CN102565403A - Magnetic particle chemiluminescence kit for detecting furaltadone and application thereof - Google Patents
Magnetic particle chemiluminescence kit for detecting furaltadone and application thereof Download PDFInfo
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- CN102565403A CN102565403A CN2010105929247A CN201010592924A CN102565403A CN 102565403 A CN102565403 A CN 102565403A CN 2010105929247 A CN2010105929247 A CN 2010105929247A CN 201010592924 A CN201010592924 A CN 201010592924A CN 102565403 A CN102565403 A CN 102565403A
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Abstract
The invention relates to a magnetic particle chemiluminescence kit for detecting furaltadone. 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 furaltadone hapten marked by an isoluminol luminescence marker; the fluorescein marker is a furaltadone 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 furaltadone in feed by use of the kit. The method has relatively high sensitivity and specificity and relatively high speed in detecting the furaltadone.
Description
Technical field
The present invention relates to a kind of direct chemical luminous detection kit and method of testing thereof, particularly detect a magnetic rate chemiluminescence detection kit of residue of furaltadone through amount in the feed sample.
Technical background
Furaltadone (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, furaltadone has obtained using widely in herding, aquaculture.
But furaltadone 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; Furaltadone and metabolic product thereof have carcinogenicity, and are prone to produce drug resistance strain, cause monster easily.China classifies furaltadone as the forbidding medicine in " primary and other compound inventories of food animal forbidding " of having the Ministry of Agriculture to issue in March, 2002.Therefore the detection method of setting up a kind of furaltadone effectively becomes task very critical in the residue of veterinary drug analysis field.
At present, the conventional sense method of residue of furaltadone through amount 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 the low characteristics HPLC method of accuracy of detection height, false positive rate and is limited to 1 μ g/kg for the lowest detection of furaltadone.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) heterogeneous 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 furaltadone detection kit, not only possesses higher sensitivity when adopting this kit to carry out the detection of furaltadone, specificity, and have higher reaction velocity.
Another object of the present invention is to provide a kind of method of testing of furaltadone, 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 furaltadone 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 furaltadone 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 furaltadone 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 furaltadone, 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 furaltadone.
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 furaltadone 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 furaltadone 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 furaltadone standard solution (0ng/ml, 0.01ng/ml, 0.03ng/m, 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/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Furaltadone 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%NaN
3, the 0.1-0.2mol/LPBS damping fluid.Said percentage composition is the quality percentage composition.
Beneficial effect of the present invention is following:
1) but kit specific detection furaltadone 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 furaltadone.
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) furaltadone is haptenic synthetic
Through derivatization, preparation has-the furaltadone haptens of COOH with furaltadone.
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 furaltadone 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 mixed anhydride method to carry out coupling furaltadone haptens and ovalbumin and obtain immunogene.
B) furaltadone 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
6The 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 furaltadone monoclonal antibody; Total protein according to desiring mark adds 0.01mg FITC 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 ℃ of lucifuge conditions; 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 furaltadone, make it contain following component:
The fluorescent marker of the furaltadone monoclonal antibody of FITC mark
The haptenic luminous marker of the furaltadone of ABEI mark
The separation agent of the paramagnetism nanometer magnetic bead of pan coating goat-anti FITC monoclonal antibody
The furaltadone standard solution (0ng/ml, 0.01ng/ml, 0.03ng/m, 0.09ng/ml, 0.27ng/ml, 0.81ng/ml), the standard items dilution is pH7.4, contains 0.05%NaN
3, the 0.1mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Furaltadone quality-control product solution concentration is respectively 0.02ng/ml, 0.5ng/ml, and the quality-control product dilution contains 0.05% thimerosal antiseptic, the 0.1mol/LTRIS 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%NaN3,0.1mol/L PBS damping fluid.Said percentage composition is the quality percentage composition.
The detection of furaltadone in embodiment three actual samples
1, feed sample pre-treatment
Take by weighing 1.0g ± 0.05g feed sample to the 50ml centrifuge tube, add 8ml ethyl acetate, add the 2ml acetonitrile again; Vibration 5min, static 10min, more than the room temperature 3000g, centrifugal 10min gets in the clean glass test tube of supernatant liquor 2ml to 10ml, flows down in 50～60 ℃ of water-bath nitrogen to dry up; Add the 1ml normal hexane,, add 1ml redissolution working fluid again, with vortex appearance whirling motion 30s with vortex appearance whirling motion 1min; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Remove upper organic phase, take off layer water 200 μ l and 200 μ l redissolution working fluid mixing; Getting 50 μ l is used for analyzing diluted sample multiple: 10.
2, detect and interpretation of result with kit
Draw 20 μ l-100 μ l standard items, sample 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 magnetic bead 80-150 μ l be coated with anti-FITC 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 furaltadone and RLU proportion relation in the sample can be through the concentration of RLU combined standard curve method calculating furaltadone.
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 furaltadone standard items (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.09ng/ml, 0.27ng/ml 0.81ng/ml) carries out plotting curves.Instrument detects according to said method and obtains a RLU value and the concentration dependent calibration curve of furaltadone, and in after this measuring, the furaltadone concentration in each sample is compared with typical curve and drawn the residue of furaltadone through 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; Furaltadone with 0.2ng/g, two concentration of 0.4ng/g adds recovery to the feed 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
Can know from table, the average recovery rate scope that two concentration of furaltadone are all added in the feed sample between 92.5-93.1%, 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 |
| Furaltadone | 100％ |
| Furaltadone | 16.3％ |
| Furazolidone | 60％ |
| Furantoin | 30％ |
| Furaxone metabolite | Less than 0.1% |
| Cistofuran metabolite | Less than 1% |
| Nitrofurazone (nitrofurazone) | Less than 1% |
| Nitrofurazone (nitrofurazone) metabolin | Less than 1% |
4, correlativity
X＝CI-2008，Y＝RIA
Y＝1.25X-2.76
R＝0.9802
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 furaltadone, 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 furaltadone 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 furaltadone 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 furaltadone 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) 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 furaltadone and RLU proportion relation in the sample can be through the concentration of RLU set calibration curve method calculating furaltadone.
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Application publication date: 20120711 |