CN104634977A - Alpha LISA detection kit for zeranol and analogue in meat product - Google Patents
Alpha LISA detection kit for zeranol and analogue in meat product Download PDFInfo
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- CN104634977A CN104634977A CN201510079070.5A CN201510079070A CN104634977A CN 104634977 A CN104634977 A CN 104634977A CN 201510079070 A CN201510079070 A CN 201510079070A CN 104634977 A CN104634977 A CN 104634977A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
The invention discloses an Alpha LISA detection method for zeranol and an analogue in a meat product and an Alpha LISA detection kit. The detection kit includes a zeranol standard substance, a beta-zeranol standard substance, an alpha-zearalanel standard substance, a beta-zearalanel standard substance, a zearalenone standard substance, a zearalenone standard substance, biotinylated zeranol, an monoclonal antibody resisting zeranol, an anti-mouse IgG coupled receptor magnetic bead, a streptavidin donor magnetic bead, a detection buffer solution, a white reaction plate, a sealing film and an operating manual. According to the detection method and the detection kit, the amount of samples is few, the sensitivity is high, no washing steps are needed and the detection period is short.
Description
Technical field
The present invention relates to the detection of the ZER in meat products, β-ZER, α-zearalenol, β-zearalenol, zearelone, zearalenone, be specifically related to AlphaLISA detection method and the kit of ZER and analog thereof in a kind of meat products.
Background technology
In meat products, the residue problem of toxic metabolite more and more causes the concern of international community, in order to realize maximizing the benefits, some illegal retailers are illegal in animal feed adds the hormone medicines such as ZER, promote the synthesis of animal body protein, improve efficiency of feed utilization, thus produce short effect of gain, obtain more economy returns.ZER and metabolic product thereof have the biologically active of estrogenic chemicals, all have inhibiting effect to promoting sexual gland hormone bind receptor, external liver hormone binding receptor.The residual meeting of estrogenic chemicals causes the disorder of human body sex hormone and affects the normal development of secondary sex characters, under external condition induction, and may be carcinogenic.The detection method of existing ZER and analog thereof has Liquid Chromatography-Tandem Mass Spectrometry, gas chromatography-mass spectrography method etc., but these methods have instrument and equipment costliness, complex operation, consuming time, the more high deficiency of personnel requirement.The present invention's research is set up AlphaLISA detection technique and is detected ZER and analog thereof in meat products, and its AlphaLISA method has that amount of samples is few, highly sensitive, the advantage such as do not need washing, sense cycle short.
Summary of the invention
In order to there be the defect in technology on overcoming, the present invention detects ZER and analog thereof in meat products by AlphaLISA detection method, provides a kind of AlphaLISA detection kit for detecting ZER and analog thereof in meat products.
The technical solution used in the present invention is: the AlphaLISA detection kit of ZER and analog thereof in meat products, comprises ZER standard items, β-ZER standard items, α-zearalenol standard items, β-zearalenol standard items, zearelone standard items, zearalenone standard items, biotinylation ZER, against murine IgG coupled receptor magnetic bead, streptavidin donor magnetic bead, anti-ZER monoclonal antibody and detects damping fluid; Wherein said against murine IgG coupled receptor magnetic bead comprise 100 μ g/mL receptor magnetic bead (final concentration), 0.05% the PBS solution of Proclin-300 and pH7.4, streptavidin donor magnetic bead comprises 100 μ g/mL donors magnetic bead (final concentration), the NaC1 solution of HEPES, 100mM of 25mM and the Proclin-300 of 0.05%.
The magnetic bead of against murine IgG coupled receptor described in mentioned reagent box is 1mg/mL, and streptavidin donor magnetic bead is 1mg/mL, and anti-ZER monoclonal antibody is 1mg/mL, biotinylation ZER 0.5mg/mL.
During use, in cumulative volume 25 μ L: wherein against murine IgG coupled receptor magnetic bead 5 μ L, streptavidin donor magnetic bead 5 μ L, anti-ZER monoclonal antibody 5 μ L, biotinylation ZER 5 μ L, testing sample 5 μ L.
During kit encapsulation of the present invention, comprise ZER and analog standard items (5000UI/mL thereof, 100 μ L/ boxes), biotinylation ZER (0.5mg/mL, 100 μ L/ boxes), anti-ZER monoclonal antibody (1mg/mL, 20 μ L/ boxes), against murine IgG bag is by receptor magnetic bead (1mg/mL, 50 μ L/ boxes), streptavidin donor magnetic bead (1mg/mL, 50 μ L/ boxes), detect damping fluid (100mL/ box), white reaction plate (384T, 1 piece/box), sealed membrane (1 piece/box), operational manual (1 part/box).
By technique scheme, the present invention has following advantage and beneficial effect at least: utilize ZER, β-ZER, α-zearalenol, β-zearalenol, zearelone, the zearalenone in kit provided by the invention detection meat products, amount of samples only uses 5 μ L less, also have highly sensitive, do not need washing, the advantage such as sense cycle is short.
Accompanying drawing explanation
Fig. 1 is the typical curve schematic diagram of ZER, β-ZER, α-zearalenol, β-zearalenol, zearelone, zearalenone;
Fig. 2 is AlphaLISA detection reaction process schematic;
In Fig. 1, A is ZER, and B is β-ZER, and C is α-zearalenol, and D is β-zearalenol, and E is zearelone, and F is zearalenone.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Anti-ZER monoclonal antibody in kit can be selected commercial, also can prepare by the conventional method preparing monoclonal antibody.Streptavidin donor magnetic bead comprises 100 μ g/mL donor magnetic beads, the NaC1 solution of HEPES, 100mM of 25mM and the Proclin-300 of 0.05%.The damping fluid in conventional sense kit selected by detection damping fluid.
One, antigen purification
The first step: pillar process
1, remove desalting column bottom sealing cap, the lid on top is unclamped (not taken off by top cap);
2, pillar is put into 1.5-2mL centrifuge tube.The centrifugal 1min of 1500g, removes conserving liquid wherein;
3, the residual liquid bottom centrifugal rear pillar is gently wiped and is blotted on paper;
4, after the centrifugal compacting of pillar, be that one side obliquely marks at resin.In centrifugation step subsequently, the placement of pillar marks outwardly all the time.The grand low desalting efficiency of improper meeting is placed in pillar position.
Second step: loading
1, pillar is put into a new centrifuge tube, removes lid, slowly add 30-130 μ L antigen to be purified in pillar center;
2, (optional) is when antigen volume to be purified is less than 70 μ L, after antigen to be purified is completely absorbed, adds 15 μ L ultrapure water or damping fluids with resin top, guarantees the maximum activation of albumen;
3, the centrifugal 2min of 1500g, collects the antigen after the purifying after desalination, discards used pillar.
3rd step: fluid exchange
Pillar process:
1, remove desalting column bottom sealing cap, the lid on top is unclamped (not taken off by top cap);
2, pillar is put into 1.5-2mL centrifuge tube.The centrifugal 1min of 1500g, removes conserving liquid wherein;
3, after the centrifugal compacting of pillar, be that one side obliquely marks at resin.In centrifugation step subsequently, the placement of pillar marks outwardly all the time.The grand low desalting efficiency of improper meeting is placed in pillar position;
4, add 1x Alphalisa buffer 300 μ L in pillar, the centrifugal 1min of 1500g, abandons supernatant;
5, repeat the 4th step 2-3 time, abandon supernatant.
Loading:
1, the pillar through processing above being put into a new centrifuge tube, removing lid, slowly add the antigen after 30 ~ 130 μ L purifying in pillar center;
2, (optional) is when the antigen volume after purifying is less than 70 μ L, after antigen is after purification completely absorbed, adds 15 μ L ultrapure water or damping fluids with resin top, guarantees the maximum activation of albumen;
3, the centrifugal 2min of 1500g, collects the antigen after the purifying after desalination, discards used pillar.
Two, the biotinylation of ZER
The first step: ZER is carried out following purification:
Use material, Thermo Zeba Desalt Spin (5mL/ props up), 15mL collection tube is applicable to the albumen of > 7000da, 500 ~ 2000 μ L sample sizes, initial concentration >=20 μ g/mL.
1, remove pillar bottom sealing cap, uncap, pillar is put into the collection tube of 15mL.
2, under 4 DEG C of conditions, the centrifugal 2min of 1500r/min, removes protection liquid.In centrifugal process, keep a direction (outwards).
3, in the pillar of 5mL, add the 1 × PBS (pH=7.4) of 2.5mL, wash post, under 4 DEG C of conditions, the centrifugal 2min of 1000r/min, repeats 2 ~ 3 times.
4, pillar is put into new collection tube, remove lid, slowly by the center of the antigen resin by injection mould after above-mentioned steps purifying.The applied sample amount of the pillar of 5mL is 500 ~ 2000 μ L.
5, (low volume sample) is in order to ensure the recovery of a small amount of sample, can add one deck ultrapure water or damping fluid on resin mold, after sample absorbs completely, can promote the recovery.In the pillar of 5mL, if sample size < 750 μ is L, the damping fluid of 100 μ L can be added.
6, under 4 DEG C of conditions, the centrifugal 2min of 1000r/min, collects the antigen after purifying, as testing sample.
7, concentration determination.
Second step: following biotin modification is carried out to ZER
Use reagent: Sol μ Link chromalink.
1. utilize desalting column, fluid exchange is become 100mM sodium phosphate, NaCl and MDB (modifying buffer, pH=8.0) of 150mM.
2. measure desalination protein concentration.MDB is adjusted to 1 ~ 2.5mg/mL.
3. prepare DMF (dimethyl formamide): the link+100 μ L dry DMF of 1 ~ 4mg, preserve 2 weeks for-20 DEG C.
4. calculate molecular proportion, add in the Biolink stock to corresponding albumen buffer of 10 ~ 20mol decile, room temperature, 90min.
5. desalination: exchanging liquid is needed for AphaLISA.
Three, the AlphaLISA reaction conditions of ZER and analog thereof
In this kit, we adopt reverse competitive mode.Principle is as follows: be first that the anti-ZER monoclonal antibody specificity of ZER in sample to be tested and analog and concentration known combines.And then add the free anti-ZER monoclonal antibody of biotinylation ZER competition binding, streptavidin donor magnetic bead and against murine IgG receptor magnetic bead is added after reaction, react and will form the donor magnetic bead-biotinylation ZER-anti-ZER monoclonal antibody-receptor magnetic bead compound shown in lower Fig. 2, and under the incident light of 680nm, there is fluorescence resonance energy transfer (FRET), free oxygen is applied in receptor magnetic bead, and simultaneous shots goes out the utilizing emitted light of 615nm.Testing process all at room temperature completes.
Through Mass Spectrometer Method, the present invention have selected proves that ZER (with other 5 kinds of analogs) 10 portions of positive meat products and 10 parts of negative meat products conditions of carrying out are groped.
ZER method of operating (other 5 kinds all similar):
1, add biotinylation ZER, 5 μ L/ holes, add anti-ZER monoclonal antibody, 5 μ L/ holes.
2, ZER standard items or testing sample is added, 5 μ L/ holes, the centrifugal 30sec of 2000r/min.
3, sealer, 23 DEG C of lucifuge reaction 1h.
4, against murine IgG bag is added by receptor magnetic bead, 5 μ L/ holes.
5, streptavidin donor magnetic bead is added, 5 μ L/ holes, the centrifugal 30sec of 2000r/min.
6, sealer, 23 DEG C of lucifuge reaction 45min.
7, reading on Enspire plate reading machine is placed in.
Under 25 μ L reaction volumes, obtain a more stable reaction system: wherein against murine IgG bag is by receptor magnetic bead 5 μ L, streptavidin donor magnetic bead 5 μ L, anti-ZER monoclonal antibody 5 μ L, biotinylation ZER 5 μ L, testing sample 5 μ L.
Four, the preparation of typical curve
It is 1 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL, 0.02 μ g/mL, 0.01 μ g/mL, five concentration that ZER, β-ZER, α-zearalenol, β-zearalenol, zearelone and zearalenone standard items dilute by respectively.Then carry out AlphaLISA detection according to above-mentioned optimal reaction system, result is as follows:
More than in table, the logarithm value of concentration is X-axis, with percentage relative intensity of fluorescence for Y-axis, and pointwise linear regression, Criterion curve, as shown in Figure 1.ZER in sample and the concentration of analog thereof can be obtained by the above-mentioned typical curve of inquiry.
Four, the Preliminary Applications of the AlphaLISA detection method of ZER and analog thereof in meat products
1, sample is detected with ZER in business-like meat products and analog activity detection kit thereof
In order to the validity of the AlphaLISA detection method of ZER in the meat products that effective evaluation has been set up and analog thereof, in the meat products of commodity in use, ZER and analog activity detection kit thereof originally carry out preliminary screening to 25 increments respectively.
1.1 Cleaning Principle
In meat products, ZER and analog activity detection kit thereof adopt indirect competitive, utilize the principle that the antibody corresponding to it of ZER residual in meat and analog contestable thereof combines, by part one Receptor recognition method Fast Measurement and ZER and analog thereof, thus detect ZER residual in meat products sample and analog thereof.
1.2 operation steps
1, take 5g beef or pork respectively in 50mL tool plug centrifuge tube, add 10mL sodium acetate buffer solution and 0.025mL beta-glucosidase acid/sulfuric ester complex enzyme, whirlpool mixes, vibration mixing 12h in 37 DEG C of water-baths.
2, add 15mL absolute ether after hydrolysis, after mechanical shaking extraction 5min, with the centrifugal 2min of 4000r/min, be transferred in concentrated bottle by supernatant, then repeat to extract once with 15mL absolute ether, merge supernatant, less than 40 DEG C spin concentration are near dry.Add 1mL methenyl choloride dissolved residue, after ultrasound wave hydrotropy 2min, proceed in 10mL centrifuge tube, then be transferred in same centrifuge tube after concentrating bottle with the rinse of 3mL sodium hydroxide solution, whirlpool mixes, and with the centrifugal 2min of 4000r/min, draws upper strata sodium hydroxide solution.Repeat rinse, extraction once with 3mL NaOH again, merge sodium hydroxide extraction liquid, add 1mL phosphoric acid-aqueous solution, to be clean after mixing.
3, respectively beef or pork extract are proceeded to HLB solid-phase extraction column.With 5mL water, the drip washing of 5mL methanol-water solution, discard; Carry out wash-out with 10mL again, collect eluent.Whole Solid phase extraction process control flow velocity is no more than 2mL/min, and eluent dries up with nitrogen below 40 DEG C, and residue 1.0mL acetonitrile dissolves, and after whirlpool mixing, crosses 0.2 μm of miillpore filter, detects for instrument.
1.3 result
After ZER in commercial meat products and analog activity detection kit thereof detect, in 3 increment product, ZER positive 3 parts, negative sample 0 part; The beautiful positive 3 parts of β-ZER, negative sample 0 part; α-zearalenol positive 3 parts, negative sample 0 part; β-zearalenol positive 3 parts, negative sample 0 part; Zearelone positive 3 parts, negative sample 0 part; Zearalenone positive 3 parts, negative sample 0 part.
The Preliminary Applications of 2.AlphaLISA detection method
The positive sample detect above-mentioned detection method and negative sample are as the preliminary assessment of AlphaLISA detection method.Detection method carries out pattern detection according to optimizing the 25 μ L detection system set up before.
Testing result finds, positive is also positive through AlphaLISA detection method testing result, and negative sample testing result is also negative.Coincidence rate reaches 100%.The consumption of the testing sample that this kit uses is few, easy and simple to handle, at room temperature just can detect.
Claims (3)
1. the AlphaLISA detection method of ZER and analog thereof and kit in meat products, it is characterized in that: comprise ZER standard items, β-ZER standard items, α-zearalenol standard items, β-zearalenol standard items, zearelone standard items, zearalenone standard items, biotinylation ZER, against murine IgG coupled receptor magnetic bead, streptavidin donor magnetic bead, anti-ZER Dan Ke falls antibody and detects damping fluid: wherein said against murine IgG coupled receptor magnetic bead comprises 100 μ g/mL receptor magnetic bead, the PBS solution of Proclin-300 and PH7.4 of 0.05%, streptavidin donor magnetic bead comprises 100 μ g/mL donor magnetic beads, the NaC1 solution of HEPES, 100mM of 25mM and the Proclin-300 of 0.05%.
2. the AlphaLISA detection kit of ZER and analog thereof in meat products according to claim 1, it is characterized in that: described against murine IgG coupled receptor magnetic bead is 1mg/mL, streptavidin donor magnetic bead is 1mg/mL, anti-ZER monoclonal antibody is 1mg/mL, biotinylation ZER 0.5mg/mL;
During use, in cumulative volume 25 μ L: wherein antibody 5 μ L, biotinylation ZER 5 μ L, testing sample 5 μ L fall in against murine IgG coupled receptor magnetic bead 5 μ L, streptavidin donor magnetic bead 5 μ L, anti-ZER Dan Ke.
3. the AlphaLISA detection method of ZER and analog thereof in meat products according to claim 1, is characterized in that: for ZER method of operating (other 5 kinds all similar):
1), add biotinylation ZER, 5 μ L/ holes, add anti-ZER monoclonal antibody, 5 μ L/ holes.
2), ZER standard items or testing sample is added, 5 μ L/ holes, the centrifugal 30sec of 2000r/min.
3), sealer, 23 DEG C of lucifuges reaction 1h.
4), against murine IgG bag is added by receptor magnetic bead, 5 μ L/ holes.
5), streptavidin donor magnetic bead is added, 5 μ L/ holes, the centrifugal 30sec of 2000r/min.
6), sealer, 23 DEG C of lucifuges reaction 45min.
7) reading on Enspire plate reading machine, is placed in.
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Cited By (8)
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| CN104865243A (en) * | 2015-06-12 | 2015-08-26 | 西南大学 | Light-stimulating chemiluminiscence immunological detection method of silkworm mature egg microsporidiosis |
| CN105974118A (en) * | 2016-06-13 | 2016-09-28 | 南京普朗医疗设备有限公司 | One-step homogeneous phase D-dimer detection kit and application thereof |
| CN106153951A (en) * | 2016-06-17 | 2016-11-23 | 天津智巧数据科技有限公司 | The visualization monomolecular detection method of Gibberella zeae alcohols in a kind of milk product |
| CN106483306A (en) * | 2016-09-29 | 2017-03-08 | 重庆出入境检验检疫局检验检疫技术中心 | The AlphaLISA detection kit of salbutamol, bromine Boot sieve and Terbutaline in animal derived musculature |
| CN106483292A (en) * | 2016-09-29 | 2017-03-08 | 重庆出入境检验检疫局检验检疫技术中心 | While detecting the AlphaLISA detection kit of Clenbuterol and Ractopamine |
| CN106771262A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | ZER detection method and kit in a kind of grain |
| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
| CN114910636A (en) * | 2017-10-26 | 2022-08-16 | 科美诊断技术股份有限公司 | Serum total IgE antibody detection kit and detection method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104865243A (en) * | 2015-06-12 | 2015-08-26 | 西南大学 | Light-stimulating chemiluminiscence immunological detection method of silkworm mature egg microsporidiosis |
| CN104865243B (en) * | 2015-06-12 | 2017-11-03 | 西南大学 | A kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method |
| CN105974118A (en) * | 2016-06-13 | 2016-09-28 | 南京普朗医疗设备有限公司 | One-step homogeneous phase D-dimer detection kit and application thereof |
| CN106153951A (en) * | 2016-06-17 | 2016-11-23 | 天津智巧数据科技有限公司 | The visualization monomolecular detection method of Gibberella zeae alcohols in a kind of milk product |
| CN106483306A (en) * | 2016-09-29 | 2017-03-08 | 重庆出入境检验检疫局检验检疫技术中心 | The AlphaLISA detection kit of salbutamol, bromine Boot sieve and Terbutaline in animal derived musculature |
| CN106483292A (en) * | 2016-09-29 | 2017-03-08 | 重庆出入境检验检疫局检验检疫技术中心 | While detecting the AlphaLISA detection kit of Clenbuterol and Ractopamine |
| CN106771262A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | ZER detection method and kit in a kind of grain |
| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
| CN114910636A (en) * | 2017-10-26 | 2022-08-16 | 科美诊断技术股份有限公司 | Serum total IgE antibody detection kit and detection method thereof |
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Application publication date: 20150520 |