperature heat and moisture preserving was placed 1 hour;
(h). wash plate three times with the PBST damping fluid;
(i). add substrate solution, every hole 200 μ L, the reaction of room temperature heat and moisture preserving lucifuge, jolting is about 15～25 minutes on micro oscillator;
(j). add 5%H
2SO
4Solution, every hole 50～100 μ L stop enzyme reaction;
(k). measure absorbancy with microplate reader;
(l). the step according to (a) to (k) replaces with sample solution with standard solution in step (e), the working sample solution absorbance obtains sample solution Phenylethanolamine A content according to the typical curve of standard substance and the absorbancy of sample solution.
Compared with prior art, the present invention has the following advantages:
1. highly sensitive: compare with the ELISA test kit of existing report, sensitivity improves 2～20 times.
2. high specificity: the cross reacting rate 0.01%～0.39% of four kinds of antibody and clenbuterol, salbutamol, Ractopamine hydrochloride, with all the other 6 kinds of medicine no cross reactions.
3. sample preparation is simple, test volume is big, testing expense is low.
4. to the mensuration of sample, ELISA and LC-MS-MS have good dependency.
Description of drawings
Fig. 1. Phenylethanolamine A derivative (PA-NH
2), the ultraviolet-visible light spectrogram of the cross-linking agent of bovine serum albumin (BSA) and Phenylethanolamine A-bovine serum albumin (PA-BSA).
Known PA-NH by Fig. 1
2The ultraviolet characteristic peak about 280nm, locate, the ultraviolet characteristic peak of BSA is also located about 280nm, and PA-BSA is except having the characteristic absorbance at the 280nm place, also has a characteristic peak at the 330nm place, this is because the N=N that albumen generates after connecting causes absorbing wavelength elongated, show that thus Phenylethanolamine A derivative is successfully crosslinked with carrier proteins.
Phenylethanolamine A derivative (PA-NH
2), oralbumin (OVA) is similar to Fig. 1 with the ultraviolet-visible light spectrogram of the cross-linking agent of Phenylethanolamine A-oralbumin (PA-OVA).
Fig. 2. the ELISA that four kinds of polyclonal antibodies are set up measures the typical curve of Phenylethanolamine A.
■ envelope antigen PA – OVA, 1:5000 (200ng/well); Antibody I, 1:50000; Goat anti-rabbit igg-horseradish peroxidase (GaRIgG-HRP), 1:10000; IC
500.049ng/mL; ● envelope antigen PA – OVA, 1:20000 (50ng/well); Antibody I I, 1:50000; GaRIgG-HRP, 1:10000; IC
500.22ng/mL; ▲ envelope antigen PA – BSA, 1:5000 (200ng/well); Antibody I II, 1:20000; GaRIgG-HRP, 1:10000; IC
500.21ng/mL; ▼ envelope antigen PA – BSA, 1:10000 (100ng/well); Antibody I V, 1:10000; GaRIgG-HRP, 1:10000; IC
500.48ng/mL.
Fig. 3 .ELISA and LC-MS-MS are to the correlation curve of the detected result of Phenylethanolamine A in 7 mark-on samples.
Embodiment
Carry out concrete description below by the present invention of embodiment; be necessary to be pointed out that at this this enforcement only is used for invention is further specified; but can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
The percentage sign that relates among the present invention " % " if do not specify, refers to mass percent; But the per-cent of solution except as otherwise herein provided, refers to contain among the solution 100ml the some grams of solute; Per-cent between the liquid refers to the ratio of capacity in the time of 20 ℃.
Embodiment 1:
1. the preparation of Phenylethanolamine A modifier
1) Phenylethanolamine A haptens (PA-NH
2) synthetic
Take by weighing PA and be dissolved in the dehydrated alcohol, add hydrazine hydrate (PA and hydrazine hydrate are by waiting mole adding) after being warming up to 80 ℃, palladium carbon is catalyzer, and 80 ℃ of back flow reaction 1～10h(are to reacting completely under the nitrogen protection condition).After reaction finished, suction filtration was spin-dried for filtrate, gets faint yellow crude product.Cross post with methylene chloride/triethylamine by the solvent ratio of (15:1:0.05), obtain pure product, product is abbreviated as PA-NH
2, its reaction formula is as follows:
2) Phenylethanolamine A haptens (PA-NH
2) sign:
1H-NMR spectrum: with the haptenic nuclear-magnetism spectrum of Bruker AMX-400 nuclear magnetic resonance analyser test Phenylethanolamine A, be solvent with deuterium for the inferior maple solution of diformazan, in be designated as TMS;
1H?NMR(DMSO-d6,400MHz):1.26(t,J=7.2Hz,3H,CH
3),1.66(s,1H,CH-H),2.00(s,1H,CH-H),2.37(s,1H,CH-H),2.53(s,1H,CH-H),2.994-3.01(m,2H,CH
2,),3.13(s,1H,CH),3.74(s,3H,CH
3O),4.82-4.88(m,2H,CH+OH),6.04(s,1H,NH),6.48(d,J=8.0Hz,2H,ArH),6.86(d,J=8.0Hz,2H,ArH),6.94(d,J=7.2Hz,2H,ArH),7.31(d,J=7.31Hz,2H,ArH),8.51(s,2H,NH
2).
HRMS: high resolution mass spectrometer test Phenylethanolamine A haptens C
19H
26N
2O
2Molecular weight.Measured value, m/z:315.2060 (M+1); Theoretical value, m/z:315.2067 (M+1).
2. the preparation of immunogen and envelope antigen
Take by weighing Phenylethanolamine A haptens (PA-NH
2) be dissolved in the water, slowly drip NaNO
2Solution, and be 1.5 with the hydrochloric acid adjust pH, 4 ℃ of reactions in dark place 1～10 hour.After rough determination diazotization is finished, slowly drip urea soln in diazotization solution, termination reaction.Above-mentioned diazotization solution dropwise joined in 0.02% the bovine serum albumin (BSA) or 0.02% oralbumin (OVA), regulating pH is 7.5,4 ℃ were slowly stirred 5 hours, dialysed 4 days, obtain two kinds of Phenylethanolamine A-protein solns, lyophilize ,-40 ℃ of preservations are stand-by: Phenylethanolamine A-bovine serum albumin (PA-BSA) and Phenylethanolamine A-oralbumin (PA-OVA) all can be used as immunogen or envelope antigen.
The ultraviolet-visible light spectrogram of PA, BSA and PA-BSA cross-linking agent as shown in Figure 1, the ultraviolet-visible light spectrogram of PA, OVA and PA-OVA cross-linking agent is similar to Fig. 1.
3. Phenylethanolamine A Polyclonal Antibody Preparation
With 4 new zealand white rabbits of two kinds of immunogen immune: take by weighing the 3mg immunogen and be dissolved in the physiological saline of 2mL, add 2mL Fu Shi Freund's complete adjuvant, be mixed into water in oil emulsion, every rabbit draws the emulsion of 1.5mL at every turn, multiple spot subcutaneous injection rabbit back after 3 weeks, carries out booster immunization to rabbit, use freund 's incomplete adjuvant, all the other are identical with immunity for the first time, after the immunity for the second time, every carrying out immunity next time 3 weeks, and for the third time, after the 4th immunity, extract 0.1～0.5mL ear blood in 6 days, detect the situation that antibody produces, after the 5th immunity, put to death rabbit in 10 days, get whole blood, blood is placed in refrigerator spent the night, draw supernatant liquid, packing, in cryogenic refrigerator, store, four kinds of antibody are arranged, be i.e. the antibody I of PA-BSA immunogen preparing, the antibody III of II and PA-OVA immunogen preparing, IV.
4. set up the ELISA adsorption analysis method (ELISA) of measuring Phenylethanolamine A
The optimization experiment condition is carried out performance characterization to prepared antibody, under optimum experiment condition, sets up the ELISA adsorption analysis method of measuring Phenylethanolamine A content.
(1) solution preparation
(a). yellow soda ash-sodium bicarbonate buffer liquid
Take by weighing 2.606g Na
2CO
310H
2O, 3.434g NaHCO
3, after the dissolving of 800mL ultrapure water mixing, regulate the pH value, add water to 1L, be made into 0.05mol/L, the yellow soda ash of pH=9.6-sodium bicarbonate buffer liquid;
(b). and phosphoric acid buffer (storing solution, PBSx10)
Take by weighing 21.961g Na
2HPO
412H
2O, 6.031g NaH
2PO
42H
2O, 87.666g NaCl adds the 800mL ultrapure water and mixes heating for dissolving; NaOH with 1mol/L regulates pH=7.4; Add ultrapure water to 1L, be made into and contain 0.15mol/L NaCl, the 0.1mol/L phosphoric acid buffer (storing solution) of pH=7.4;
(c). casein solution
Take by weighing the casein heating for dissolving in the PBS of 0.01mol/L, be made into 0.5～2% casein solution;
(d). and phosphoric acid buffer-tween storing solution (the 0.1mol/L phosphoric acid buffer storing solution that contains 1%Tween20, PBSTx10, pH=7.4).
(e). substrate solution (20mL pure water; The 1mL sodium-acetate buffer; 200 μ l tetramethyl benzidines (TMB) (1%); 20 μ l hydrogen peroxide (5%))
1.. sodium-acetate buffer: take by weighing 3.450g CH
3COONa3H
2O with the dissolving of 100mL ultrapure water, uses the 1mol/L citric acid (to take by weighing 21.031g C again
6H
8O
7H
2O is dissolved in the 100mL water) regulate pH=5.8 after, add water again in the 250mL volumetric flask, be made into the 0.1mol/L sodium-acetate buffer;
2. .TMB: take by weighing 0.0717g tetramethyl benzidine (TMB), use the 7.17mL dmso solution, mixing is made into 1%, v/v;
3.. hydrogen peroxide: the hydrogen peroxide of getting 20 μ L30% adds in the 100 μ L ultrapure waters, and mixing is made into 5%;
(f) .H
2SO
4Solution: pipette the dense H of 25mL
2SO
4, be dissolved in the ultrapure water of 475mL, be made into 5%H
2SO
4Solution;
(2) key instrument
Wash plate machine: 1575, Bio-Rad, USA; Microplate reader: A2082, Tecan, Austria; Using high performance liquid chromatography tandem mass spectrum instrument: Shimadzu HPLC, Japan; AB SCIEX QTRAP4000, Applied Biosystem Analytical Technologies, USA
(3) indirect competitive ELISA step
(a). add certain density envelope antigen in enzyme plate, the 50-200ng/ hole,, every hole 200 μ L, refrigerator spends the night for 4 ℃;
(b). wash plate three times with PBST damping fluid (PBST storing solution, 1:10 dilution, 350 μ L/ holes);
(c). add casein and blockade, every hole 300 μ L, the room temperature heat and moisture preserving was placed 1 hour; (d). wash plate three times with the PBST damping fluid;
(e). add 100 μ L standardized solution and 100 μ L successively respectively by the antibody of 1:10000 to 1:50000 dilution in every hole of enzyme plate, the room temperature heat and moisture preserving was placed 1 hour;
(f). wash plate three times with the PBST damping fluid;
(g). and the adding ELIAS secondary antibody (goat anti-rabbit igg-horseradish peroxidase, GaRIgG-HRP), every hole 200 μ L, the room temperature heat and moisture preserving was placed 1 hour;
(h). wash plate three times with the PBST damping fluid;
(i). add substrate solution (facing with newly joining), every hole 200 μ L, the reaction of room temperature heat and moisture preserving lucifuge, jolting is about 15～25 minutes on micro oscillator;
(j). add 5%H
2SO
4Solution, every hole 50～100 μ L stop enzyme reaction;
(k). measure absorbancy with microplate reader, make typical curve, carry out interpretation of result and discussion.
(4) optimal conditions of ELISA
Table 1ELISA optimal conditions parameter comprises concentration, the antibody of various combination, the envelope antigen of antigen-antibody
The extent of dilution of extent of dilution, goat anti-rabbit igg-horseradish peroxidase (GaRIgG-HRP)
(5) typical curve of ELISA and sensitivity
Fig. 2 is the typical curve that the ELISA that sets up of four kinds of polyclonal antibodies measures Phenylethanolamine A.As shown in Figure 2, four kinds of antibody all can be used for the immunoassay of Phenylethanolamine A, the IC of the ELISA that sensitivity is the highest
50Be 0.049ng/mL, the IC of other three kinds of ELISAs
50Be respectively 0.22,0.21 and 0.48ng/mL.
(6) specificity of ELISA
The ELISA specificity can be represented with the size of cross reacting rate.The IC of cross reacting rate (CR%)=Phenylethanolamine A
50The IC of/test substances
50* 100%.Cross reacting rate is more little, and the specificity of ELISA is more high.
The chemical structure of table 2 cross reacting material
The cross reaction result of four kinds of antibody of table 3
The present invention selects Ractopamine hydrochloride and other the 8 kinds medicines that can make an addition in the animal-feed to carry out the cross reaction experiment, and the chemical structure of cross reacting material and cross reaction experimental result are shown in table 2, table 3.The cross reacting rate 0.01%～0.39% of four kinds of antibody and Ractopamine hydrochloride, clenbuterol and salbutamol with all the other 6 kinds of medicine no cross reactions, illustrates the ELISA method high specificity of setting up.
5.ELISA to Phenylethanolamine A Determination on content in the mark-on sample
Select four kinds of samples: pig kidney, pork liver, pig feed and pork carry out mark-on and reclaim experiment; Same sample is got two parts, and an amount of Phenylethanolamine A standard reserving solution of a adding is a as blank sample; (vortex is behind the supersound extraction 30min for 0.1:100, v/v) extracting solution to add formic acid methyl alcohol in sample, 10000g/15min is centrifugal, gets supernatant liquor and crosses the Solid-Phase Extraction column purification, collects elutriant, nitrogen dries up, and adds phosphoric acid buffer, directly measures with ELISA behind the dilute with water.The result is as shown in table 4, and recovery of standard addition is 92.2 – 113.7%, and relative standard deviation (RSD) is 3.8 – 10.9% (n=3) in batch.
Table 4ELISA measures the rate of recovery and the relative standard deviation (RSD) of Phenylethanolamine A in the mark-on sample
* blank is negative sample
6.ELISA the comparison with LC-MS-MS
The HPLC condition determination is: chromatographic condition: C
18Post (50mm * 2.1mm, 3 μ m); Mobile phase A (0.1% formic acid), Mobile phase B (acetonitrile), condition of gradient elution: 0～0.5min, 5%B～5%B, 0.5～2min, 5%B～80%B, 2～3.5min, 80%B～80%B, 3.5～3.6min, 80%B～5%B, 3.6～6min, 5%B～5%B; Column temperature: 30 ° of C; Flow velocity is 0.3mL/min; Sample size is 10 μ L.Mass spectrum condition: electric spray ion source; Positive ion scanning; Multiple-reaction monitoring MRM; Spray voltage: 4500V; Ion source temperature: 500 ° of C; Qualitative ion pair m/z345.2 → 327.2 and 345.2 → 150.1, corresponding impact energy is respectively 19eV and 32eV; Quota ion is to m/z345.2 → 150.1.Phenylethanolamine A concentration of standard solution is: 0.5,1,5,10,50,100,200ng/mL, sample extracting solution directly measure after with the membrane filtration of 0.45 μ m.
The reliability of the ELISA method of setting up is further verified with the LC-MS-MS method.Two kinds of samples add the Phenylethanolamine A(pig feeds of different amounts: add scalar and be respectively 1,5 and 20ng/g; Pork: 2,10,50 and 100ng/g), (0.1:100 v/v) measures with ELISA method and LC-MS-MS method respectively behind the extraction and cleaning mark-on sample with formic acid methyl alcohol.Measurement result with LC-MS-MS is X-coordinate, and the ELISA measurement result is the ordinate zou mapping, gets both correlation curves, as shown in Figure 3, regression curve is that (R=0.9956 n=7), shows that ELISA and LC-MS-MS have good dependency to Y=0.97266X-1.48719.
Claims (10)
1. Phenylethanolamine A haptens, it has the structural formula shown in the formula (I):
2. Phenylethanolamine A antigen, it is the described haptens of claim 1 and carrier protein couplet mixture.
3. Phenylethanolamine A antibody, it makes as the immunogen immune animal for the described antigen of claim 2.
4. Phenylethanolamine A detection kit, it comprises the described antigen of claim 2 and/or the described antibody of claim 3.
5. detection kit according to claim 4 is characterized in that, it comprises: wrap by the enzyme plate of the described antigen of claim 2 the described antibody of claim 3; And be selected from one or more of following reagent:
A) ELIAS secondary antibody;
B) substrate solution;
C) stop buffer;
D) standard solution;
E) confining liquid;
F) washings.
6. detection kit according to claim 5 is characterized in that, described antigen is the coupled complex of the described haptens of claim 1 and bovine serum albumin or oralbumin; Described antibody is that the coupled complex of the described haptens of claim 1 and bovine serum albumin or oralbumin is the polyclonal antibody that immunogen makes; Described ELIAS secondary antibody is the horseradish peroxidase-labeled goat anti-rabbit igg; Described substrate solution is TMB solution; Described stop buffer is H
2SO
4Solution; Described standard solution is Phenylethanolamine A solution; Described confining liquid is casein solution; Described washings is the PBST damping fluid.
7. the application of each described detection kit of claim 4～6 in detecting Phenylethanolamine A.
8. method that detects Phenylethanolamine A content, this method comprises the steps:
(a). add the described antigen of claim 2 in enzyme plate, the 50-200ng/ hole,, every hole 200 μ L, refrigerator spends the night for 4 ℃;
(b). use the PBST damping fluid, with 350 μ L/ holes, wash plate three times;
(c). add casein and blockade, every hole 300 μ L, the room temperature heat and moisture preserving was placed 1 hour;
(d). wash plate three times with the PBST damping fluid;
(e). add the antibody of 100 μ L Phenylethanolamine A standard solutions and 100 μ L1:10000 to 1:50000 dilution in every hole of enzyme plate successively respectively, the room temperature heat and moisture preserving was placed 1 hour;
(f). wash plate three times with the PBST damping fluid;
(g). add the horseradish peroxidase-labeled goat anti-rabbit igg, every hole 200 μ L, the room temperature heat and moisture preserving was placed 1 hour;
(h). wash plate three times with the PBST damping fluid;
(i). add substrate solution, every hole 200 μ L, the reaction of room temperature heat and moisture preserving lucifuge, jolting is about 15～25 minutes on micro oscillator;
(j). add 5%H
2SO
4Solution, every hole 50～100 μ L stop enzyme reaction;
(k). measure absorbancy with microplate reader;
(l). the step according to (a) to (k) replaces with sample solution with standard solution in step (e), the working sample solution absorbance obtains sample solution Phenylethanolamine A content according to the typical curve of standard substance and the absorbancy of sample solution.
9. the preparation described Phenylethanolamine A of claim 1 haptenic method, this method is that Phenylethanolamine A is dissolved in the dehydrated alcohol, adds hydrazine hydrate, palladium carbon after being warming up to 80 ℃, the following 80 ℃ of back flow reaction of nitrogen protection condition obtain.
10. the method for preparing the described Phenylethanolamine A of claim 2 antigen, this method are that the described Phenylethanolamine A of claim 1 haptens is dissolved in the water, and slowly drip NaNO
2Solution, and be 1.5 with the hydrochloric acid adjust pH, 4 ℃ of reactions in dark place 1～10 hour, after rough determination diazotization is finished, slowly drip the urea soln termination reaction, above-mentioned diazotization solution is dropwise joined in 0.01～0.02% the carrier proteins solution, regulating pH is 6～9,4 ℃ were slowly stirred 1～10 hour, dialyse 1～5 day namely.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152455A (en) * | 2014-07-30 | 2014-11-19 | 暨南大学 | Phenethylamine aptamer and aptamer electrochemical biosensor for detecting phenethylamine |
| CN104558183A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting phenolethanolamine A |
| CN110646381A (en) * | 2018-06-27 | 2020-01-03 | 北京中龙益诚科技有限公司 | Surface plasma resonance immunization method for detecting beta 2 receptor stimulant in pig urine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152455A (en) * | 2014-07-30 | 2014-11-19 | 暨南大学 | Phenethylamine aptamer and aptamer electrochemical biosensor for detecting phenethylamine |
| CN104152455B (en) * | 2014-07-30 | 2016-07-06 | 暨南大学 | The aptamers electrochemica biological sensor of gram rumba amine aptamers and detection Ke Lunba amine |
| CN104558183A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting phenolethanolamine A |
| CN110646381A (en) * | 2018-06-27 | 2020-01-03 | 北京中龙益诚科技有限公司 | Surface plasma resonance immunization method for detecting beta 2 receptor stimulant in pig urine |
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| Publication number | Publication date |
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| CN103265440B (en) | 2014-10-22 |
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