CN102565402A - Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof - Google Patents
Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof Download PDFInfo
- Publication number
- CN102565402A CN102565402A CN2010105912744A CN201010591274A CN102565402A CN 102565402 A CN102565402 A CN 102565402A CN 2010105912744 A CN2010105912744 A CN 2010105912744A CN 201010591274 A CN201010591274 A CN 201010591274A CN 102565402 A CN102565402 A CN 102565402A
- Authority
- CN
- China
- Prior art keywords
- kit
- furaxone metabolite
- fluorescein
- metabolite
- add
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a magnetic particle chemiluminescence kit for detecting furazolidone metabolite. The kit comprises the following reagents: a luminescence marker, a fluorescein marker, a standard substance, a quality control substance and a separation reagent. The luminescence marker is a furazolidone metabolite hapten marked by an isoluminol luminescence marker; the fluorescein marker is a furazolidone metabolite monoclonal antibody marked by fluorescein or derivative thereof; and the separation reagent refers to paramagnetic nano micro-beads coated by a goat anti-FITC (fluorescein isothiocyanate) monoclonal antibody. The invention also relates to a method for detecting furazolidone metabolite in animal derived food by use of the kit. The method has relatively high sensitivity and specificity and relatively high speed in detecting the furazolidone metabolite.
Description
Technical field
The present invention relates to a kind of chemiluminescence detection kit and method of testing thereof, particularly detect honey, egg, aquatic products, organize the magnetic particle of Furaxone metabolite residual quantity in the equal samples to compete direct chemical luminous detection kit.
Technical background
Furazolidone (Frazolidone) is a kind of broad spectrum antibiotic of synthetic; Efficacy stability can suppress or kill multiple Grain-positive and negative bacterium, and some protozoon, fungi are had certain effect; Because efficient cheap, furazolidone has obtained using widely in herding, aquaculture.
But furazolidone residual in animal derived food can pass to the mankind through food chain; Long-term absorption can give rise to diseases; According to Food and Agricultural Organization of the United Nations (FAO) and The World Health Organization's food additives joint specialist committee report; Furazolidone and metabolic product thereof have carcinogenicity, and are prone to produce drug resistance strain, cause monster easily.China classifies furazolidone as the forbidding medicine in March, 2002 " primary and other compound inventories of food animal forbidding " of Ministry of Agriculture issue being arranged, furazolidone gets into understands very soon in the animal body that metabolism becomes Furaxone metabolite and Furaxone metabolite has the harm same with furazolidone to human body.Therefore the detection method of setting up a kind of Furaxone metabolite effectively becomes task very critical in the residue of veterinary drug analysis field.
At present, the conventional sense method of Furaxone metabolite residual quantity mainly contains high performance liquid chromatography (HPLC), LC/MS (LC-MS) and enzyme linked immunosorbent assay (ELISA) etc.
1, high performance liquid chromatography (HPLC) has accuracy of detection height, characteristics that false positive rate is low.The major defect of HPLC method is that instrument costs an arm and a leg, operate more loaded down with trivial details, length consuming time, testing cost is expensive, and testing staff's specialty is had relatively high expectations, the sample pre-treatment is complicated.
2, LC/MS (LC-MS), sample does not need derivatization treatment, can detect urine, blood, liver, hair and eyeball sample.The LC-MS/MS coupling can further improve signal to noise ratio (S/N ratio), so can be used for the affirmation means to positive findings.But no matter LC-MS or LC-MS/MS fail to solve instrument and involve great expense, complex operation step, and the sample complex pretreatment requires problems such as height to operating personnel's specialty attainment.
3, enzyme linked immunosorbent assay (ELISA) is to occur the seventies in 20th century; Be used for the detection of micro substance; Be applied to Clinical detection such as infectious disease, tumor markers, hormonal readiness the earliest; Beginning the nineties to apply in China food safety detection field, rely on the kit of elisa technique, the leading products that test strips has become food security fast detecting field at present, also is simultaneously the major product type that my company researches and develops and sells.The enzyme linked immunosorbent detection method is based on the specific reaction of Ag-Ab; Detection sensitivity is higher, specificity is better; Technical operation is simple and easy; Easy master, qualitative, the quantitative test work that have solved a large amount of former insoluble many micro-small-molecule substances such as microbiotic, hormone, residues of pesticides etc. have really been played positive facilitation to the development of food security Fast Detection Technique.But there are many defectives that self can't overcome in the ELISA method, and mainly shows:
1) homogeneous reaction: be separated free thing and bond in the testing process, need multistep to wash the plate process, time and effort consuming is difficult to improve the automation of operation degree.
2) enzymic catalytic reaction: by substrate for enzymatic activity colour developing, assaying reaction liquid absorbance.There are considerable influence in the time and the temperature of reaction to enzyme activity, and reagent stability is poor.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of Furaxone metabolite detection kit, not only possesses higher sensitivity when adopting this kit to carry out the detection of Furaxone metabolite, specificity, and have higher reaction velocity.
Another object of the present invention is to provide a kind of method of testing of Furaxone metabolite, and this method is simple to operate, and is highly sensitive, and specificity is good.
Realize above-mentioned purpose, the present invention provides a kind of Furaxone metabolite detection kit, and its main agents that comprises has: luminous marker, fluorescein-labelled thing, standard items, quality-control product, separation agent.
Described separation agent is the paramagnetism nano microsphere that is coated with goat-anti FITC monoclonal antibody.
Described paramagnetism nano microsphere, surface contain-OH ,-COOH or-NH
2The microballon of reactive group is used to encapsulate goat-anti FITC monoclonal antibody, and its inner core is Fe
3O
4Or γ-Fe
2O
3, make microballon have paramagnetism.
Described luminous marker is the Furaxone metabolite haptens of different luminol derivant mark.Described different luminol derivant is ABEI, AHEI or ABEN.
Described kit also comprises standard items, quality-control product and concentrated washing lotion.
The Furaxone metabolite monoclonal antibody that described fluorescein-labelled thing is the FITC mark.
The present invention also provides a kind of method of utilizing kit to detect Furaxone metabolite, comprises the following steps:
1) draw 20 μ l-100 μ l standard items or samples respectively, add 20 μ l-100 μ l luminous markers then, add the fluorescein-labelled thing of 20 μ l-100 μ l again, behind the abundant mixing, 37 ℃ of incubation 15min;
2) add the paramagnetism nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min;
3) separate 5min with the magnetic separator frame, precipitate with cleaning fluid 300-500 μ l flushing compound after abandoning supernatant;
4) above-mentioned cleaning step repeats 2-4 time;
5) 4) compound of gained separator well is directly put into the measurement camera bellows; Add and excite substrate 1 and excite substrate 2; Postpone to detect the relative light intensity (RLU) that sends behind the 3-5s; Content in the sample and RLU proportion relation can be through the concentration of RLU set calibration curve method calculating Furaxone metabolite.
The principal ingredient of described luminous substrate is NaOH and H
2O
2
The used analyser of analysis test method is among the present invention: comprise power circuit, automatic syringe pump 1 and 2, measuring chamber, illuminated chamber, photomultiplier counter and output system; Also dispose the Windows Control Software of computing machine and Chinese interface simultaneously; Can carry out that data typing, result gather, quality control, the result stores and function such as result queries; Can accomplish the programming of multiple analytical model, quantitative or qualitative reporting the result generates and storage, update functions automatically; Automatically revise typical curve at 2, the system that is adopted is the CI-2008 system.
Separation agent of the present invention is to encapsulate goat-anti FITC monoclonal antibody, and the content of its surface group is 0.1-0.3eqm/g, and it is stored in pH7.1-7.5; Contain the 3-5% casein; The 0.1-0.3% Tween-20,0.02-0.04% thimerosal antiseptic is in the 0.03-0.05mol/LTRIS damping fluid.Described FITC is a fluorescein isothiocynate.Said percentage composition is the quality percentage composition.
Described luminous marker is the Furaxone metabolite haptens of different luminol derivant ABEI, AHEI or ABEN mark, and it is stored in pH7.2-7.6, contains the 0.1-0.3% Tween-20,0.02-0.04%NaN
3The TRIS damping fluid in.Said percentage composition is the quality percentage composition.
The Furaxone metabolite monoclonal antibody that described fluorescein-labelled thing is the FITC mark, it is stored in pH7.2-7.6, contains the 8-10% ovalbumin, 0.02-0.04% thimerosal antiseptic, 0.1-0.2mol/LPBS damping fluid.Said percentage composition is the quality percentage composition.
The Furaxone metabolite standard solution (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml), the standard items dilution is pH7.2-7.6,0.03-0.05% thimerosal antiseptic, 0.05-0.1mol/L TRIS damping fluid.Said percentage composition is the quality percentage composition.
Furaxone metabolite quality-control product solution concentration is respectively 0.02ng/ml, 0.5ng/ml, quality-control product dilution pH7.2-7.4,0.03-0.05% thimerosal antiseptic, 0.05-0.1mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Described concentrated washing lotion is PH7.0-7.4,0.2-0.4% Tween-20,0.2-0.4% sodium azide, 0.1-0.2mol/L PBS damping fluid.Said percentage composition is the quality percentage composition.
Beneficial effect of the present invention is following:
1) but kit specific detection Furaxone metabolite of the present invention does not have intersection to other drug.
2) sensitivity of kit of the present invention is higher, can reach 0.01ng/ml to the detection sensitivity of Furaxone metabolite.
3) detection of kit of the present invention is quick, is lower than 20min detection time.
Embodiment
The preparation of the concrete component of embodiment 1 kit
1, the preparation of luminous marker
A) haptenic synthetic
With the Furaxone metabolite derivatization, obtain the furazolidone derivative hapten.
B) luminous marker preparation
Get 4.5mmol/L ABEI, be dissolved in the 4ml distilled water, the 5.0mmol/L N-hydroxy-succinamide is dissolved in 0.5ml N, in the dinethylformamide, and room temperature reaction 3-4h behind the two abundant mixing.Get the Furaxone metabolite haptens 15mg of above-mentioned preparation, to 1.5ml, add the ABEI solution of above-mentioned activation with the pH7.4PBS adjusted volume then, fully room temperature reaction spends the night behind the mixing, crosses G-25 gel column purifying.
2, fluorescent marker preparation
A) immunogenic preparation:
Adopt the diazotising method to carry out coupling Furaxone metabolite derivative hapten and bovine serum albumin(BSA) and obtain immunogene.
B) Furaxone metabolite MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: with immunogene the Balb/c mouse is carried out immunity, immunizing dose is 150 μ g/, makes it produce antiserum.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, merge, obtain the hybridoma cell strain of monoclonal antibody in 9: 1 ratios and SP2/0 myeloma cell.
Cell cryopreservation and recovery: hybridoma is processed 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: the Balb/c mouse peritoneal is only injected sterilization paraffinum liquidum 0.5ml/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
5Individual/as only, to gather ascites after 7 days.Carry out purifying, obtain monoclonal antibody with sad-saturated ammonium sulfate method.
C) fluorescent marker preparation
Use 0.025mol/L, the carbonate buffer solution of pH9.0 is diluted to 1% mass percent concentration with the Furaxone metabolite monoclonal antibody; Total protein according to desiring mark adds 0.01mgFITC by every milligram of immunoglobulin (Ig), accurately takes by weighing required FITC powder with analytical balance.With the solution that FITC is made into 0.1mg/ml,, sneak into the FITC dilution with same damping fluid by 3-5 times of above-mentioned antibody-solutions volume; Electromagnetic agitation, mark 30-48h under 4～℃ lucifuge condition; With label solution 3000r/min, the centrifugal 20min of room temperature removes wherein a spot of sediment, and in the bag filter of packing into, the PBS damping fluid of pH7.4 dialysis is 2-3 days again, during change dislysate at least 3 times; Get the label of dialysed overnight, through SephadexG-25 or G-50 post, the separated free luciferin is collected the fluorescent marker of mark and is identified that packing is stored in 4 ℃ of refrigerators.
3, separation agent preparation
A) magnetic bead activation
The magnetic bead of surface-COOH group (purchase in DYNAL, particle diameter is 2.8 μ m), its content is 0.15eq/g; Get 100 μ l magnetic beads, with 100 μ l 25mmol/L, pH5.0,0.05%Tween-20MES solution washing twice, magnetic removes supernatant after separating; Before the use, dispose EDC, the NHS solution of 50mmol/L respectively with the 25mmol/L MES solution of 4 ℃ of storages; Add new EDC that disposes of 50 μ l and NHS solution respectively in the centrifuge tube that magnetic bead is housed, vortex mixing, room temperature activation 30min; Centrifuge tube places on the magnetic separator frame magnetic to separate 4min, removes supernatant, adds 100 μ l, 25mmol/L, pH5.0, MES clean get final product after 2-3 time the magnetic bead of surperficial activated carboxylic.
B) magnetic bead coupling goat-anti FITC monoclonal antibody
50-100 μ g goat-anti FITC monoclonal antibody is dissolved into 60 μ l, and 25mmol/L among the pH5.0MES, regulates cumulative volume to 100 μ l with said MES solution, soft mixing magnetic bead and goat-anti FITC monoclonal antibody; At least coupling 30min or 4 ℃ of coupling 2h under the room temperature condition, vortex appearance capable of using makes magnetic bead keep the mixing state during this period; Centrifuge tube places magnetic separation 3-5min on the magnetic separator frame, removes supernatant; For cancellation unreacted-COOH, can add 100 μ l, pH7.4, TRIS reaction 15min or 100 μ l, pH8.0 contains the PBS sealing magnetic bead of 50mmol/L monoethanolamine; With 100 μ l, 0.1-0.3%BSA, the PBS of 0.1%Tween-20 or TRIS clean the good magnetic bead of sealing 3-5 time; At last magnetic bead is redissolved in containing 0.1-0.5%BSA, 0.01-0.1%Tween-20,0.02%NaN
3PBS or TRIS damping fluid in, 2-8 ℃ of preservation.
The establishment of embodiment two kits
Set up the magnetic granule chemoluminescence detection kit that detects Furaxone metabolite, make it contain following component:
The fluorescent marker of the Furaxone metabolite monoclonal antibody of FITC mark
The haptenic luminous marker of the Furaxone metabolite of ABEI mark
The separation agent of the paramagnetism nano microsphere of pan coating goat-anti FITC monoclonal antibody
The Furaxone metabolite standard solution (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml), the standard items dilution is pH7.4, contains 0.05% thimerosal antiseptic, the 0.1mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Furaxone metabolite quality-control product solution concentration is respectively 0.02ng/ml, 0.5ng/ml, and the quality-control product dilution contains 0.05% thimerosal antiseptic, 0.1mol/L TRIS damping fluid for pH7.4.Said percentage composition is the quality percentage composition.
Concentrated washing lotion is pH7.2,0.4% Tween-20,0.3% sodium azide, 0.1mol/L PBS damping fluid.Said percentage composition is the quality percentage composition.
The detection of Furaxone metabolite in embodiment three actual samples
1, sample pre-treatment
(1) muscle, liver organization pre-treating method
With homogenizer homogeneous sample; Take by weighing the tissue samples (liver appearance/meat appearance) behind 1.0 ± 0.05g homogeneous, add deionized water, 0.5ml 1M hydrochloric acid solution and the 100 μ l derivatization reagents of 4ml, with the oscillator 2min that fully vibrates; At 37 ℃ of night incubation (approximately 16h); Add 5ml 0.1M dipotassium hydrogen phosphate solution, 0.4ml1M sodium hydroxide solution and 5ml ethyl acetate respectively, with oscillator thermal agitation 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Get in 2.5ml ethyl acetate to the 10ml dry glass test tube, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add 1ml normal hexane (or normal heptane),, add 1ml redissolution working fluid again, with the abundant mixing of vortex appearance whirling motion 1min with vortex appearance whirling motion 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Remove upper organic phase, take off layer water 50 μ l and be used for analyzing.Sample extension rate: 2.
(2) aquatic products pre-treating method
With homogenizer homogeneous sample; Take by weighing in equal pledge (fish/shrimp) to the 50ml polystyrene centrifuge tube of 1.0 ± 0.05g, add the 4ml deionized water, 0.5ml 1M hydrochloric acid solution and 100 μ l derivatization reagents, 2min fully vibrates; At 37 ℃ of night incubation (approximately 16h); Add 5ml 0.1M dipotassium hydrogen phosphate solution respectively, 0.4ml 1M NaOH and 5ml ethyl acetate, thermal agitation 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Take out the 2.5ml upper organic phase to the clean glass test tube of 10ml, 50～60 ℃ of water-bath nitrogen flow down and dry up; Dissolve dry thing with 1ml normal hexane (or normal heptane), fully mix with 1ml redissolution liquid working fluid; (higher fatty acid sample can strengthen the normal hexane consumption, with the dry thing of 3ml n-hexane dissolution) be (20-25 ℃) centrifugal 10min more than the 3000g at room temperature; Remove upper organic phase, get 50 μ l lower floor waters and be used for analyzing sample extension rate: 2.
(3) honey pre-treating method
Take by weighing in 1.0 ± 0.05g honey to the 50ml polystyrene centrifuge tube; Add the 4ml deionized water, vibrate to honey with oscillator and all dissolve; Add 0.5ml 1M hydrochloric acid solution and 100 μ l derivatization reagents, fully vibrate with oscillator; At 37 ℃ of night incubation (approximately 16h); Add 5ml 0.1M dipotassium hydrogen phosphate solution, 0.4ml 1M sodium hydroxide solution and 5ml ethyl acetate respectively, with oscillator thermal agitation 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Get in 2.5ml ethyl acetate to the 10ml dry glass test tube, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add 1ml normal hexane (or normal heptane),, add 1ml redissolution working fluid again, with the abundant mixing of vortex appearance whirling motion 1min with vortex appearance whirling motion 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Remove upper organic phase, take off layer water 50 μ l and be used for analyzing sample extension rate: 2.
2, detect and interpretation of result with kit
Draw 20 μ l-100 μ l standard items or samples respectively, add 20 μ l-100 μ l luminous markers then, add the fluorescein-labelled thing of 20 μ l-100 μ l again, behind the abundant mixing, 37 ℃ of incubation 15min; Add the paramagnetism Nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min; Separate 5min with the magnetic separator frame, precipitate with cleaning fluid 300-500 μ l flushing compound after abandoning supernatant; The compound of gained separator well is directly put into the measurement camera bellows; Add and excite substrate 1 and excite substrate 2; Postpone to detect the relative light intensity (RLU) that sends behind the 3-5s; The content of Furaxone metabolite and RLU proportion relation in the sample can be through the concentration of RLU combined standard curve method calculating Furaxone metabolite.
Exciting substrate 1 is NaOH, and exciting substrate 2 is H
2O
2Before substrate loads, clean the substrate pump 10-20 time, behind the remaining water mark, more corresponding substrate is directly put into instrument in the emptying pipe, pump line is inserted in the substrate bottle with distilled water; With substrate flushing pipe 5 times, and measure the RLU value of substrate, under the normal condition, the RLU value of substrate should be above 1200.If surpass 1200, need with distilled water pipeline and substrate pump to be carried out more times cleaning again, in blank value is reduced to zone of reasonableness.Do not do experiment if surpass three days, need unloading substrate bottle, and cover lid, with vaporization prevention.Use the distilled water pipe blow-through subsequently, and emptying, in order to avoid strong base solution corrosion substrate pump.
The present invention adopt 6 Furaxone metabolite standard items (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml),
Carry out plotting curves.Instrument detects according to said method and obtains a RLU value and the concentration dependent calibration curve of Furaxone metabolite, and in after this measuring, the Furaxone metabolite concentration in each sample is compared with typical curve and drawn the Furaxone metabolite content in the sample.
The mensuration of embodiment four kit quality
1, the sensitivity of kit
Being defined as of kit sensitivity: measure 20 times the zero standard article, the mean value of mensuration adds 3 times of standard deviations.The sensitivity of this kit is 0.01ng/ml.
2, the accuracy of sample and precision
Accuracy is meant the matching degree between measured value and true value, and the kit accuracy recovery commonly used is represented.Precision is claimed repeatability again, and the coefficient of variation commonly used is represented.
Sample extraction method according to embodiment three; Furaxone metabolite with 0.03ng/g (ml), two concentration of 0.06ng/g (ml) adds recovery to chicken, shrimp, honey sample; Every kind of each 4 of each concentration of sample are parallel; Measure with three batches of kits, calculate the average recovery rate and the precision of sample.Experimental result sees the following form.
Table 1 accuracy and precision are measured ng/g (ml)
Can know from table, the average recovery rate scope that two concentration of Furaxone metabolite are all added in chicken, shrimp, the honey sample between 88.3-101.3%, in batch, batch between all the coefficient of variation less than 15%.
3, specificity
Cross reacting rate is meant the ability that antibody combines with structure different antigens determinant.
Table 2 kit cross reacting rate
| Medicine name | Cross reacting rate |
| Furazolidone | 100％ |
| AMOZ | Less than 0.1% |
| Cistofuran metabolite | Less than 0.1% |
| Nitrofurazone (nitrofurazone) metabolin | Less than 0.1% |
| Furazolidone | 15.3％ |
| Furaltadone | Less than 1% |
| Furantoin | Less than 1% |
| Nitrofurazone (nitrofurazone) | Less than 1% |
[0091]4, correlativity
X＝CI-2008，Y＝RIA
Y＝1.23X-2.58
R＝0.9862
X is CI-2008 system measurement result, and Y is that the result is measured in the aspect.
Claims (8)
1. magnetic granule chemoluminescence kit that detects Furaxone metabolite, the reagent that comprises has: luminous marker, fluorescein-labelled thing, standard items, quality-control product, separation agent.
2. kit according to claim 1 is characterized in that: described separation agent is the paramagnetism nano microsphere that is coated with goat-anti FITC monoclonal antibody.
3. kit according to claim 2 is characterized in that: described paramagnetism nano microsphere is that the inside is coated with Fe
3O
4Or γ-Fe
2O
3, the surface contains-OH ,-COOH or-NH
2The microballon of reactive group.
4. according to each described kit of claim 1-3, it is characterized in that: described luminous marker is the Furaxone metabolite haptens of different luminol derivant mark.
5. kit according to claim 4 is characterized in that: described different luminol luminous marker is ABEI, AHEI or ABEN.
6. according to each described kit of claim 1-3, it is characterized in that: described kit also comprises titer, quality-control product and concentrated washing lotion.
7. according to the described kit of one of claim 1-3, it is characterized in that: the Furaxone metabolite monoclonal antibody that described fluorescein-labelled thing is the FITC mark.
8. a method of utilizing each described kit of claim 1-7 to detect Furaxone metabolite comprises the following steps:
1) draw 20 μ l-100 μ l standard items or samples respectively, add 20 μ l-100 μ l luminous markers then, add the fluorescein-labelled thing of 20 μ l-100 μ l again, behind the abundant mixing, 37 ℃ of incubation 15min;
2) add the paramagnetism nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min;
3) separate 5min with the magnetic separator frame, precipitate with cleaning fluid 300-500 μ l flushing compound after abandoning supernatant;
4) above-mentioned cleaning step repeats 2-4 time;
5) 4) compound of gained separator well is directly put into the measurement camera bellows; Add and excite substrate 1 and excite substrate 2; Postpone to detect the relative light intensity (RLU) that sends behind the 3-5s; The content of Furaxone metabolite and RLU proportion relation in the sample can be through the concentration of RLU set calibration curve method calculating Furaxone metabolite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105912744A CN102565402A (en) | 2010-12-16 | 2010-12-16 | Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105912744A CN102565402A (en) | 2010-12-16 | 2010-12-16 | Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102565402A true CN102565402A (en) | 2012-07-11 |
Family
ID=46411359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105912744A Pending CN102565402A (en) | 2010-12-16 | 2010-12-16 | Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102565402A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539413A (en) * | 2010-12-16 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005103698A2 (en) * | 2004-04-19 | 2005-11-03 | Charm Sciences, Inc. | Methods and antibodies for nitrofuran detection |
| CN1888901A (en) * | 2006-04-21 | 2007-01-03 | 深圳市新产业生物医学工程有限公司 | Magnetic separating direct chemical illuminating reagent and testing method using the same reagent |
| CN101324578A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Immune radiometric analysis reagent kit for detecting tumour sign object CA50 and use method thereof |
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN101419234A (en) * | 2008-10-17 | 2009-04-29 | 杭州博日科技有限公司 | Chemiluminescence detection kit based on corpuscle |
| CN101806796A (en) * | 2010-03-25 | 2010-08-18 | 中国农业科学院上海兽医研究所 | Detection kit and method of furazolidone metabolin |
| CN102539413A (en) * | 2010-12-16 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit |
-
2010
- 2010-12-16 CN CN2010105912744A patent/CN102565402A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005103698A2 (en) * | 2004-04-19 | 2005-11-03 | Charm Sciences, Inc. | Methods and antibodies for nitrofuran detection |
| CN1888901A (en) * | 2006-04-21 | 2007-01-03 | 深圳市新产业生物医学工程有限公司 | Magnetic separating direct chemical illuminating reagent and testing method using the same reagent |
| CN101324578A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Immune radiometric analysis reagent kit for detecting tumour sign object CA50 and use method thereof |
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN101419234A (en) * | 2008-10-17 | 2009-04-29 | 杭州博日科技有限公司 | Chemiluminescence detection kit based on corpuscle |
| CN101806796A (en) * | 2010-03-25 | 2010-08-18 | 中国农业科学院上海兽医研究所 | Detection kit and method of furazolidone metabolin |
| CN102539413A (en) * | 2010-12-16 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit |
Non-Patent Citations (1)
| Title |
|---|
| 高红军 等: "化学发光免疫分析法检测血清NSE的室间比对结果分析", 《中国卫生检验杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539413A (en) * | 2010-12-16 | 2012-07-04 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102928413A (en) | Magnetic particle chemiluminescence kit for detecting tetracyclines, and applications thereof | |
| CN101113981A (en) | Sulfonamides direct-competition ELISA detecting reagent kit | |
| CN100501409C (en) | ELISA kit for detecting chloramphenicols in animal derived food | |
| CN101013130A (en) | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof | |
| CN102539412A (en) | Magnetic particle chemical luminous kit for detecting clenbuterol and application thereof | |
| CN102928417A (en) | Magnetic particle chemiluminescence kit for detecting sulfanilamide drugs, and applications thereof | |
| CN102768278B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN101358967B (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN101013131B (en) | Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof | |
| CN102608316B (en) | Kit or test strip for detecting quinoxaline compound | |
| CN103364553B (en) | The enzyme linked immunological kit of detection nitroimidazoles medicine and application thereof | |
| CN102539413A (en) | Magnetic particle chemiluminescence kit for detecting frazolidone metabolites and application of magnetic particle chemiluminescence kit | |
| CN102928410A (en) | Magnetic particle chemiluminescence kit for detecting ractopamin, and applications thereof | |
| CN102928407A (en) | Magnetic particle chemiluminescence kit for detecting avermectins, and applications thereof | |
| CN102928411A (en) | Magnetic particle chemiluminescence kit for detecting streptomycin, and applications thereof | |
| CN102928409A (en) | Magnetic particle chemiluminescence kit for detecting lincomycin, and applications thereof | |
| CN102565400A (en) | Magnetic granule chemiluminescence kit for detecting melamine and application of magnetic granule chemiluminescence kit | |
| CN103592435A (en) | Magnetic-particle chemiluminescence kit used for detecting monensin and applications of the kit | |
| CN102565032A (en) | Magnetic granule chemiluminescence kit for detecting nitrofurantoin metabolite and application of magnetic granule chemiluminescence kit | |
| CN102928406A (en) | Magnetic particle chemiluminescence kit for detecting sudan I, and applications thereof | |
| CN102539763A (en) | Magnetic particle chemiluminescent kit for detecting furaltadone metabolite and application thereof | |
| CN102565402A (en) | Magnetic particle chemiluminescence kit for detecting furazolidone metabolite and application thereof | |
| CN102707073A (en) | Enzyme linked immunosorbent assay kit for detecting metandienone and using method of enzyme linked immunosorbent assay kit | |
| CN102928592A (en) | Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120711 |