CN104849476A - Enzyme-labeled antigen for protein anabolic hormone immunoassay and analysis method - Google Patents
Enzyme-labeled antigen for protein anabolic hormone immunoassay and analysis method Download PDFInfo
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Abstract
The invention provides an enzyme-labeled antigen for protein anabolic hormone immunoassay and an analysis method and belongs to the immunochemical analysis technology. The enzyme-labeled antigen for protein anabolic hormone immunoassay is prepared by connecting nandrolone hapten and horseradish peroxidase, and the nandrolone hapten is prepared by introducing carboxyl on the basis of hydroxide radical of nandrolone. The enzyme-labeled antigen can be applied to protein anabolic hormone multi-residue chemiluminescence immunoassays of nandrolone, trenbolone, propionic acid nandrolone, Stanolone and mestanolone. The analysis method overcomes the defect of low sensitivity of the traditional Enzyme linked immunosorbent analysis method and improves the accuracy and detection efficiency of immunodetection.
Description
Technical field
The invention belongs to micromolecular compound immunochemistry and multi-residue analysis technical field, especially relate to a kind of enzyme-labelled antigen for protein anabolic hormone immunoassay and analytical approach.
Background technology
Protein anabolic hormone (anabolic steroid), also known as anabolic hormone, is steroid hormone.It mainly comprises nandrolone (17 β-19-nortestosterone), Trenbolone (β-trenbolone), testobolin (19-nortestosterone propionate), allodihydrotestosterone (stanolone), Mestanlone (mestanolone) and Tibolone (tibolone) etc.
Protein anabolic hormone too much can cause harmful effect to body many-side at people's body accumulation.Such as, in the dysfunction of liver of people, protein anabolic hormone effect can make glutamic-oxalacetic transaminease, alkaline phosphatase enzyme, cholerythrin and gpt activity improve, and produces toxic and side effect to liver, form hepatohemia tumour, even cancer.And show according to reliable relevant report, the coronary heart disease in cardiovascular disease is too high by LDL-C in protein anabolic hormone and cause.In addition, the steroids in protein anabolic hormone can extreme influence Development of Reproductive System.Children or juvenile edible too much protein anabolic hormone there will be the existing of bad growth.Common performance has of short and small stature, and muscle is not independently twitched.
But because protein anabolic hormone is often illegally added in the middle of the feed of animal edible, in order to promote that animal increases, cause this type of medicament residue phenomenon in China's food to exist in a large number, very disruptive food products market, have impact on food security.For hitting this behavior, China clear stipulaties forbids to use protein anabolic hormone in edible animal is raised.Meanwhile, must not detect in animal derived food and functional food.
At present, more common high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), GC-MS(gas chromatography-mass spectrography) (GC-MS), Liquid Chromatography-Mass Spectrometry (LC-MS) and enzyme linked immunosorbent assay (ELISA) are used to detect protein anabolic hormone.There is complex pretreatment in traditional instrument method, instrument and corresponding support expense costliness, cannot carry out the shortcomings such as Duplicate Samples detects simultaneously on a large scale.And the minimum detectability of enzyme linked immunological often cannot reach the detection limit requirement of China for this type of material.Therefore in the urgent need to developing easier, quick, sensitive analytical technology.
Chemiluminescence immune analysis method (chemiluminescence immunoassay, CLIA) combine traditional euzymelinked immunosorbent assay (ELISA) advantage in the quick context of detection of batch samples and chemical luminescent detecting technology high sensitivity two, in the immune analysis method exploitation of Small molecular hazardous compound, become study hotspot in recent years.
But different from large molecule, micromolecular compound immunoassay has own characteristic:
(1) micromolecular compound (MW≤1000dolton) does not generally have immunogenicity, specific antibody can not be produced, the haptens of outstanding molecule stereo structure specific site must be synthesized by direct immunization animal, and connecting and composing binding element with macromolecular carrier, ability immune animal produces the specific antibody for this target micromolecular compound.The bond of this haptens and macromolecular carrier is called artificial antigen.The preparation of artificial antigen is not arbitrary, comprise binding site, combination, kind of carrier and haptens and any structural difference of target analytes as the factors of size, shape, composition, configuration, conformation, polarity, cloud density etc., all greatly may affect the character of corresponding antibodies, therefore they are the keys determining to produce its specific antibody and set up immune analysis method.
(2) although micromolecular compound does not have immunogenicity, there is reactionogenicity, namely there is the ability with corresponding antibodies generation immunological response, and can Quantitative in vitro be carried out, follow the mass action law.
Immuno analytical method is introduced into Small molecular residual point of folding field, become a kind of quantitative analysis tech having development and application potential most it-, paid attention to widely.The key of this technical research is the preparation of haptenic MOLECULE DESIGN, synthesis and artificial holoantigen and antibody.Therefore, target analyte molecule immunological characteristic, and how by chemistry or biochemical technology outstanding and utilize these characteristics, be the important research contents in this field.This technology has become a brand-new field of microanalysis research at present, can with traditional analysis side by side as a new analysis approach.
(3) chemiluminescence immunoassay is on the basis of immune analysis method, determines each component concentration in reaction according to chemiluminescence reaction in the luminous intensity put sometime (such as peak strength) or luminous total amount.Compare traditional enzyme-linked immune analytic method, its sensitivity can improve greatly.
The general broad spectrum activity of protein anabolic hormone immune analysis method of current bibliographical information is poor, generally can detect one to three kind of protein anabolic hormone medicine, and there is not been reported for more than three kinds protein anabolic hormone medicament residue chemiluminescence immune analysis methods simultaneously.Protein anabolic hormone medicament categories is various, widespread use and have a certain curative effect also have tens kinds.If adopt these analytical approachs existing to detect actual sample, some protein anabolic hormone medicines are often caused to be detected, the situation that in testing result and sample, protein anabolic hormone residual quantity difference is larger really.Therefore be necessary to provide a kind of analytical approach that simultaneously can detect multiple protein anabolic hormone, solve the problem, improve immune detection accuracy and detection efficiency.
Summary of the invention
In view of this, the present invention is intended to propose a kind of enzyme-labelled antigen for protein anabolic hormone immunoassay and analytical approach, with overcome the low shortcoming of traditional enzyme-linked immune analytic method sensitivity and solve current protein anabolic hormone medicine multi-residue determination method lack problem, the immune analysis method of five kinds of protein anabolic hormones such as nandrolone, Trenbolone, testobolin, allodihydrotestosterone and Mestanlone can be detected in conjunction with chemiluminescence simultaneously.
For achieving the above object, technical scheme of the present invention is achieved in that
For an enzyme-labelled antigen for protein anabolic hormone immunoassay, to be connected with horseradish peroxidase by nandrolone haptens and to synthesize, its preparation comprises the steps;
1) the haptenic preparation of nandrolone, taking 0.5 ~ 2.0g nandrolone is dissolved in 10 ~ 25mL anhydrous tetrahydro furan, and add 0.5 ~ 2.0g succinic anhydride, 0.6 ~ 2.0mL triethylamine and 0.2 ~ 1.5g4-dimethylamino naphthyridine, sustained response 8 ~ 17h at 62 ~ 70 DEG C of temperature, be spin-dried for by solvent subsequently, product volume ratio is that the sherwood oil of 1:2 ~ 5:6 and the mixed liquor of ethyl acetate dissolve; Take volume ratio as the sherwood oil of 1:2 ~ 5:6 and the mixed liquor of ethyl acetate be developping agent, carry out purifying and obtain white crystal, be nandrolone haptens;
2) by active ester method, with hydroxyl CO=OH for bridge, make nandrolone artificial semiantigen be connected in horseradish peroxidase, namely obtain enzyme-labelled antigen.
Preferably, step 1) in, taking 1.316g nandrolone is dissolved in 14.50mL anhydrous tetrahydro furan, and add 0.960g succinic anhydride, 1.32mL triethylamine and 0.584g4-dimethylamino naphthyridine, sustained response 12h at 60 DEG C of temperature, be spin-dried for by solvent subsequently, product volume ratio is that the sherwood oil of 1:1 and the mixed liquor of ethyl acetate dissolve; Take volume ratio as the sherwood oil of 1:1 and the mixed liquor of ethyl acetate be developping agent, carry out purifying and obtain white crystal, be nandrolone haptens.
Preferably, step 2) in, the preparation of enzyme-labelled antigen comprises the steps,
A) precise 0.03 ~ 0.04mmol nandrolone haptens is dissolved in 250 ~ 350 μ LN-N dimethyl formamide solutions, add 3.5 ~ 5.5mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and 2 ~ 3mgN-N-Hydroxysuccinimide lucifuge, 5 ~ 6 DEG C of stirring reaction 8 ~ 12h respectively, obtain first liquid;
B) precise 11 ~ 12mg horseradish peroxidase, join in the sodium bicarbonate buffer liquid of 3 ~ 5mL, the pH of buffer solution is 6 ~ 8, treat to dissolve completely, obtain second liquid, under condition of ice bath, first liquid is slowly joined in second liquid, after the entry to be completely, 3 ~ 6 DEG C of lucifuges stir 8 ~ 12h, then by reactant liquor 3 ~ 6 DEG C, pH dialyses 2 ~ 4 days in the phosphate buffered solution of 6 ~ 8, namely obtains enzyme-labelled antigen solution.
Preferably, step 2) in, the preparation of enzyme-labelled antigen comprises the steps,
A) precise 0.02mmol is dissolved in 300 μ LN-N dimethyl formamide solutions, add 4.78mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and 2.75mgN-N-Hydroxysuccinimide lucifuge 4 DEG C of stirring reaction about 10h respectively, obtain first liquid;
B) precise 10.00mg horseradish peroxidase, join in the sodium bicarbonate buffer liquid of 4mL, the pH of buffer solution is 7.4, treat to dissolve completely, obtain second liquid, under condition of ice bath, first liquid is joined in second liquid lentamente, after the entry to be completely, 4 DEG C of lucifuges stir 12h, then by reactant liquor 4 DEG C, pH dialyses 3 days in the phosphate buffered solution of 7.4, namely obtains enzyme-labelled antigen solution.
Synthesized the haptens of Small molecular target analytes in the present invention, and with carrier protein coupling, prepare effective enzyme-labelled antigen.The key of this technical research is the preparation of haptenic MOLECULE DESIGN, synthesis and enzyme-labelled antigen.Wherein haptenic design, synthesis are the keys affecting the success or failure of protein anabolic hormone immunoassay.
In nandrolone structure, not there is suitable group, therefore will transform the structure of nandrolone, the hydroxyl of nandrolone introduces carboxyl, form the arm that can be connected with carrier protein.Therefore, the present invention is when design and synthesis nandrolone artificial semiantigen, nandrolone and succinic anhydride is adopted to react, introduce active side chain, synthesis nandrolone haptens, so both maintain five kinds of protein anabolic hormones similarity structurally such as nandrolone haptens and nandrolone, Trenbolone, testobolin, allodihydrotestosterone and Mestanlone, make again hapten molecule be provided with the suitable construction be connected with carrier protein.
Above-mentioned nandrolone artificial semiantigen is utilized to be combined with carrier protein, obtained artificial antigen, antibody prepared by immune animal.Protein A-sepharose4B affinity column is used to carry out purifying to containing anti-protein anabolic hormone class drug antiserum.
The specific binding rate of purified nandrolone antibody and nandrolone, Trenbolone, testobolin, allodihydrotestosterone and Mestanlone with suppress antibody Bmax 50% needed for the concentration of nandrolone and IC
50value represents, several protein anabolic hormone class medicine is to the IC of antibody of the present invention
50value is respectively, nandrolone 0.050ng/mL, Trenbolone 0.094ng/mL, testobolin 0.66ng/mL, allodihydrotestosterone 1.40ng/mL and Mestanlone 2.40ng/mL.By above-mentioned IC
50value compares with the protein anabolic hormone class medicine maximum residue limit value specified both at home and abroad, is not difficult to find out, the IC of above-mentioned five kinds of sulfonamides
50value, all far below the maximum residue limit value of regulation, when illustrating that antibody of the present invention is used for chemiluminescence immune assay, can detect the residual content of five kinds of protein anabolic hormone medicines specifically simultaneously.
Present invention also offers a kind of use as above for the analytical approach of the enzyme-labelled antigen of protein anabolic hormone immunoassay, comprise the steps,
1) bag quilt, the nandrolone antibody treating bag quilt is first diluted by phosphate buffered solution, the nandrolone antibody diluent diluted is added in the micropore of ELISA Plate, and every micropore adds 80 ~ 120 μ L cleansing solution PBST damping fluids, adds rear ELISA Plate to be placed in reaction 10 ~ 15h under 20 ~ 35 DEG C of conditions, discard liquid in hole, in ELISA Plate, add 200 ~ 300 μ L cleansing solution PBST damping fluids, earthquake device shakes 1 ~ 4min, discards cleansing solution, be and wash plate 1 time, repeat to wash plate 2 ~ 5 times; Preferably, the nandrolone antibody diluent diluted is added in the micropore of ELISA Plate, every micropore adds 100 μ L cleansing solution PBST damping fluids; Add and rear ELISA Plate is placed in reaction 10 ~ 15h under 25 DEG C of conditions, discard liquid in hole, in ELISA Plate, add the cleansing solution PBST damping fluid of 250 μ L, earthquake device shakes 1 ~ 4min; Discard cleansing solution, be and wash plate 1 time, repeat to wash plate 2 ~ 5 times, preferably, repeat to wash plate 3 times;
2) close: in each micropore of ELISA Plate, add 150 ~ 250 μ L confining liquids, add rear ELISA Plate is placed in 20 ~ 35 DEG C of conditions under, take out ELISA Plate after 0.5 ~ 1.5h and discard confining liquid, repeat to wash plate 2 ~ 5 times; Preferably, in each micropore of ELISA Plate, add 200 μ L confining liquids, add rear ELISA Plate is placed in 37 DEG C of conditions under, take out ELISA Plate after 1h and discard confining liquid, repeat to wash plate 4 times; Described confining liquid is be the phosphate buffered solution of 0.5% skimmed milk power containing mass concentration;
3) competitive reaction: each micropore to ELISA Plate first adds the good determinand sample solution of 50 ~ 150 μ L gradient dilutions or sample extracting solution, and then add the enzyme-labelled antigen solution of 50 ~ 150 μ L dilutions wherein, enzyme-labelled antigen dilutes by phosphate buffered solution, after ELISA Plate hatches 0.5 ~ 1.5h under being placed in 20 ~ 35 DEG C of conditions, repeat to wash plate 2 ~ 5 times; Preferably, each micropore to ELISA Plate first adds the good determinand sample solution of 50 μ L gradient dilutions or sample extracting solution, and then adds the enzyme-labelled antigen solution of 50 μ L dilutions wherein; After ELISA Plate hatches 1h under being placed in 37 DEG C of conditions, repeat to wash plate 5 times;
4) luminous value is detected: the ELISA Plate that previous step is washed is placed in fluorogenic chemiluminescence analyser, set corresponding program, instrument adds 50 ~ 150 μ L chemical luminous substrates in each micropore of trend ELISA Plate, and after 10 ~ 40s, instrument measures the luminous intensity values of each micropore immediately; Preferably, in each micropore of ELISA Plate, add 100 μ L chemical luminous substrates, after 30s, instrument measures the luminous intensity values of each micropore immediately.
Preferably, step 1) in, described nandrolone antibody is through purification process, and purification step is as described below,
1) balance pillar: with the phosphate buffer flushing line of pH6 ~ 9, flow velocity 5 ~ 8mL/min, 1 ~ 3min, the phosphate buffer balance pillar again with pH6 ~ 9 after dress post, flow velocity 0.5 ~ 2mL/min, until baseline values; Preferably, with the phosphate buffer flushing line of pH7.4, flow velocity 6.5mL/min, 2min, the phosphate buffer balance pillar again with pH7.4 after dress post, flow velocity 1mL/min, until baseline values;
2) loading: after baseline values, by melamine antiserum loading after the phosphate buffer dilution of equal-volume pH6 ~ 9, preferably, pH 7.4;
3) upper prop, flow velocity is 0.2 ~ 0.8mL/min, after end of the sample, rush pillar with the phosphate buffer of pH6 ~ 9, flow velocity 0.5 ~ 2.0mL/min, nandrolone antibody by specific adsorption on filler site, other foreign proteins flow out with damping fluid, until baseline values; Preferably, flow velocity is 0.5mL/min, after end of the sample, rushes pillar with the phosphate buffer of pH7.4, flow velocity 1mL/min, until baseline values;
4) wash-out: with the antibody that the elution buffer wash-out pillar of pH 1.5 ~ 3.5 combines, wash out destination protein, flow velocity is 0.2 ~ 1.5mL/min; Preferably, with the antibody that the elution buffer wash-out pillar of pH2.7 combines, wash out destination protein, flow velocity is 0.5mL/min;
5) measure: under 200 ~ 350nm ultraviolet, detect eluent protein concentration; When absorbance is greater than 0.1 ~ 0.3, collect eluent, after collection, adjust pH to 6.0 ~ 9.0 with 0.5 ~ 2.0mol/L Tris rapidly; Preferably, under 280nm ultraviolet, detect eluent protein concentration, when absorbance is greater than 0.2, collect eluent, after collection, adjust pH to 7.0 with 1mol/L Tris rapidly; Described Tris is trishydroxymethylaminomethane;
6) dialysis of antibody and preservation: dialyse gained antibody with pH 6 ~ 9 antibody dislysate, 2 ~ 6 DEG C of dialysis 2 ~ 4 days, change 2 ~ 4 dialysis every day, the rear taking-up of dialysis, adds the NaN of 0.05 ~ 0.2% (W/V)
3, 3 ~ 5 DEG C store for future use; Preferably, with pH7.4 antibody dislysate dialysis gained antibody, dialyse 3 days, change 3 dialysis every day for 4 DEG C; Take out after dialysis, add the NaN of 0.1% (W/V)
3; 4 DEG C store for future use; Described antibody dislysate is phosphate buffer.
Preferably, step 4) in, described chemistry substrate of giving out light is that luminol sodium, reinforcing agent phenothiazine-10-base-propyl sulfonic acid sodium salt and 4-morpholine pyridine are dissolved in Tris-HCl, before carrying out detection luminous intensity values, takes out a part and H
2o
2corresponding ratio mixing, namely can be used for experiment; The storage temperature of luminous substrate is 4 DEG C.
Analytical approach as above, its sensitivity (IC
50) and detectability (IC
15) be respectively: nandrolone IC
50=0.050 μ g/L, IC
15=0.00378 μ g/L, Trenbolone IC
50=0.094 μ g/L, IC
15=0.00330 μ g/L, testobolin IC
50=0.66 μ g/L, IC
15=0.259 μ g/L, allodihydrotestosterone IC
50=1.40 μ g/L, IC
15=0.0398 μ g/L, Mestanlone IC
50=2.40 μ g/L, IC
15=0.078 μ g/L.
The present invention measures chemiluminescence immunoassay method and has carried out technological improvement, and its mensuration comprises: nandrolone antibody purification; Standard antibody package amount; Carry out chemiluminescence immunoassay reaction; With the absorption parameter of fluorogenic chemiluminescence analyser to the ripple of certain wavelength; Make protein anabolic hormone standard items and fluorogenic chemiluminescence analyser checks Parameter Map; The fluorogenic chemiluminescence analyser detected parameters of sample is compared with the fluorogenic chemiluminescence analyser detected parameters figure of standard items and obtains the total amount of nandrolone in sample, Trenbolone, testobolin, allodihydrotestosterone, Mestanlone.
Relative to prior art, a kind of enzyme-labelled antigen for protein anabolic hormone immunoassay of the present invention and analytical approach, have following advantage:
(1) analytical approach of the present invention has good specificity and broad spectrum activity for multiple protein anabolic hormone medicine, and compared with traditional ELISA adsorption analysis method, sensitivity improves 5 ~ 8 times.
(2) artificial semiantigen synthetic method of the present invention is not only easy, and primary raw material succinic anhydride price used comparatively cheap, easily obtain, all can buy in general chemical reagents corporation.Because combined coefficient is high, reactions steps is few, haptens only needs single step reaction to synthesize, thus improves the controllability of reaction.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is the chemiluminescence immune analysis method typical curve of the nandrolone described in the embodiment of the present invention one;
Fig. 2 is the chemiluminescence immune analysis method typical curve of the Trenbolone described in the embodiment of the present invention one;
The chemiluminescence immune analysis method typical curve of the testobolin described in Fig. 3 embodiment of the present invention one;
Fig. 4 is the chemiluminescence immune analysis method typical curve of the allodihydrotestosterone described in the embodiment of the present invention one;
Fig. 5 is the chemiluminescence immune analysis method typical curve of the Mestanlone described in the embodiment of the present invention one.
Embodiment
It should be noted that, when not conflicting, the embodiment in the present invention and the feature in embodiment can combine mutually.
Below with reference to the accompanying drawings and describe the invention in detail in conjunction with the embodiments.
Embodiment one
The haptenic synthesis of nandrolone
Taking 1.316g nandrolone is dissolved in 14.50mL anhydrous tetrahydro furan, and add 0.960g succinic anhydride, 1.32mL triethylamine and 0.584g4-dimethylamino naphthyridine (DMAP), sustained response 12h at 60 DEG C of temperature, subsequently solvent is spin-dried for, product organic solvent petrol ether/ethyl acetate (1/1, v/v) is dissolved.With petrol ether/ethyl acetate (1/1, v/v) for developping agent, carry out purifying and obtain white crystal, be nandrolone haptens.
The synthesis of enzyme-labelled antigen
Active ester method: precise 0.02mmol (7.47mg) is dissolved in 300 μ LN-N dimethyl formamide (DMF) solution, add 4.78mg (0.02mmol) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and 2.75mg (0.02mmol) N-hydroxy-succinamide (NHS) lucifuge 4 DEG C of stirring reaction about 10h respectively, obtain first liquid.
Precise 10.00mg horseradish peroxidase (HRP), join (pH7.4) in the sodium bicarbonate buffer liquid of 4mL, treat to dissolve completely, obtain second liquid, under condition of ice bath, first liquid is slowly joined in second liquid, after the entry to be completely, 4 DEG C of lucifuges stir 12h, then by reactant liquor 4 DEG C, dialysis 3 days in the phosphate buffered solution (PBS) of pH 7.4, the then volume of accurate measuring enzyme-labelled antigen solution, measure concentration and add thimerosal, 4 DEG C of preservations.
Antiserum purification nandrolone antibody
Concrete steps are as follows:
1) periodic monitor animal antibody titer, when endpoint titers reaches more than 200,000, adopts whole blood by arteria carotis.At 4 DEG C, leave standstill 8h, the then centrifugal 10min of 3500 turns/min by after the whole blood 20 DEG C of standing 2h gathered, collect supernatant in-20 DEG C of preservations.The serum of centrifugal rear acquisition adopts proteinA-Sepharose-4B immune affinity chromatographic column to be further purified, preparation IgG antibody.Balance pillar: with phosphate buffer (Binding buffer) flushing line of pH7.4, flow velocity 6.5mL/min, 2min.The phosphate buffer of pH7.4 (Binding buffer) is used to balance pillar, flow velocity 1mL/min, until baseline values again after dress post.
2) loading: after baseline values, by melamine antiserum with after the phosphate-buffered (Binding buffer) dilution (1:1, V/V) of equal-volume pH7.4.
3) upper prop, flow velocity is 0.5mL/min.After end of the sample, rush pillar with pH7.4Binding buffer, flow velocity 1mL/min.Antibody is by specific adsorption on filler site, and other foreign proteins flow out with damping fluid, until baseline values.
4) wash-out: with the antibody that elution buffer (Elution buffer) the wash-out pillar of pH 2.7 combines, wash out destination protein, flow velocity is 0.5mL/min.
5) measure: under 280nm ultraviolet, detect eluent protein concentration, as absorbance A 280 > 0.2, collect eluent, after collection, adjust pH to 7.0 with 1mol/L Tris rapidly.
6) dialysis of antibody and preservation: to dialyse gained antibody with pH 7.4 antibody dislysate (phosphate buffer, PB), 4 DEG C of dialysis three days, change three dialysis every day, after dialysis, taking-up, adds the NaN of 0.1% (W/V)
3, 4 DEG C store for future use.
Five kinds of protein anabolic hormone many chemical residue chemiluminescence immunoassay method detecting steps such as nandrolone, Trenbolone, testobolin, allodihydrotestosterone and Mestanlone:
(1) coated antibody
When coated antibody, need first dilute by phosphate buffered solution the nandrolone antibody treating bag quilt, the antibody diluent diluted is joined (100 μ L/well) in 96 micropores in ELISA Plate, add rear ELISA Plate is placed in 4 DEG C of conditions under, spend the night, next day, discard liquid in hole, in ELISA Plate, add the cleansing solution PBST damping fluid of 250 μ L, earthquake device shakes 2min, discard cleansing solution, be and wash plate once, repeat to wash plate 3 times.
(2) close
Immediately previous step, sealase target, adds 200 μ L confining liquids in each micropore of ELISA Plate, add rear ELISA Plate is placed in 37 DEG C of conditions under, take out ELISA Plate after 1h and discard confining liquid, repeat to wash plate 4 times.
(3) enzyme-labelled antigen is added
After step (2), the good determinand sample solution of 50 μ L gradient dilutions or sample extracting solution is first added to each micropore in ELISA Plate, and then add 50 μ L enzyme-labelled antigen solution wherein, enzyme-labelled antigen dilutes by phosphate buffered solution, after ELISA Plate hatches 1h under being placed in 37 DEG C of conditions, repeat to wash plate 5 times;
(4) luminous value is read
The ELISA Plate that previous step is washed is placed in fluorogenic chemiluminescence analyser, and set corresponding program, instrument adds 100 μ L luminous substrate in each micropore of trend ELISA Plate, and after 30s, instrument measures the luminous intensity values of each micropore immediately.
The chemiluminescence immune analysis method typical curve of nandrolone, Trenbolone, testobolin, allodihydrotestosterone and Mestanlone is set up respectively, as shown in Fig. 1 ~ Fig. 5 according to luminous intensity values.
Use analytical approach corresponding in embodiment one, the sensitivity of nandrolone is 0.050 μ g/L, minimum detectability is 0.00378 μ g/L, the sensitivity of Trenbolone is 0.094 μ g/L, minimum detectability is 0.00330 μ g/L, the sensitivity of testobolin is 0.66 μ g/L, minimum detectability is 0.259 μ g/L, the sensitivity of allodihydrotestosterone is 1.40 μ g/L, minimum detectability is 0.0398 μ g/L, the sensitivity of Mestanlone is 2.40 μ g/L, and minimum detectability is 0.078 μ g/L.
Compliance test result test one, accurately take pork sample 1g (being accurate to 0.01g) and add 2mL methanol solution homogenate mechanical shaking extraction 6min, 4 DEG C, 9000rpm, centrifugal 12min, get whole supernatant in test tube, after nitrogen dries up, redissolve with 1mL methyl alcohol, dilute 60 times with PBS and prepare standard series drafting extraction standard curve afterwards, the average recovery rate of the nandrolone drawn is 80 ~ 100%.
Compliance test result test two, accurately take chicken and beef sample 1g (being accurate to 0.01g) adds 2mL methanol solution homogenate mechanical shaking extraction 6min, 4 DEG C, 9000rpm, centrifugal 12min, get whole supernatant in test tube, after nitrogen dries up, redissolve with 1mL methyl alcohol, dilute 60 times with PBS and prepare standard series drafting extraction standard curve afterwards, the average recovery rate of the nandrolone drawn is 80 ~ 100%.
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (7)
1. for an enzyme-labelled antigen for protein anabolic hormone immunoassay, it is characterized in that: to be connected with horseradish peroxidase by nandrolone haptens and to synthesize, its preparation comprises the steps;
1) the haptenic preparation of nandrolone, taking 0.5 ~ 2.0g nandrolone is dissolved in 10 ~ 25mL anhydrous tetrahydro furan, and add 0.5 ~ 2.0g succinic anhydride, 0.6 ~ 2.0mL triethylamine and 0.2 ~ 1.5g4-dimethylamino naphthyridine, sustained response 8 ~ 17h at 62 ~ 70 DEG C of temperature, be spin-dried for by solvent subsequently, product volume ratio is that the sherwood oil of 1:2 ~ 5:6 and the mixed liquor of ethyl acetate dissolve; Take volume ratio as the sherwood oil of 1:2 ~ 5:6 and the mixed liquor of ethyl acetate be developping agent, carry out purifying and obtain white crystal, be nandrolone haptens;
2) by active ester method, with hydroxyl CO=OH for bridge, make nandrolone artificial semiantigen be connected in horseradish peroxidase, namely obtain enzyme-labelled antigen.
2. the enzyme-labelled antigen for protein anabolic hormone immunoassay according to claim 1, it is characterized in that: step 1) in, taking 1.316g nandrolone is dissolved in 14.50mL anhydrous tetrahydro furan, and add 0.960g succinic anhydride, 1.32mL triethylamine and 0.584g4-dimethylamino naphthyridine, sustained response 12h at 60 DEG C of temperature, be spin-dried for by solvent subsequently, product volume ratio is that the sherwood oil of 1:1 and the mixed liquor of ethyl acetate dissolve; Take volume ratio as the sherwood oil of 1:1 and the mixed liquor of ethyl acetate be developping agent, carry out purifying and obtain white crystal, be nandrolone haptens.
3. the enzyme-labelled antigen for protein anabolic hormone immunoassay according to claim 1, is characterized in that: step 2) in, the preparation of enzyme-labelled antigen comprises the steps,
A) precise 0.03 ~ 0.04mmol nandrolone haptens is dissolved in 250 ~ 350 μ LN-N dimethyl formamide solutions, add 3.5 ~ 5.5mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and 2 ~ 3mgN-N-Hydroxysuccinimide lucifuge, 5 ~ 6 DEG C of stirring reaction 8 ~ 12h respectively, obtain first liquid;
B) precise 11 ~ 12mg horseradish peroxidase, join in the sodium bicarbonate buffer liquid of 3 ~ 5mL, the pH of buffer solution is 6 ~ 8, treat to dissolve completely, obtain second liquid, under condition of ice bath, first liquid is slowly joined in second liquid, after the entry to be completely, 3 ~ 6 DEG C of lucifuges stir 8 ~ 12h, then by reactant liquor 3 ~ 6 DEG C, pH dialyses 2 ~ 4 days in the phosphate buffered solution of 6 ~ 8, namely obtains enzyme-labelled antigen solution.
4. the enzyme-labelled antigen for protein anabolic hormone immunoassay according to claim 3, is characterized in that: step 2) in, the preparation of enzyme-labelled antigen comprises the steps,
A) precise 0.02mmol is dissolved in 300 μ LN-N dimethyl formamide solutions, add 4.78mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and 2.75mgN-N-Hydroxysuccinimide lucifuge 4 DEG C of stirring reaction about 10h respectively, obtain first liquid;
B) precise 10.00mg horseradish peroxidase, join in the sodium bicarbonate buffer liquid of 4mL, the pH of buffer solution is 7.4, treat to dissolve completely, obtain second liquid, under condition of ice bath, first liquid is slowly joined in second liquid, after the entry to be completely, 4 DEG C of lucifuges stir 12h, then by reactant liquor 4 DEG C, pH dialyses 3 days in the phosphate buffered solution of 7.4, namely obtains enzyme-labelled antigen solution.
5. use an analytical approach for the enzyme-labelled antigen for protein anabolic hormone immunoassay as described in Claims 1 to 4, it is characterized in that: comprise the steps,
1) bag quilt, the nandrolone antibody treating bag quilt is first diluted by phosphate buffered solution, the nandrolone antibody diluent diluted is joined in the micropore of ELISA Plate, and every micropore adds 80 ~ 120 μ L cleansing solution PBST damping fluids, adds rear ELISA Plate to be placed in reaction 10 ~ 15h under 20 ~ 35 DEG C of conditions, discard liquid in hole, in ELISA Plate, add 200 ~ 300 μ L cleansing solution PBST damping fluids, earthquake device shakes 1 ~ 4min, discards cleansing solution, be and wash plate 1 time, repeat to wash plate 2 ~ 5 times; Preferably, the nandrolone antibody diluent diluted is joined in the micropore of ELISA Plate, every micropore adds 100 μ L cleansing solution PBST damping fluids; Add and rear ELISA Plate is placed in reaction 10 ~ 15h under 25 DEG C of conditions; Then, discard liquid in hole, in ELISA Plate, add the cleansing solution PBST damping fluid of 250 μ L, earthquake device shakes 1 ~ 4min; Discard cleansing solution, be and wash plate 1 time, repeat to wash plate 2 ~ 5 times, preferably, repeat to wash plate 3 times;
2) close: in each micropore of ELISA Plate, add 150 ~ 250 μ L confining liquids, add rear ELISA Plate is placed in 20 ~ 35 DEG C of conditions under, take out ELISA Plate after 0.5 ~ 1.5h and discard confining liquid, repeat to wash plate 2 ~ 5 times; Preferably, in each micropore of ELISA Plate, add 200 μ L confining liquids, add rear ELISA Plate is placed in 37 DEG C of conditions under, take out ELISA Plate after 1h and discard confining liquid, repeat to wash plate 4 times; Described confining liquid is be the phosphate buffered solution of 0.5% skimmed milk power containing mass concentration;
3) competitive reaction: each micropore to ELISA Plate first adds the good determinand sample solution of 50 ~ 150 μ L gradient dilutions or sample extracting solution, and then add the enzyme-labelled antigen solution of 50 ~ 150 μ L dilutions wherein, enzyme-labelled antigen dilutes by phosphate buffered solution, after ELISA Plate hatches 0.5 ~ 1.5h under being placed in 20 ~ 35 DEG C of conditions, repeat to wash plate 2 ~ 5 times; Preferably, each micropore to ELISA Plate first adds the good determinand sample solution of 50 μ L gradient dilutions or sample extracting solution, and then adds the enzyme-labelled antigen solution of 50 μ L dilutions wherein; After ELISA Plate hatches 1h under being placed in 37 DEG C of conditions, repeat to wash plate 5 times;
4) luminous value is detected: the ELISA Plate that previous step is washed is placed in fluorogenic chemiluminescence analyser, set corresponding program, instrument adds 50 ~ 150 μ L chemical luminous substrates in each micropore of trend ELISA Plate, and after 10 ~ 40s, instrument measures the luminous intensity values of each micropore immediately; Preferably, in each micropore of ELISA Plate, add 100 μ L chemical luminous substrates, after 30s, instrument measures the luminous intensity values of each micropore immediately.
6. the analytical approach of use enzyme-labelled antigen according to claim 5, is characterized in that: step 1) in, described nandrolone antibody is through purification process, and purification step is as described below,
1) balance pillar: with the phosphate buffer flushing line of pH6 ~ 9, flow velocity 5 ~ 8mL/min, 1 ~ 3min, the phosphate buffer balance pillar again with pH6 ~ 9 after dress post, flow velocity 0.5 ~ 2mL/min, until baseline values; Preferably, with the phosphate buffer flushing line of pH7.4, flow velocity 6.5mL/min, 2min, the phosphate buffer balance pillar again with pH7.4 after dress post, flow velocity 1mL/min, until baseline values;
2) loading: after baseline values, by melamine antiserum loading after the phosphate buffer dilution of equal-volume pH6 ~ 9, preferably, pH 7.4;
3) upper prop, flow velocity is 0.2 ~ 0.8mL/min, after end of the sample, rush pillar with the phosphate buffer of pH6 ~ 9, flow velocity 0.5 ~ 2.0mL/min, nandrolone antibody by specific adsorption on filler site, other foreign proteins flow out with damping fluid, until baseline values; Preferably, flow velocity is 0.5mL/min, after end of the sample, rushes pillar with the phosphate buffer of pH 7.4, flow velocity 1mL/min, until baseline values;
4) wash-out: with the antibody that the elution buffer wash-out pillar of pH 1.5 ~ 3.5 combines, wash out destination protein, flow velocity is 0.2 ~ 1.5mL/min; Preferably, with the antibody that the elution buffer wash-out pillar of pH 2.7 combines, wash out destination protein, flow velocity is 0.5mL/min;
5) measure: under 200 ~ 350nm ultraviolet, detect eluent protein concentration; When absorbance is greater than 0.1 ~ 0.3, collect eluent, after collection, adjust pH to 6.0 ~ 9.0 with 0.5 ~ 2.0mol/L Tris rapidly; Preferably, under 280nm ultraviolet, detect eluent protein concentration, when absorbance is greater than 0.2, collect eluent, after collection, adjust pH to 7.0 with 1mol/L Tris rapidly; Described Tris is trishydroxymethylaminomethane;
6) dialysis of antibody and preservation: dialyse gained antibody with pH 6 ~ 9 antibody dislysate, 2 ~ 6 DEG C of dialysis 2 ~ 4 days, change 2 ~ 4 dialysis every day, the rear taking-up of dialysis, adds the NaN of 0.05 ~ 0.2% (W/V)
3, 3 ~ 5 DEG C store for future use; Preferably, with pH7.4 antibody dislysate dialysis gained antibody, dialyse 3 days, change 3 dialysis every day for 4 DEG C; Take out after dialysis, add the NaN of 0.1% (W/V)
3; 4 DEG C store for future use; Described antibody dislysate is phosphate buffer.
7. the analytical approach of use enzyme-labelled antigen according to claim 5, it is characterized in that: step 4) in, described chemistry substrate of giving out light is that luminol sodium, reinforcing agent phenothiazine-10-base-propyl sulfonic acid sodium salt and 4-morpholine pyridine are dissolved in Tris-HCl, before carrying out detection luminous intensity values, take out a part and H
2o
2corresponding ratio mixing, namely can be used for experiment; The storage temperature of luminous substrate is 3 ~ 5 DEG C, preferably, and 4 DEG C.
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