CN102565031A - Magnetic granule chemiluminescence kit for detecting chloramphenicol and application thereof - Google Patents
Magnetic granule chemiluminescence kit for detecting chloramphenicol and application thereof Download PDFInfo
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- CN102565031A CN102565031A CN2010105902329A CN201010590232A CN102565031A CN 102565031 A CN102565031 A CN 102565031A CN 2010105902329 A CN2010105902329 A CN 2010105902329A CN 201010590232 A CN201010590232 A CN 201010590232A CN 102565031 A CN102565031 A CN 102565031A
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Abstract
The invention relates to a magnetic granule chemiluminescence kit for detecting chloramphenicol, which comprises the following reagents: a luminescence marker, a fluorescein marker, a standard preparation, a quality control product and a separation reagent, wherein the luminescence marker is chloramphenicol hapten labeled by an isoluminol luminescence marker; the fluorescent marker is a chloramphenicol monoclonal antibody labeled by fluorescein or a derivative thereof; and the separation reagent is a paramagnetic nano microbead coated with a goat anti-FITC (fluorescein isothiocyanate) monoclonal antibody. The invention also relates to a method for detecting chloramphenicol in animal-derived food by adopting the kit. The method has higher sensitivity, specificity and detection speed when used for detecting chloramphenicol.
Description
Technical field
The present invention relates to a kind of chemical luminescence reagent kit and method of testing thereof, particularly detect the magnetic granule chemoluminescence detection kit of chloramphenicol residue in animal tissue, urine, the feed equal samples.
Technical background
(Chloramphenicol CAP) is widely used antibiotic to chloromycetin, in livestock and poultry control and treatment, has played vital role.But because there is serious adverse in chloromycetin; Can suppress mitochondrial protein synthesis in the bone marrow cell; Cause bone marrow cell and hepatocellular toxicity, thereby cause people's reproducibility aplastic anemia, agranulocytosis; Diseases such as neonate, premature's ash baby syndrome, developed countries such as America and Europe forbid in succession or strictness bans use of.In Dec, 2002, the China Ministry of Agriculture announced the literary composition regulation No. 235, and chloromycetin and salt thereof, ester (comprising Chloramphenicol Succinate) must not detect in all edible tissues of all food animals.But because chloromycetin low price and be broad-spectrum antibiotic, illegal use is still very general.Therefore strengthen the residue detection of chloromycetin in the animal food is very important.
At present, mainly adopting methods such as LC/MS (LC-MS), gas chromatography-mass spectrography (GC-MS), ELISA (ELISA) and microbial method to be used for the animal derived food residual chloromycetin detects.
1, gas chromatography-mass spectrography (GC-MS) sensitivity is very high, and false positive rate is low.The major defect of this method is that sample needs derivatization to handle complicated like this early stage.Though can in mass spectrum, provide more structural information through the sample of derivatization treatment, derivatization can produce a plurality of different products and cause the partial loss of sample, causes the deviation of experimental result.
2, LC/MS (LC-MS), sample does not need derivatization treatment, can detect urine, blood, liver, hair and eyeball sample.This method reaches 0.007～0.100 μ g/kg to the LDL of chloromycetin, and 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, the instrument detecting method is the same with GC-MS, and fails to solve instrument and involve great expense, complex operation step, the sample complex pretreatment requires problems such as height to operating personnel's specialty attainment.
3, ELISA (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.
Though 4, the microorganism detection method is economical, easy and simple to handle, when in sample, having other microbial inhibitors to exist, its sensitivity and specificity are restricted.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of chloromycetin detection kit, not only possesses higher sensitivity when adopting this kit to carry out the detection of chloromycetin, specificity, and have higher reaction velocity.
Another object of the present invention is to provide a kind of method of testing of chloromycetin, 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 chloromycetin 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 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 chloromycetin 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.
Described fluorescein-labelled thing is a FITC mark chloromycetin monoclonal antibody.
The present invention also provides a kind of method of utilizing kit chlorine detection mycin, 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 chloromycetin.
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 content is to be dissolved in containing 0.1-0.5%BSA 0.01-0.1%Tween-20,0.02%NaN when being its preservation
3, in the PBS damping fluid of pH7.2-7.6.Described FITC is a fluorescein isothiocynate.Said percentage composition is the quality percentage composition.
Described luminous marker is that hapten-marked its of chloromycetin of different luminol derivant ABEI, AHEI or ABEN mark is stored in and contains pH7.2-7.6,0.1-0.3%Tween-20,0.01%NaN
3The PBS damping fluid in.Said percentage composition is the quality percentage composition.
The chloromycetin monoclonal antibody that described fluorescein-labelled thing is the FITC mark, it is stored in pH7.2-7.6, contains 5-8%BSA, 0.02-0.04%NaN
3The PBS damping fluid.Said percentage composition is the quality percentage composition.
The chloromycetin standard solution (0ng/ml, 0.025ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.35ng/ml), the standard items dilution is pH7.4,0.03%NaN
3, 0.05mol/L TRIS damping fluid.Said percentage composition is the quality percentage composition.
Chloromycetin quality-control product solution concentration is respectively 0.02ng/ml, 1.0ng/ml, quality-control product dilution pH7.4,0.03%NaN
3, the 0.05mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Described concentrated washing lotion is PH7.2-7.8,0.2-0.4%Tween-20,0.02-0.04%NaN
3, 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 chloromycetin of the present invention does not have intersection to other drug.
2) sensitivity of kit of the present invention is higher, can reach 25ng/ml to the detection sensitivity of chloromycetin.
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 one kit
1) haptenic synthetic
The chloromycetin haptens that adopts the glutaric anhydride acidylate to obtain the alcoholic extract hydroxyl group in the chloromycetin drug molecular structure.
2) immunogenic preparation
Adopt the water-soluble carbodiimide method to carry out coupling chloromycetin haptens and thyroprotein and all arrive immunogene.
3) MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: with immunogene the Balb/c mouse is carried out immunity, immunizing dose is 100 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 the cell suspension of 1 * 109/ml with cryopreserving liquid, preserve in that liquid nitrogen is medium-term and long-term.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/, and pneumoretroperitoneum injection hybridoma was 5 * 107/in 7 days, gathered ascites after 7 days.Carry out purifying, obtain monoclonal antibody with sad-saturated ammonium sulfate method.
4) preparation of luminous marker
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 chloromycetin 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.
5) fluorescent marker preparation
Use 0.025mol/L, the carbonate buffer solution of pH9.0 is diluted to 1% mass percent concentration with the chloromycetin 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～℃ 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.
6) 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 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, in the PBS of 0.02%NaN3 or the TRIS damping fluid, 2-8 ℃ of preservation.
The establishment of embodiment two kits
Set up the magnetic particle direct competitive chemiluminescence detection kit of chlorine detection mycin, make it contain following component:
The fluorescent marker of the chloromycetin monoclonal antibody of FITC mark
The haptenic luminous marker of the chloromycetin of ABEI mark
The separation agent of the paramagnetism nano microsphere of pan coating goat-anti FITC monoclonal antibody
The chloromycetin standard solution (0ng/ml, 0.025ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.35ng/ml), the standard items dilution is pH7.4, contains 0.03%NaN
3, the 0.05mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Chloromycetin quality-control product solution concentration is respectively 0.02ng/ml, 1.0ng/ml, and quality-control product dilution pH7.4 contains 0.03%NaN
3, the 0.05mol/LTRIS damping fluid.Said percentage composition is the quality percentage composition.
Concentrated washing lotion is pH7.6,0.4%Tween-20,0.02%NaN
3, 0.1mol/L PBS damping fluid.Said percentage composition is the quality percentage composition.
The detection of chloromycetin in embodiment three actual samples
1, sample pre-treatment
(1) chicken is organized pre-treating method
With homogenizer homogeneous structure sample; Take by weighing in the equal pledge of 3.0 ± 0.05g to the 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, with the oscillator 10min that vibrates, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Pipette 4ml upper organic phase (sample that is equivalent to 2g approximately) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex appearance whirling motion 30s, add 1ml redissolution working fluid again, 1min changes in the 2ml centrifuge tube with the whirling motion of vortex appearance, more than the 3000g, and the centrifugal 15min of room temperature (20-25 ℃); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.Diluted sample multiple: 0.5.
(2) this pre-treating method of chicken blood sample
Gather chicken blood sample this (suggestion blood sampling syringe is also used the liquaemin rinse) with the centrifuge tube that is added with liquaemin (20-30 unit/ml blood), this room temperature of blood sample leaves standstill 1h, after waiting to separate out blood plasma, and more than the 3000g, 15 ℃ of centrifugal 10min; Get in 1ml blood plasma to the 10ml polystyrene centrifuge tube, add 2ml ethyl acetate vibration 1min; Leave standstill under the room temperature (20-25 ℃) and treat water and the complete layering of organic phase or more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃); Pipette upper strata institute organic phase to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add 1ml redissolution working fluid, with the dry residue of vortex appearance whirling motion 2min dissolving; Getting 50 μ l is used for analyzing serum plasma sample extension rate: 1
(3) urine pre-treating method
With urine more than 3000g, the centrifugal 5min of room temperature (20-25 ℃), limpid until urine specimen; Pipette in the limpid urine specimen of 2ml to the 50ml polystyrene centrifuge tube, add 0.5ml pH4.8100mM sodium-acetate buffer and mix; (Merck Art.No.4114) to the urine specimen of dilution, puts 37 ℃ of hydrolysis 2h (or spending the night) at least, takes out room temperature (20-25 ℃) and places to add 40 μ l glucuronidases; This solution returns to room temperature (20-25 ℃) back and adds 8ml ethyl acetate mixing 1min; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃) gets in 4ml supernatant liquid to the 10ml dry glass test tube, flows down in 50～60 ℃ of water-bath nitrogen to dry up; Add 1ml redissolution working fluid, the dry residue of whirling motion 2min dissolving; Getting 50 μ l is used for analyzing urine sample extension rate: 1
(4) casing pre-treating method
With homogenizer homogeneous sample; Take by weighing in sample to the 50ml polystyrene centrifuge tube behind 1.0 ± 0.05g homogeneous, add 10ml ethyl acetate, vibration 10min, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Get 5ml upper organic phase (sample that is equivalent to 0.5g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, whirling motion 30s adds 0.5ml again and redissolves the working fluid 1min that vibrates strongly and change in the 2ml centrifuge tube, more than the 3000g, and the centrifugal 5min of room temperature (20-25 ℃); Remove upper organic phase, take off layer 50 μ l and be used for analyzing casing diluted sample multiple: 1.
(5) milk sample pre-treating method
Get the milk sample, more than the 3000g, 10 ℃ of centrifugal 10min absorb upper strata fat; Get 5ml and remove in fatty milk sample to the 10ml polystyrene centrifuge tube, add 150 μ l C liquid and deposition occurs, vibration 15s adds 150 μ l D liquid again, and vibration 1min fully mixes; More than the 3000g, 15 ℃ of centrifugal 10min pipette upper strata liquid; With redissolving working fluid with equal-volume dilution upper strata liquid (1 part of upper strata liquid+1 part redissolution working fluid); Getting 50 μ l is used for analyzing diluted sample multiple: 2.
(6) eggs pre-treating method
With homogenizer low speed homogeneous egg sample, make egg white and yolk fully mix; Take by weighing in egg sample to the 50ml polystyrene centrifuge tube behind 3.0 ± 0.05g homogeneous, add the 9ml acetonitrile-aqueous solution, vibration 10min, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Get 3ml upper strata liquid and fully mix, add 4.5ml ethyl acetate, fully mix 5min with 3ml distilled water, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Pipette upper organic phase to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane,, add 2ml redissolution working fluid again, fully mix 1min with the vortex appearance and change in the 4ml centrifuge tube with vortex appearance whirling motion 30s dissolving dried residue, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃); Remove upper organic phase, take off layer 50 μ l and be used for analyzing eggs diluted sample multiple: 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 chloromycetin and RLU proportion relation in the sample can be through the concentration of RLU combined standard curve method calculating chloromycetin.
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 chloromycetin standard items (0ng/ml, 0.025ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml 1.35ng/ml) carries out plotting curves.Instrument detects according to said method and obtains a calibration curve that the RLU value is relevant with chloramphenicol concentration, and in after this measuring, the chloramphenicol concentration in each sample is compared with typical curve and drawn the chloromycetin 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 0.025ng/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; Chloromycetin with 0.05ng/g (ml), two concentration of 0.1ng/g (ml) adds chicken, chicken blood, pig urine, casing sample respectively; Chloromycetin with 0.1ng/g (ml), two concentration of 0.2ng/g (ml) adds recovery to milk, egg sample respectively; 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 chloromycetin are all added in chicken, chicken blood, pig urine, casing, milk, the egg sample between 79.5-102.4%, 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 |
| Chloromycetin | 100％ |
| Chloramphenicol palmitate | Less than 1% |
| Thiamphenicol | Less than 1% |
| Florfenicol | Less than 1% |
| Acyl chlorides mycin | Less than 1% |
4, correlativity
X＝CI-2008，Y＝RIA
Y＝1.24X-2.66
R＝0.9755
X is CI-2008 system measurement result, and Y is that the result is measured in the aspect.
Claims (8)
1. the magnetic granule chemoluminescence kit of a chlorine detection mycin, 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 chloromycetin 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 chloromycetin monoclonal antibody that described fluorescein-labelled thing is the FITC mark.
8. a method of utilizing each described kit chlorine detection mycin of claim 1-7 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 chloromycetin and RLU proportion relation in the sample can be through the concentration of RLU set calibration curve method calculating chloromycetin.
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Cited By (1)
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| CN108088992A (en) * | 2017-12-22 | 2018-05-29 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of chloramphenicol and preparation method thereof |
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