CN108426996A - A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained - Google Patents
A kind of quick detection kit and its preparation method and application that 3- Jia based quinoxalines -2- is carboxylic acid remained Download PDFInfo
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Abstract
The invention belongs to fluoroimmunoassay technical fields, are related to a kind of quick detection kit and its preparation method and application that 3 Jia based quinoxalines 2 are carboxylic acid remained.The kit includes detection card and fluorescent reagent micropore, test strips in detection card include bottom plate, sample pad, chromatographic film and water absorption pad, and the detection zone for being coated with 3 Jia based quinoxalines, 2 carboxylic acid carrier protein couplet object and the quality control region for being coated with sheep anti mouse secondary antibody are provided on the base material of chromatographic film；Fluorescent reagent micropore includes the time-resolved fluorescence microballoon and resealable container of 3 Jia based quinoxalines, 2 carboxylic acid labeling of monoclonal antibody.It whether carboxylic acid remained containing 3 Jia based quinoxalines 2 can be detected within a short period of time in animal tissue's sample using the kit, not need expensive device, be suitable for promoting.In addition, detection method high sensitivity, detection limit maintains an equal level up to 0.2 μ g/kg or is higher than ELISA method, and detection time is shorter.
Description
Technical field
The invention belongs to fluoroimmunoassay technical fields, are related to a kind of for quickly detecting olaquindox metabolite 3- methyl
Oxoquinoxaline-2-carboxylic acid remained kit, preparation method, and its 3- Jia based quinoxalines -2- in detection animal tissue sample
Application in carboxylic acid remained.
Background technology
Olaquindox (also known as olaquindox), trade name olaquindox, Olaquindox, No. CAS is 23696-28-8, and molecular formula is
C12H13N3O4, entitled 2- [N- (2- hydroxyls) ethyl] carbamoyl -3- methyl-quinoxaline -1 of chemistry, 4- dioxide is one
Kind feed addictive promotes livestock growth although can improve food conversion ratio, and has apparent teratogenesis to most animals
Effect also has potential three-induced effect (mutagenesis, carcinogenic, teratogenesis) to people.Therefore, olaquindox is all banned in USA and EU
Only be used as feed addictive, 2005 editions《Chinese veterinary pharmacopoeia》Also clear stipulaties：Forbid olaquindox being used for poultry and aquaculture.
Olaquindox is unstable in animal body, and more than ten kinds of metabolin, 3- first based quinoxaline -2- carboxylics can be generated in the short time
Acid (also known as 3- methylquinoxaline-2-carboxylic acids, MQCA) is then one of its main metabolites.The compound is relatively steady in vivo
It is fixed, it is one of the mark residue that Codex Alimentary Commission (CAC) is assert.It is provided in No. 235 bulletin of the Ministry of Agriculture of China：
Maximum residue limit in animal muscle and liver organization is respectively 4 μ g/kg and 50 μ g/kg.
Currently, mainly having instrument detection method and immunology fast for the remaining detection methods of MQCA in animal tissue's sample
Fast detection method.Instrument detection method, which generally requires expensive instrument and professional operator, current method, mainly agriculture
The measurement high performance liquid chromatography of olaquindox metabolite residual quantity, GB/T in the bulletin -5-2008 aquatic products of industry portion the 1077th
20746-2006 oxen, pig liver and muscle in carbadox and olaquindox and the measurement liquid chromatography-tandem of metabolite residue amount
Mass spectrography etc., lowest detection are limited to 0.5 μ g/kg.Only ELISA method can reach 0.2 μ g/ to immunology quick detection method at present
The detection of kg limits, and there is still a need for large-scale instruments such as microplate reader, and the detection operating time is long.The equally glue based on immunology principle
Although body gold detection method has the advantages that operating method simplicity, its remolding sensitivity ELISA is poor, and testing requirements are not achieved, because
And it to be not yet used for the detection of MQCA at present.
Invention content
When, detection limit for height high for instrument dependency degree existing for the residual detecting methods of MQCA in the prior art, complicated for operation, operation
Between it is long the problems such as, the present invention is intended to provide remaining quick detection kits of a kind of MQCA and its preparation method and application.This hair
It is bright to utilize time resolved fluoro-immunoassay (time-resolved fluoroimmunoassay, TRFIA) technical substitution colloid
Golden labeling method, and immunochromatography technique is combined, it successfully develops for the remaining quick detection kits of MQCA, Ke Yijian
Just, the MQCA rapidly in detection animal tissue sample is remained, and can reach the detection limit of 0.2 μ g/kg.
Specifically, the present invention adopts the following technical scheme that：
On the one hand, the present invention provides a kind of remaining quick detection kits of MQCA comprising detection card and fluorescence examination
Agent micropore, wherein：
The detection card includes test strips and the shell for accommodating the test strips, and the test strips include bottom plate, sample
Product pad, chromatographic film and water absorption pad, wherein：
Be fixed with the sample pad, chromatographic film and water absorption pad on the bottom plate successively, the beginning of the sample pad with it is described
The beginning of bottom plate is aligned, and the end of the sample pad is connected with the beginning of the chromatographic film, the end of the chromatographic film with it is described
The beginning of water absorption pad is connected, and the end of the water absorption pad is aligned with the end of the bottom plate；
The detection zone (also known as T lines) for being coated with MQCA- carrier protein couplet objects is provided on the base material of the chromatographic film
With the quality control region (also known as C lines) for being coated with sheep anti mouse secondary antibody, the detection zone is located adjacent to the side of the sample pad, described
Quality control region is located remotely from the side of the sample pad；Preferably, the detection zone and quality control region are ribbon, the two parallel
Arrange and length direction perpendicular to the chromatographic film length direction；
It is provided with well and peep hole on the shell；It is described to add when accommodating the test strips in the shell
Sample hole is located at the top of the sample pad, and the peep hole is located at the top of the chromatographic film, and passes through the peep hole energy
Enough observe the detection zone and the quality control region；
The fluorescent reagent micropore includes the time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies and for accommodating
State the resealable container of the time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies.
In above-mentioned quick detection kit, the bottom plate is made of polyvinyl chloride (PVC) or polystyrene (PS).
In above-mentioned quick detection kit, the sample pad is by glass fibre, polyester fiber, absorbent filter or hemofiltration film
It is made.
In above-mentioned quick detection kit, the base material of the chromatographic film is made of nitrocellulose (NC) film.It is existing
NC films include mainly Sartorius NC140, NC95, Millipore 135,180, PALL vivid170, WHATMAN
FF125, Prima 40,60,85 etc..
In above-mentioned quick detection kit, the water absorption pad is made of blotting paper.The thickness of blotting paper is 0.4~2mm,
Grammes per square metre is 200~500g/m2。
In above-mentioned quick detection kit, the MQCA- carrier protein couplets object is by MQCA, ethylenediamine and carrier protein
It is made through coupling reaction, the carrier protein is bovine serum albumin(BSA) (BSA), ovalbumin (OVA), keyhole limpet hemocyanin
(KLH) or poly-D-lysine (PLL).
In above-mentioned quick detection kit, the shell is by polyvinyl chloride (PVC), polystyrene (PS), makrolon
(PC), polypropylene (PP) or polyethylene (PE) are made.
In above-mentioned quick detection kit, the MQCA monoclonal antibodies with MQCA- carrier protein couplet objects by being made
Mouse is immunized for immunogene and is obtained.
In above-mentioned quick detection kit, the time-resolved fluorescence microballoon of the MQCA labeling of monoclonal antibodies is surface
It is coated with the time-resolved fluorescence microballoon of MQCA monoclonal antibodies；The time-resolved fluorescence microballoon is that commercially available internal package contains
Europium (Eu3+) complex, outside modification carboxyl (- COOH) functional group polystyrene microsphere；The time-resolved fluorescence microballoon is logical
The carboxyl for crossing outside modification is combined with the amino in MQCA monoclonal antibodies to complete the coating process of monoclonal antibody；The time resolution
A diameter of 100~300nm of fluorescent microsphere；The excitation wavelength of the time-resolved fluorescence microballoon is 365nm, and launch wavelength is
610nm。
In above-mentioned quick detection kit, the resealable container is the enzyme mark being sealed using masking foil adhesive sticker
Plate, preferably removable ELISA Plate (the removable existing light transmission of ELISA Plate, also have lighttight, and lighttight removable ELISA Plate was both
Have white, also there is black), the more preferable lighttight removable ELISA Plate of black, the most preferably lighttight 96 hole (8* of black
12) removable ELISA Plate.
On the other hand, the present invention provides the preparation methods of above-mentioned quick detection kit comprising the following steps：
1) preparation of MQCA- carrier protein couplets object：By coupling reaction, using ethylenediamine by MQCA and carrier protein into
Row coupling, obtains MQCA- carrier protein couplet objects；
2) preparation of MQCA monoclonal antibodies：Using the MQCA- carrier protein couplets object that is obtained in step 1) as immunogene
Mouse, acquisition MQCA monoclonal antibodies is immunized；
3) preparation of the time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies：Pass through the amino on monoclonal antibody surface
The MQCA monoclonal antibodies obtained in step 2) are coated on time resolution by the amidation process between the carboxyl of microsphere surface
On fluorescent microsphere, the time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies is obtained；
4) assembly of fluorescent reagent micropore：By the time-resolved fluorescence of the MQCA labeling of monoclonal antibodies obtained in step 3)
Microballoon diluted is transferred in resealable container and carries out vacuum freeze drying, then seals container, completes fluorescence examination
The assembly of agent micropore；
5) preparation of chromatographic film；The MQCA- carrier protein couplet objects obtained in step 1) are coated on detection zone, and will
Sheep anti mouse secondary antibody is coated on quality control region, obtains chromatographic film；
6) assembly of detection card：The chromatographic film obtained in sample pad, step 5), water absorption pad are sequentially fixed on bottom plate,
Test strips are obtained, then test strips are cut into scheduled size, are fitted into shell, the assembly of detection card is completed.
In another aspect, the present invention provides above-mentioned quick detection kits in detection animal tissue sample in MQCA residuals
Application, particularly provide it is a kind of using above-mentioned quick detection kit detect the remaining methods of MQCA comprising the following steps：
1) sample pre-treatments：
2) it is detected using the quick detection kit；
3) fluoroscopic examination card readout instrument testing result is used.
Compared with prior art, there are following advantages using the present invention of above-mentioned technical proposal：
(1) whether detection method of the invention is convenient and efficient, can within a short period of time detect in animal tissue's sample and contain
There are MQCA residuals, does not need the equipment and instrument of the costliness such as microplate reader, be suitable for popularization and application；
(2) compared with traditional colloidal gold immunity chromatography, Timed resolved fluoroimmunoassay has higher sensitive
Degree；
(3) the detection limit of detection method of the invention can reach 0.2 μ g/kg, maintain an equal level or higher than ELISA method, Er Qiejian
It is shorter to survey the time.
Description of the drawings
Fig. 1 is the sectional view of detection card along its length in the quick detection kit of the present invention.
Fig. 2 is the vertical view of the detection card in the quick detection kit of the present invention.
Fig. 3 is the schematic diagram of the fluorescent reagent micropore in the quick detection kit of the present invention.
Fig. 4 is the SDS-PAGE schematic diagrames of BSA and MQCA-BSA, wherein left side sample is BSA, right side sample is MQCA-
BSA。
Specific implementation mode
Technical scheme of the present invention is made below in conjunction with attached drawing and specific embodiment further elucidated above.Unless another
Other than being described, reagent, material, instrument used in the following example etc. can be obtained by routine business means.
Embodiment one：The composition of quick detection kit.
The remaining quick detection kits of MQCA of the present invention include detection card 1 and fluorescent reagent micropore 2.
As depicted in figs. 1 and 2, detection card 1 includes shell 11, bottom plate 12, sample pad 13, chromatographic film 14 and water absorption pad 15.
Sample pad 13, chromatographic film 14 and water absorption pad 15, beginning and the bottom plate 12 of sample pad 13 are fixed on bottom plate 12 successively
Beginning alignment, the end of sample pad 13 is connected with the beginning of chromatographic film 14, the beginning of the end and water absorption pad 15 of chromatographic film 14
It is connected, the end of water absorption pad 15 is aligned with the end of bottom plate 12.
Detection zone 141 and quality control region 142 are provided in chromatographic film 14, detection zone 141 is located adjacent to the side of sample pad 13
And it is coated with MQCA- carrier protein couplets object (preferably MQCA- bovine serum albumin(BSA)s conjugate), quality control region 142 is located remotely from
The side of sample pad 13 and be coated with sheep anti mouse secondary antibody, both for ribbon, arranged in parallel and length direction perpendicular to
The length direction of test strips.
Well 111 and peep hole 112 are provided on shell 11, well 111 is located at the top of sample pad 13, peep hole
112 are located at the top of chromatographic film 14, and are able to observe that detection zone 141 and quality control region 142 by peep hole 112.
As shown in figure 3, fluorescent reagent micropore 2 includes the time-resolved fluorescence microballoon 21 of MQCA labeling of monoclonal antibodies and uses
(preferably use the black that is sealed of masking foil adhesive sticker impermeable in the resealable container 22 for the fluorescent microsphere for accommodating antibody label
The removable ELISA Plate in 96 holes of light).
Embodiment two：The preparation of quick detection kit.
1, the preparation of MQCA- bovine serum albumin(BSA)s conjugate (MQCA-BSA)：
MQCA (1.65mg, CAS are weighed respectively：74003-63-7), ethylenediamine (0.9 μ L) and BSA (20mg), magnetic agitation
It is completely dissolved in the 0.05M MES buffer solutions (4mL) of pH=4.5,2% is then added dropwise under the conditions of magnetic agitation
EDCHCl aqueous solutions (0.5mL), room temperature, which is protected from light, to be stirred overnight.The solution after reaction is collected, it is heavy that 6000rpm centrifuges 5min removals
It forms sediment, supernatant is fitted into the bag filter handled well, dialyses in 0.01M PBS, is changed the liquid once per 8h, changes liquid in total 6 times.It collects saturating
Liquid in bag is analysed, it is dense to measure albumen using commercialization BCA determination of protein concentration kit (BCA Protein Assay Kit)
Degree, in -20 DEG C of preservations after packing.
The identification of MQCA-BSA is carried out using SDS-PAGE, separation gel 5%, separation gel 12%, the results are shown in Figure 4.
As shown in Figure 4, the molecular weight of MQCA-BSA bands ratio BSA bands is increased, it was demonstrated that MQCA-BSA is successfully prepared.
2, the preparation of MQCA monoclonal antibodies：
Balb/C female mices are immunized as immunogene in comlete antigen.It is using sterile saline that comlete antigen is dilute
It releases to 1mg/mL, takes appropriate dilution, mixed with isometric tachysynthesis adjuvant, after shaken well, according to the agent of 10 μ g/ only
Amount carries out muscle single-point injection.After three weeks, it carries out second according to same dosage and mode to be immunized, and right in immune latter seven days
Mouse carries out tail portion blood sampling and detects potency.
By the mouse tail blood system of acquisition from serum after, the potency of serum is measured using indirect ELISA and indirect competitive ELISA
And inhibition.The mouse that selection potency is high, has inhibited carries out cell fusion.
Mouse impact in 18 days after last time is immune is immune, removes spleen by sterile working after three days, prepares spleen list
Cell suspension, according to 10:1 ratio is mixed with the murine myeloma cell SP2/0 cells that logarithmic phase is grown.Centrifugation removal
Cell is shaken after dissipating, cell fusion is carried out by polyethylene glycol method by supernatant fluid.The 3rd day and the 5th day after fusion, carry out respectively
50%, 90%HAT culture solutions change liquid, the screening for carrying out cell conditioned medium in the 7th~9 day.Positive cell conditioned medium is selected, is utilized
ELISA, which is measured, inhibits situation, and selects to inhibit best cell hole to be subcloned.
Subclone uses limiting dilution assay, per 0.5~2, hole cell, is subcloned in triplicate.Subclone uses for the first time
HT culture solutions, after use 1640 culture medium twice, serum content is 15% in all culture solutions, finally obtains required Dan Ke
Grand antibody cell strain.After obtained cell strain is expanded culture, conservation is frozen, remaining cell continues to cultivate, and passes through internal ascites
Method prepares monoclonal antibody, is purified using sad ammonium sulfate method or Protein G method.
3, the preparation of the time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies：
(1) selection of time-resolved fluorescence microballoon：
The time-resolved fluorescence microballoon of label is internal polyphenyl of the package containing europium complex, outside modification carboxyl functional group
Ethylene microballoon, microsphere diameter are 100~300nm, excitation wavelength 365nm, launch wavelength 610nm.
(2) preparation of related solution：
Activation buffer：2- (N- morpholines) ethanesulfonic acid buffer (MES) of the 0.01M of pH=4.5.
Coupling buffer：The phosphate buffer (PBS) of the 0.01M of pH=9.0.
Block buffer：The phosphate buffer (PBS) (BSA for including 100mg/mL) of the 0.01M of pH=7.4.
Storage buffer solution：The phosphate buffer (PBS) of the 0.01M of pH=7.4 (includes BSA and the anticorrosion of 0.2%wt
Agent (such as Proclin300, thimerosal, sodium azide etc.)).
Activator：Water-soluble carbodiimide (EDAC)/n-hydroxysuccinimide (NHS).
Cleaning solution：The phosphate buffer (Tween-20 for including 0.2%wt) of the 0.01M of pH=7.4.
(3) it is prepared by the amidation process between the EDAC and NHS microsphere surface carboxyls mediated and protein surface amino
The time-resolved fluorescence microballoon of MQCA labeling of monoclonal antibodies, specific preparation process are as follows：
Take the ultrapure aqueous suspensions (solid content 1%wt, 10mg) of 1mL time-resolved fluorescence microballoons, 12000rpm centrifugations
5min is discarded supernatant, and is cleaned 3 times, is finally suspended in 1.8mL activation buffers using ultra-pure water, and microsphere suspension is formed；Make
Activator solution is prepared with activation buffer, the wherein mass concentration of NHS is 20mg/mL, and the mass concentration of EDAC is 10mg/
mL；It respectively takes 100 μ L activator solutions to be added drop-wise in microsphere suspension, vibrates mixing, it is (living in 37 DEG C of (or room temperature) revolving reactions
Change) 4h, 12000rpm centrifuge 5min, discard supernatant, using ultra-pure water clean 3 times, be finally suspended in 0.8mL coupling buffers
In, vibrate mixing；100 μ LMQCA monoclonal antibody solutions (a concentration of 1.5mg/mL of albumen) are added, mixing are vibrated, in 37 DEG C
100 μ L Block buffers are added in (or room temperature) revolving reaction (coupling) 4h, overnight in 37 DEG C (or room temperature) rotation (closing),
12000rpm centrifuges 5min, discards supernatant, and is cleaned 3 times using cleaning solution, obtains the time resolution of MQCA labeling of monoclonal antibodies
Fluorescent microsphere.
(4) identification of fluorescent microsphere：The solution of the 0.01MPBS of the pH=7.4 containing MQCA-BSA, BSA is respectively configured,
Above-mentioned MQCA-BSA, BSA solution is coated on the detection zone (T lines) on nitrocellulose filter, package amount 1.0 using film instrument is drawn
μL/cm；Sheep anti mouse secondary antibody (being purchased from Jackson ImmunoResearch) is diluted to using the 0.01M PBS of pH=7.4
Above-mentioned secondary antibody diluent is coated on the quality control region (C lines) on nitrocellulose filter by 0.2mg/mL using film instrument is drawn, and package amount is
1.0μL/cm.By the nitrocellulose filter after coating in 37 DEG C of dry 12h to get test chromatographic film.By sample pad (glass
Cotton), chromatographic film, water absorption pad (blotting paper) be pasted onto successively on PVC bottom plates, the beginning of sample pad is aligned with the beginning of bottom plate, sample
The end of product pad is connected with the beginning of chromatographic film, and the end of chromatographic film is connected with the beginning of water absorption pad, the end and bottom of water absorption pad
The end of plate is aligned, and the detection zone in chromatographic film is located adjacent to one end of sample pad, and quality control region is located remotely from sample pad
One end.The test strips being consequently formed are cut into the small item that width is 4mm with strip cutting machine, be fitted into plastics get stuck it is middle compacting to get
Detection card.
By the fluorescent microsphere of preparation, 5 μ L are taken to be added in empty micropore respectively, the 0.01M PBS for adding pH=7.4 are molten
Liquid (150 μ L) is sucked out after mixing, is added in the detection card of coating MQCA-BSA, BSA respectively, ultraviolet lamp is used after 5min
(365nm) irradiation observation.It is coated with the detection card of MQCA-BSA, C/T lines have red fluorescence, are coated with the detection card of BSA, only C
Line has red fluorescence, T lines not to have.It is possible thereby to prove, fluorescent microsphere marks successfully.
4, the assembly of fluorescent reagent micropore：
(1) preparation of dilution：
By potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KCl), poly- second
Glycol -6000 (PEG-6000), sucrose and Tween-20 (Tween-20) are dissolved in ultra-pure water, obtain dilution, wherein：Phosphorus
The mass concentration of acid dihydride potassium is 0.27g/L, and the mass concentration of disodium hydrogen phosphate is 1.42g/L, and the mass concentration of sodium chloride is
The mass concentration of 8.0g/L, potassium chloride are 2.0g/L, and the mass concentration of polyethylene glycol-6000 is 25g/L, the mass concentration of sucrose
Volumetric concentration for 50g/L, Tween-20 is 10mL/L.
(2) antibody label fluorescent microsphere dilution, be lyophilized and seal up for safekeeping：
After the fluorescent microsphere that monoclonal antibody marks is diluted 100 times respectively with dilution, it is added to using eight channel pipettors black
In the micropore of the lighttight removable ELISA Plate in 96 hole of color, per 50 μ L of hole.The removable ELISA Plate that microballoon dilution has been added is placed in
In freeze dryer, and according to the freeze-drying program vacuum freeze-drying in table 1, in dry conditions utilize masking foil adhesive sticker seal to get
Fluorescent reagent micropore.
The freeze-drying program of 1. microballoon dilution of table
5, the preparation of chromatographic film：
MQCA- bovine serum albumin(BSA)s conjugate (MQCA-BSA) is diluted to by 0.3mg/ using the 0.01MPBS of pH=7.4
Above-mentioned MQCA-BSA dilutions are coated on the detection zone (T lines) on nitrocellulose filter by mL using film instrument is drawn, and package amount is
1.0μL/cm；Sheep anti mouse secondary antibody is diluted to by 0.2mg/mL using the 0.01MPBS of pH=7.4, using drawing film instrument by above-mentioned secondary antibody
Dilution is coated on the quality control region (C lines) on nitrocellulose filter, and package amount is 1.0 μ L/cm.By the nitrocellulose after coating
Film is in 37 DEG C of dry 12h to get chromatographic film.
6, the assembly of detection card：
Sample pad (mineral wool), chromatographic film, water absorption pad (blotting paper) are pasted onto successively on PVC bottom plates, the beginning of sample pad
End is aligned with the beginning of bottom plate, and the end of sample pad is connected with the beginning of chromatographic film, the end of chromatographic film and the beginning of water absorption pad
It is connected, the end of water absorption pad is aligned with the end of bottom plate, and the detection zone in chromatographic film is located adjacent to one end of sample pad, matter
Control area is located remotely from one end of sample pad.The test strips being consequently formed are cut into the small item that width is 4mm with strip cutting machine, are packed into
Plastics get stuck middle compacting to get detection card.
Embodiment three：The application of quick detection kit.
Animal tissue's sample to be measured is gone to allowance for bark, fat, takes lean meat part homogenizer homogeneous, weigh 2g and be placed in
In 50mL centrifuge tubes, 5mL ethyl acetate and 1mL deionized waters is added, vibrates 5min with oscillator, adds 0.5mL sulfuric acid
(2mol/L), vibrates 30s, and 4000rpm centrifuges 5min；It draws 2mL supernatants to be placed in 7mL centrifuge tubes, be blown in 60 DEG C of nitrogen
It is dry, 2mL n-hexanes are added and 0.5mL redissolves liquid (the 0.1M PBS of the Tween-20 containing 0.5%v/v), acutely vibrate 30s,
4000rpm centrifuges 1min or standing, removes upper layer n-hexane phase, takes 0.15mL lower liquids as prepare liquid.
Prepare liquid is added in the micropore of 96 hole elisa Plates of fluorescent reagent micropore, it will be to be measured with dropper after waiting 1min
The fluorescent microsphere that liquid is marked with monoclonal antibody in micropore mixes well, then is all added dropwise to the liquid in micropore with dropper after waiting 2min
In the well for detecting card, then start timing, after reacting at room temperature 5min, is inserted into hand-held fluorescence detection card readout instrument and sentences
It reads.
Testing result is weighed with the fluorescence intensity ratio of detection line T and nature controlling line C.It is dense that different mark-ons are measured respectively
The ratio of degree, each concentration 20 parts of samples of each measurement, spiked levels and C/T are as shown in table 2.
Table 2. detects the fluoroscopic examination result of animal tissue's sample using quick detection kit
| Spiked levels (μ g/kg) | C/T mean values | CV |
| 0 | 0.50 | 0.6% |
| 0.05 | 0.61 | 1.6% |
| 0.1 | 0.75 | 1.2% |
| 0.2 | 0.98 | 0.8% |
| 0.3 | 1.18 | 0.8% |
| 0.5 | 1.35 | 0.7% |
From Table 2, it can be seen that with the raising of spiked levels, C/T is in gradually rise trend.In view of instrument detection side
The sensitivity factor of method and actual demand, definition detection are limited to 0.2 μ g/kg, and detection limits corresponding 95% credibility intervals C/T and is
0.75±0.017.Fluorescent reader interpretation threshold value is set as 0.70, can distinguish the spiked levels of 0.1 and 0.2 μ g/kg.
Select 20 parts of negative findings and 20 parts of positives (instrument detection level is higher than 0.2 μ g/kg) as a result, using fluoroscopic examination
Card is tested, and test result is shown：Negative sample 100% meets, and positive sample 100 meets, and false positive rate, false negative rate are
0。
Claims (10)
1. a kind of quick detection kit that 3- Jia based quinoxalines -2- is carboxylic acid remained comprising detection card and fluorescent reagent micropore,
Wherein：
Detection card includes test strips and the shell for accommodating the test strips, the test strips include bottom plate, sample pad,
Chromatographic film and water absorption pad, wherein：
The sample pad, chromatographic film and water absorption pad, the beginning of the sample pad and the bottom plate are fixed on the bottom plate successively
Beginning alignment, the end of the sample pad is connected with the beginning of the chromatographic film, the end of the chromatographic film and the water suction
The beginning of pad is connected, and the end of the water absorption pad is aligned with the end of the bottom plate；
The detection for being coated with 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object is provided on the base material of the chromatographic film
Area and quality control region for being coated with sheep anti mouse secondary antibody, the detection zone are located adjacent to the side of the sample pad, the quality control region
It is located remotely from the side of the sample pad；
It is provided with well and peep hole on the shell；When accommodating the test strips in the shell, the well
Positioned at the top of the sample pad, the peep hole is located at the top of the chromatographic film, and can be seen by the peep hole
Observe the detection zone and the quality control region；
The fluorescent reagent micropore include 3- Jia based quinoxaline -2- carboxylic acid labeling of monoclonal antibodies time-resolved fluorescence microballoon and
Resealable container for the time-resolved fluorescence microballoon for accommodating the 3- Jia based quinoxalines -2- carboxylic acid labeling of monoclonal antibodies.
2. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 1, it is characterised in that：
The shell is made of polyvinyl chloride, polystyrene, makrolon, polypropylene or polyethylene；
The bottom plate is made of polyvinyl chloride or polystyrene；
The sample pad is made of glass fibre, polyester fiber, absorbent filter or hemofiltration film；
The base material of the chromatographic film is made of nitrocellulose filter；
The water absorption pad is made of blotting paper.
3. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 1, it is characterised in that：
The 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object is by 3- Jia based quinoxaline -2- carboxylic acids, ethylenediamine and carrier
Albumen is made through coupling reaction.
4. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 3, it is characterised in that：
The carrier protein is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin or poly-D-lysine.
5. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 1, it is characterised in that：
The 3- Jia based quinoxalines -2- carboxylic acids monoclonal antibody passes through with 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object
Mouse is immunized as immunogene and is obtained.
6. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 1, it is characterised in that：
The time-resolved fluorescence microballoon is that internal polystyrene of the package containing europium complex, outside modification carboxyl functional group is micro-
Ball.
7. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 6, it is characterised in that：
A diameter of 100~300nm of the time-resolved fluorescence microballoon, excitation wavelength 365nm, launch wavelength 610nm.
8. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to claim 1, it is characterised in that：
The resealable container is the ELISA Plate being sealed using masking foil adhesive sticker.
9. a kind of quick detection reagent that 3- Jia based quinoxalines -2- according to any one of claim 1 to 8 is carboxylic acid remained
The preparation method of box comprising the following steps：
1) preparation of 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object：By coupling reaction, using ethylenediamine by 3- first
Based quinoxaline -2- carboxylic acids are coupled with carrier protein, obtain 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object；
2) preparation of 3- Jia based quinoxalines -2- carboxylic acid monoclonal antibodies：With the 3- Jia based quinoxaline -2- carboxylics obtained in step 1)
Mouse is immunized as immunogene in acid-carrier protein couplet object, obtains 3- Jia based quinoxaline -2- carboxylic acid monoclonal antibodies；
3) preparation of the time-resolved fluorescence microballoon of 3- Jia based quinoxalines -2- carboxylic acid labeling of monoclonal antibodies：It is anti-by monoclonal
Amidation process between the amino in body surface face and the carboxyl of microsphere surface, the 3- Jia based quinoxalines -2- that will be obtained in step 2)
Carboxylic acid monoclonal antibody is coated on time-resolved fluorescence microballoon, obtains 3- Jia based quinoxaline -2- carboxylic acid labeling of monoclonal antibodies
Time-resolved fluorescence microballoon；
4) assembly of fluorescent reagent micropore：By the 3- Jia based quinoxaline -2- carboxylic acid labeling of monoclonal antibodies obtained in step 3)
Time-resolved fluorescence microballoon diluted, is transferred in resealable container and carries out vacuum freeze drying, then that container is close
Envelope completes the assembly of fluorescent reagent micropore；
5) preparation of chromatographic film；The 3- Jia based quinoxalines -2- carboxylic acids-carrier protein couplet object obtained in step 1) is coated on inspection
Area is surveyed, and sheep anti mouse secondary antibody is coated on quality control region, obtains chromatographic film；
6) assembly of detection card：The chromatographic film obtained in sample pad, step 5), water absorption pad are sequentially fixed on bottom plate, obtained
Test strips, then test strips are cut into scheduled size, it is fitted into shell, completes the assembly of detection card.
10. quick detection kit carboxylic acid remained 3- Jia based quinoxalines -2- according to any one of claim 1 to 8
Application during 3- Jia based quinoxalines -2- is carboxylic acid remained in detection animal tissue sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710079789.8A CN108426996B (en) | 2017-02-15 | 2017-02-15 | Rapid detection kit for 3-methyl quinoxaline-2-carboxylic acid residues and preparation method and application thereof |
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| CN201710079789.8A CN108426996B (en) | 2017-02-15 | 2017-02-15 | Rapid detection kit for 3-methyl quinoxaline-2-carboxylic acid residues and preparation method and application thereof |
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Cited By (2)
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| CN110927375A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
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