CN107478643A - A kind of chemiluminescence detection kit of sulfaquinoxaline and preparation method thereof - Google Patents
A kind of chemiluminescence detection kit of sulfaquinoxaline and preparation method thereof Download PDFInfo
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- CN107478643A CN107478643A CN201710720629.7A CN201710720629A CN107478643A CN 107478643 A CN107478643 A CN 107478643A CN 201710720629 A CN201710720629 A CN 201710720629A CN 107478643 A CN107478643 A CN 107478643A
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- G01—MEASURING; TESTING
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention discloses a kind of chemiluminescence detection kit of sulfaquinoxaline and preparation method thereof.The kit includes：Coupling has magnetic particle, acridinium ester label, sulfaquinoxaline serial standards, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B, the cleaning fluid of sulfaquinoxaline antigen or antibody.Compared with the technology of existing detection sulfaquinoxaline, this kit has a high sensitivity, and signal to noise ratio is high, the advantages of quick detection.Its sensitivity for analysis is up to 0.025 μ g/L.
Description
Technical field
The present invention relates to technical field of food detection, the chemiluminescence detection examination of sulfaquinoxaline in specifically a kind of food
Agent box and preparation method thereof.
Background technology
Sulfaquinoxaline, belong to disulfonamide thing, its structure is：
Sulfaquinoxaline belongs to disulfonamide thing, is the special sulfa drug of anticoccidial, is widely used in coccidiosis of livestock and poultry.By
It is similar to aminobenzoic acid (PABA) in the basic structure of sulfonamides, thus dihydrofolate synthetase can be fought for mutually, and shadow
Dihydrofoilic acid is rung to be formed, it is final to influence nucleoprotein synthesis, so as to suppress the growth and breeding of bacterium and coccidia.Sulfaquinoxaline is oral
After can absorb rapidly, but drain more slow, cause its residence time in histoorgan and egg very long, can after people is edible
Various bacteria is caused to produce drug resistance to it, thereby increases and it is possible to have potential carcinogenicity.In addition, the cost of the compound of the type compared with
Low, cheap, unreasonable serious using phenomenon in husbandry sector, therefore, it is remained in animal food causes the mankind
The potential threat of health hazard by various countries' common concern, English, U.S. etc. country to sulfa drugs in meat and dairy products
Maximum permission quantity be limited to 100 μ g/kg；Provided in the command of European Union the 675/92nd, the disulfonamide in meat and milk
The maximum residue limit of thing must not exceed 100 μ g/kg；The residual limit amount of highest that Japan requires is more strict, single sulfa drugs
Residual limit amount is 20 μ g/kg, and maximum residual total amount must not exceed 100 μ g/kg；And China to sulfa drugs in meat and
Maximum permission quantity in dairy products is limited to 100 μ g/kg.
At present, detecting the method for sulfaquinoxaline has high performance liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry, quantum dot
Fluorescent immune method, enzyme linked immunosorbent assay, colloidal gold method etc..Wherein, liquid phase chromatographic analysis method lacks highly sensitive detector, instrument
Device is expensive, cumbersome, and complex pretreatment, time-consuming.Enzyme-linked immunoassay method, using HRPO or alkaline phosphatase
Label, its enzyme marker easy in inactivation, chromogenic substrate are shown in that light easily decomposes, and sensitivity is low, and the compound similar to structure has one
Fixed cross reaction, cause test result inaccurate.
CN 106501200A（2017.03）A kind of kit for detecting seven kinds of sulfa drugs is disclosed, the kit is adopted
With ELIAS secondary antibody, the colour developing of TEM substrates, its shortcoming be mainly separated in detection process educt with during conjugate, it is necessary to which multistep is washed
Plate, take time and effort；And it mainly develops the color by enzymatic TEM substrates, reaction time, acid-base value, thimerosal etc. all can be right
There is considerable influence in enzyme activity, cause reagent stability poor.CN 101144819A（2008.03）Sulphur is detected using colloidal gold method
The residual quantity of amine quinoxaline, the last testing result of its kit are characterized with macroscopic color, and error is larger, and
Operate cumbersome, flow is more, is more easy to error occur, and its sensitivity is low.
CN 101936984 A（2011.01）Disclose a kind of quantum dot fluorescence immunoreagent for detecting sulfonamides compound
Box, the stabilization of kit is by environmental condition（PH, ionic strength, temperature etc.）, especially pH's has a great influence, and causes test to be tied
Fruit is inaccurate, and it has the shortcomings that cost is high, the cycle is long.A kind of and sulfaquinoxaline chemiluminescence examination of our company's exploitation
Agent box, using acridinium ester as label, there is the advantages of detection is stable, measurement is quick, high sensitivity.
The simplicity that chemiluminescence method is to detect compared with the advantages of other method.
The advantages of system uses Magneto separate system, and the chemiluminescence of acridinium ester label is detected be：Acridinium ester
The presence of catalyst is not required to as luminous agent, can be lighted in the dilute alkaline soln for have hydrogen peroxide；Acridinium ester molecular weight is small, keeps away
Exempt to cover antibody combining site, system overall sensitivity can be improved, be swift in response；Background is low, signal to noise ratio is high, is a kind of effective
Chemiluminescent labels.
The content of the invention
It is an object of the invention to provide one kind is stable, detection speed is fast, has higher sensitivity and specific immune
Magnetic microparticle chemiluminescence detection method, facility is provided to detect sulfaquinoxaline in food.
To achieve the above object, the present invention adopts the following technical scheme that：
A certain amount of sample to be tested is added in reaction cup, magnetic particle coupling suspension and acridinium ester label is then added, mixes
It is even, it is incubated several minutes in 37 DEG C, is finally separating, washs, add chemiluminescence preexciting liquid A and exciting liquid B, determines corresponding hair
Luminous intensity, sulfaquinoxaline examination criteria curve is compareed, obtain the concentration for the sulfaquinoxaline treated in test sample.
Chemiluminescent labels in the present invention are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
Acridinium ester label in the present invention includes：Acridinium ester label is sulfaquinoxaline antigen or antibody, wherein a word used for translation
The concentration range of pyridine ester solution is 2-85 mmol/L, and its preferred concentration is 6.5 mmol/L.
Buffer solution is pH 4.5-6.5 in the present invention, and concentration is 0.1 mol/L, and the MES by 0.22 μm of filter filtering delays
Fliud flushing.
Heretofore described sulfaquinoxaline series of calibration product concentration is respectively：0 μg/L、0.02 μg/L、0.1 μg/
L, 0.5 μ g/L, 2.5 μ g/L, 12.5 μ g/L, its buffer solution are containing 0.5-5.0% BSA and 0.1-0.5% PC300
Tris-HCl。
Chemiluminescence preexciting liquid A in the present invention is：H2O2And HNO3Mixed liquor, wherein H2O2Mass fraction be
0.05-5% mol/L, HNO3Concentration is 0.05-2.5 mol/L.
Chemiluminescence exciting liquid B in the present invention is：Triton X-100 and NaOH mixed liquor, wherein Triton X-
100 concentration is that 0.05-2.0 mol/L, NaOH concentration are 0.05-1.0 mol/L.
Cleaning fluid in the present invention is：PH 7.0-9.0, the Tris-HCl solution that concentration is 5.0-50.0 mmol/L, its
In containing concentration be 0.05-0.50 mol/L NaCl and 0.01-0.25% Tween-20.
Compared with prior art, the advantage of the invention is that：The stabilization of kit of the present invention, detection are fast, high sensitivity and
Specificity is good.
Embodiment
The technical scheme in the present invention will clearly and detailedly be illustrated below.Experimental method is such as without special in example
Illustrate, be conventional method.
Embodiment 1：The establishment of kit 1 and its preparation of concrete component
1. the establishment of kit
A kind of chemiluminescence detection kit of sulfaquinoxaline, makes it contain following component：
The sulfaquinoxaline antigen of acridinium ester label；
Carboxyl magnetic bead is coupled sulfaquinoxaline antibody；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Sulfaquinoxaline serial standards solution, standard concentration are respectively：0 μg/L、0.02 μg/L、0.1 μg/L、0.5 μ
G/L, 2.5 μ g/L, 12.5 μ g/L, its buffer solution are the Tris-HCl containing 0.5-5.0% BSA and 0.1-0.5% PC300；
Cleaning fluid, specially pH 7.2, the Tris-HCl solution that concentration is 25 mmol/L, wherein being 0.15 mol/L containing concentration
NaCl and 0.05% Tween-20.
2. the preparation of magnetic bead coupled antibody
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds a certain amount of 0.1 mol/L MES buffer solutions, is vortexed mixed
It is even, it is placed on magnetic frame, standing 5 min makes magnetic particle be separated with liquid, abandoning supernatant, washs 3 times, adds a certain amount of
MES（PH value is 6.0）Buffer solution, it is vortexed.
（2）Add 15 μ L（15 μg）Sulfaquinoxaline antibody, it is vortexed, revolving reaction pipe, is incubated at room temperature 30 min.
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL to be vortexed, revolving reaction pipe, be incubated at room temperature 2 h.
（4）Supernatant is removed, adds a certain amount of cleaning buffer solution（TBS+0.05%Tween-20）, wash 3 times.
（5）Closed with the buffer solution containing 1%BSA, repeatedly closing 4 times, every time 10 min.By the magnetic particle suspension
It is placed in 2-8 DEG C of preservation.
3. the preparation of MES buffer solutions（0.1 mol/L）
19.52 g MES solids are taken, are dissolved in 500 mL deionized water, it is 6.0 to arrive pH with 1 mol/L NaOH regulations；
The solution mixed up is transferred in 1 L volumetric flask, constant volume.
4. coupling reagent EDC preparation（Concentration is 10 mg/mL）
1 g EDC is taken in 50 mL beaker, adds the MES buffer solutions of precooling, stirs, treats its solution transfer to 100 mL's
In volumetric flask, constant volume.
5. the preparation of TBS-T buffer solutions（Concentration is 25 mmol/L）
Weigh 3.05 g Tris, in 8.775 g NaCl to 500 mL beaker, add 1mL 0.05% Tween-20, magnetic
It is 7.2 to adjust pH with 6 mol/L HCl under conditions of power stirring, is moved in 1000 mL volumetric flask, constant volume.
6. the preparation of acridinium ester label antigen
（1）A certain amount of sulfaquinoxaline is placed in bag filter, and bag filter is placed in the mark buffer solution not less than 1 L
Dialysis, during which buffer solution is at least changed 3 times, last time dialysed overnight, and mark buffer solution is that pH 9.5, concentration are 0.1 mol/
L Na2CO3-NaHCO3Buffer solution.
（2）1.7 mg acridinium ester NSP-DMAE-NHS is weighed, is dissolved in 447 μ L anhydrous dimethyl formamides DMF, matches somebody with somebody
Into 6.5 mmol/L NSP-DMAE-NHS DMF solutions.
（3）Antigenic solution after dialysis is placed in 500 μ L centrifuge tubes, adds a certain amount of 6.5 mmol/L NSP-
The molar ratio of DMAE-NHS DMF solutions, acridinium ester and antigen is 10:1, add 200 μ L mark buffer solutions, room temperature reaction 45
Min, the μ L of 10 g/L lysines 100 are added, continue to react 15 min, make mark reaction terminating.
（4）Label NSP-DMAE-NHS-Ag and free NSP-DMAE-NHS passes through Sephadex G-50 posts（1×
25cm）Separation, the PBS for being 0.1mol/L with purification buffer pH 6.3, concentration are balanced and are eluted chromatographic column.
（5）Protein peak is detected with chromatograph in separation process, measures the chemiluminescence intensity and 280 nm of efflux respectively
Absorbance.
（6）The eluent that shading value is high and absorbance is big is collected, adds 1%BSA（Volume）After dispense stored frozen.
7. the preparation of elution buffer（The Na that pH is 9.5, concentration is 0.1mol/L2CO3-NaHCO3）
A liquid（0.1 mol/L, Na2CO3）：Take 10.62 g Na2CO3Add distilled water to 1 L,
B liquid（0.1 mol/L, NaHCO3）：Take 8.4 g NaHCO3Add distilled water to 1 L,
The mL of A liquid 3 mL, B liquid 7 is taken to mix, i.e. A liquid：B liquid=3:7 ratio mixing.
8. chemiluminescence exciting liquid A, B preparation
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, H2O2Mass fraction is 1.5%, HNO3Concentration is 0.1
Mol/L, 20 mL/ branch are distributed into brown bottle, 2-8 DEG C saves backup.
（2）Chemiluminescence exciting liquid B is made up of Triton X-100 and NaOH mixed liquor.Wherein, Triton X-100
Concentration is 0.1 mol/L, and NaOH concentration is 0.35 mol/L, is distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
Embodiment 2：The establishment of kit 2 and its preparation of component
1. the establishment of kit
A kind of chemiluminescence detection kit of sulfaquinoxaline, makes it contain following component：
The sulfaquinoxaline antibody of acridinium ester label；
Carboxyl magnetic bead is coupled sulfaquinoxaline antigen；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Sulfaquinoxaline serial standards solution, standard concentration are respectively：0 μg/L、0.02 μg/L、0.1 μg/L、0.5 μ
G/L, 2.5 μ g/L, 12.5 μ g/L, its buffer solution are the Tris-HCl containing 0.5-5.0% BSA and 0.1-0.5% PC300；
Cleaning fluid, specially pH 7.2, the Tris-HCl solution that concentration is 25 mmol/L, wherein being 0.15 mol/L containing concentration
NaCl and 0.05% Tween-20.
2. the preparation of magnetic bead coupled antigen
（1）1mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds a certain amount of 0.1 mol/L MES buffer solutions, is vortexed mixed
It is even, it is placed on magnetic frame, standing 5min makes magnetic particle be separated with liquid, abandoning supernatant, washs 3 times, adds a certain amount of MES
（PH value is 5.0）Buffer solution, it is vortexed.
（2）Add 18 μ L（18 μg）Sulfaquinoxaline antigen, be vortexed, revolving reaction pipe, be incubated at room temperature 30 min.
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL to be vortexed, revolving reaction pipe, be incubated at room temperature 2 h.
（4）Supernatant is removed, adds 200 μ L cleaning buffer solution（TBS+0.05% Tween-20）, wash 3 times.
（5）Closed with the buffer solution containing 1%BSA, repeatedly closing 4 times, every time 10 min.By the magnetic particle suspension
It is placed in 2-8 DEG C of preservation.
3. the preparation of acridinium ester label antibody
（1）A certain amount of sulfaquinoxaline antibody is placed in bag filter, and the mark that bag filter is placed in not less than 1 L buffers
Dialysed in liquid, during which buffer solution is at least changed 3 times, last time dialysed overnight, mark buffer solution be pH value be 10.1, concentration be
0.1 mol/L Na2CO3-NaHCO3Buffer solution.
（2）1.7 mg acridinium ester NSP-DMAE-NHS is weighed, is dissolved in 447 μ L anhydrous dimethyl formamides DMF, matches somebody with somebody
Into 6.5 mmol/L NSP-DMAE-NHS DMF solutions.
（3）Antibody-solutions after dialysis are placed in 500 μ L centrifuge tubes, add a certain amount of 6.5 mmol/L NSP-
The molar ratio of DMAE-NHS DMF solutions, acridinium ester and antibody is 7.4:1,200 μ L mark buffer solutions are added, at room temperature instead
45 min are answered, add the μ L of 10 g/L lysines 100, continues to react 15 min, makes mark reaction terminating.
（4）Label NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS passes through Sephadex G-50 posts（1×
25cm）Separation, balanced with the PBS that purification buffer pH values are 6.3, concentration is 0.1mol/L and elute chromatographic column.
（5）Protein peak is detected with chromatograph in separation process, the chemiluminescence intensity and 280nm for measuring efflux respectively are inhaled
Shading value.
（6）The eluent that shading value is high and absorbance is big is collected, adds 1%BSA（Volume）After dispense stored frozen.
Embodiment 3：The detection of kit
1. Sample pretreatment
（1）The acquisition of milk test sample solution：The μ L of fresh milk 150 are taken in 500 μ L centrifuge tubes, 4 DEG C of 10 min of centrifugation
（3000 r/min）, discard upper-layer fat.The μ L of milk sample 25 after centrifugation are pipetted in clean teat glass, add 950
μ L concentration is that 0.02 mol/L phosphate buffer is diluted.
（2）Take the g of honey 1 to add in 25 mL centrifuge tubes, add the mol/L hydrochloric acid solutions of 2 mL 0.5, vibrated with oscillator
All dissolve, warmed in 55-75 DEG C of hot water several minutes to honey, add the hydroxide that 5 mL concentration are 0.2 mol/L
Sodium, regulation pH value range are 4.0-6.0, and vibration mixes.8 mL ethyl acetate are added, after spinning upside down 5 min of vibration, centrifugation
10min（3000 r/min）.Take the mL of upper strata organic solvent 5 and in clean glass tube, dried up in air, 0.5 mL is added dropwise
Concentration is 0.02 mol/L phosphate buffers, is mixed, to be checked.
2. course of reaction
（1）By μ L of sample to be tested 100, μ L of coupling magnetic particle suspension 150, the μ L of acridinium ester label 150, sequentially add anti-
Ying Guanzhong, vibration mix, 37 DEG C of 15 min of incubation.
（2）Separation, washing 5 times.
（3）Reaction vessel after washing, which is fully vibrated, makes magnetic particle be uniformly dispersed.
（4）100 μ L chemiluminescence exciting liquid B are added after adding 100 μ L chemiluminescence preexciting liquid A, 1 s, measure it
Relative luminous intensity, the content of sulfaquinoxaline and its luminous intensity proportion relation in sample.
Embodiment 4：The performance indications of kit
（1）The sensitivity of kit
20 retests are carried out to zero standard solution, the average value for taking zero standard solution to determine adds 3 times of standard deviation, i.e.,
For the sensitivity of this kit.Sensitivity of this kit to sulfaquinoxaline is 0.025 μ g/L.
（2）The specificity of kit
The competition medicine similar to sulfaquinoxaline structure or function：Sulfamethazine, 5-methoxysulfadiazine, sulfanilamide (SN)
Pyrimidine.By kit step operation, it is separately added into：Sulfaquinoxaline, sulfamethazine, 5-methoxysulfadiazine, sulfanilamide (SN)
Pyrimidine, suppression curve is made, 50% inhibition concentration of each medicine is calculated according to linear equation.Cross reacting rate is antibody to sulphur
The IC of amine quinoxaline50IC with antibody to sulfaquinoxaline competitor50The ratio between percentage.As a result show：Kit is to sulfanilamide (SN)
Quinoxaline has higher specificity, pair with sulfaquinoxaline structure or it is intimate competition the equal no cross reaction of medicine.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (9)
1. the chemiluminescence detection kit and its composition of a kind of sulfaquinoxaline, it is characterised in that including：Coupling has sulfanilamide (SN) quinoline
Dislike the magnetic particle suspension of quinoline antibody, the sulfaquinoxaline antigen of acridinium ester label, sulfaquinoxaline serial standards, chemistry hair
Light preexciting liquid A, chemiluminescence exciting liquid B, cleaning fluid.
2. the chemiluminescence detection kit of a kind of sulfaquinoxaline according to claim 1, it is characterised in that described
Chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
A kind of 3. chemiluminescence detection kit of sulfaquinoxaline according to claim 1, it is characterised in that acridinium ester
Mark is sulfaquinoxaline antigen.
4. the chemiluminescence detection kit of a kind of sulfaquinoxaline according to claim 1, it is characterised in that described
Magnetic particle and Streptavidin or can be coupled, while use biotin mark by magnetic particle directly with sulfaquinoxaline antibody coupling
Remember sulfaquinoxaline antibody.
5. the chemiluminescence detection kit of a kind of sulfaquinoxaline according to claim 1, it is characterised in that described
Chemiluminescence preexciting liquid A is by H2O2And HNO3 Mixed liquor composition, chemiluminescence exciting liquid B is by Triton X-100 and NaOH
Mixed liquor composition.
6. the chemiluminescence detection kit of a kind of sulfaquinoxaline according to claim 1, it is characterised in that described
Calibration object is using the Tris-HCl buffer solutions containing 0.5-5.0% BSA and 0.1-0.5% PC300 as matrix, adds sulfanilamide (SN) quinoline and dislikes
The calibration object solution for the series concentration gradient that the configuration of quinoline sterling forms.
A kind of 7. chemiluminescence detection kit of sulfaquinoxaline, it is characterised in that including：Coupling has sulfaquinoxaline antigen
Magnetic particle suspension, acridinium ester label be sulfaquinoxaline antibody, sulfaquinoxaline serial standards, chemiluminescence are pre- swashs
Lotion A, chemiluminescence exciting liquid B, cleaning fluid.
A kind of 8. chemiluminescence detection kit of sulfaquinoxaline according to claim 7, it is characterised in that acridinium ester
Mark is sulfaquinoxaline antibody.
9. the chemiluminescence detection kit of a kind of sulfaquinoxaline according to claim 7, it is characterised in that described
Magnetic particle directly can be coupled with sulfaquinoxaline antigen, or magnetic particle and Streptavidin are coupled, while use biotin mark
Remember sulfaquinoxaline antigen.
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| CN102079788A (en) * | 2009-11-27 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
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