CN107490574A - A kind of chemiluminescence detection kit of melamine and preparation method thereof - Google Patents
A kind of chemiluminescence detection kit of melamine and preparation method thereof Download PDFInfo
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- CN107490574A CN107490574A CN201710683708.5A CN201710683708A CN107490574A CN 107490574 A CN107490574 A CN 107490574A CN 201710683708 A CN201710683708 A CN 201710683708A CN 107490574 A CN107490574 A CN 107490574A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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 melamine chemiluminescence detection kit and preparation method thereof.The kit includes：Acridinium ester label, coupling have magnetic particle, calibration object solution, cleaning fluid, chemiluminescence preexciting liquid A, the chemiluminescence exciting liquid B of antigen or antibody.Kit of the present invention is using Magneto separate chemiluminescence as detection means, in combination with acridinium ester label technology.Kit of the present invention is simple to operate, and reaction condition is gentle, and light value stabilization, is influenceed by external condition few；Compared with prior art, there is high sensitivity, detection is quick and convenient, accuracy is high, reproducible, high specificity.
Description
Technical field
The invention belongs to technical field of immune assay, is related to food safety detection technology, and in particular to a kind of trimerization
Chemiluminescence detection kit of cyanamide and preparation method thereof.
Background technology
Melamine (Melamine), is commonly called as melamine, extract of protein, and IUPAC is named as " 1,3,5-triazines -2,4,6-
Triamido ".Its structure is：
Relative molecular weight is 126.12, is a kind of triazines nitrogen heterocyclic ring organic compound, is white monoclinic crystal, tasteless, low
Poison.Melamine is a kind of widely used Organic Chemicals, and main purposes is condensed with aldehyde, produces plastics.Food work
In industry, generally use Kjeldahl nitrogen determination nitrogen content calculates protein content.Because the average nitrogen content of protein is about
16%, and the nitrogen content of melamine is up to 66%, it is therefore, often illegal by criminal far above the nitrogen content of usual protein
It is added in food and animal feed, to cause protein content illusion up to standard.And melamine has stronger stickiness, hold
The lithogenous oxalic acid of shape is easily adsorbed in vivo, and is deposited in urinary system.Long-term intake melamine can cause urinary system
Infringement, produce kidney, vesical calculus.Therefore, safety of the efficient detection for guarantee food especially dairy products is carried out to melamine
Tool is of great significance.
Method currently used for detecting melamine mainly has high performance liquid chromatography, gas chromatography-mass spectrometry, liquid
Phase chromatography-mass spectroscopy/mass spectrography, colloidal gold method, enzyme-linked immunosorbent assay, chemiluminescence immunoassay etc..Wherein, efficient liquid phase
Chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrography/mass spectrography need large-scale precision instrument, cumbersome time-consuming, detection
High cost, Finite Samples are detected, are not suitable with the detection of high-volume sample；The detection of collaurum method is with macroscopic face
Color is characterized, and it is larger error to be present, it is cumbersome, flow is more, sensitivity is low, precision is not high the shortcomings of；CN
102086230 A（2011.06）A kind of enzyme linked immunological kit for detecting melamine and its application, the kit is disclosed to adopt
Detected with enzyme-linked immunoassay method, enzyme-linked immunosorbent assay has that sensitivity is low, is not easy to realize full-automatic, detection time length
Deng the shortcomings that；CN 105181680 A（2015.12）Disclose a kind of Magneto separate chemiluminescence immunoassay detection side of melamine
Method, for this method using alkaline phosphatase as label, the shortcomings that being marked using alkaline phosphatase is enzymatic by environment shadow
Ring big：As thimerosal, acid-base value, ion can all impact to it, stability is weaker, so as to influence measurement result.
The present invention is detected using Magneto separate chemiluminescence as detection means in combination with acridinium ester label technology.
Acridine substituent is used for immunoassay tool as chemiluminescent labels and had many advantages, and mainly has：1. chemistry
Reaction is simple, it is quick, without catalyst, having H2O2Dilute alkaline solution in can light；2. background luminescence is low, signal to noise ratio is high,
Luminescence-producing reaction disturbing factor is few；3. emission type lights for flash type, the quick concentration of light release, luminous efficiency are high, luminous intensity
Greatly；4. acridinium ester molecular weight is small, avoids covering antibody combining site, system overall sensitivity can be improved；5. it is easy to and protein
Photon yield is not reduced after being coupled and being coupled；6. label is stable, is not influenceed by ambient oxidant, can be preserved at 2-8 DEG C
Several months long.Therefore acridine substituent is a kind of very effective chemiluminescent labels.
Magneto separate immunoassay technology is a kind of a kind of novel immune established using magnetic particle as solid phase carrier of separating
Detection method, this method can be such that the association reaction of antigen, antibody is carried out under conditions of approximate liquid phase, thus rapid reaction, thorough
Bottom.
The content of the invention
The technical problems to be solved by the invention are overcome the deficiencies in the prior art, there is provided a kind of easy to operate, sensitivity
Higher, rapid reaction, the magnetic microparticle chemiluminescence measure kit for quantitative determining melamine stably, exactly.
To achieve the above object, the present invention can take following technical proposals：
The chemical luminescence reagent kit and its composition of a kind of melamine of the present invention, including following components：Acridinium ester label
Antigen or antibody, coupling have the magnetic particle of antigen or antibody, calibration object solution, cleaning fluid, chemiluminescence preexciting liquid A and
Chemiluminescence exciting liquid B.
Described chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS；Wherein, acridinium ester
The antigen of mark or the ratio of antibody are 5-100:1.
Described acridinium ester label buffer solution is that pH 8.0-11.0, concentration are 0.05-0.50 mol/L Na2CO3-
NaHCO3Buffer solution.
Described magnetic particle directly can be coupled with melamine antibody or antigen, or magnetic particle and Streptavidin is even
Connection, while use biotin labeling melamine antibody or antigen.
By the calibration object solution composition of 6 various concentrations, calibration object solution specifically resists described calibration object by melamine
Former and calibration object buffer solution composition.
The described different calibration object concentration gradients of melamine 6 be 0 ng/mL, 0.01 ng/mL, 0.1 ng/mL,
1ng/mL、10 ng/mL、100 ng/mL。
Described calibration object buffer solution is the Tris-HCl bufferings containing 0.5-5.0% BSA and 0.1-0.5% PC300
Liquid.
Described chemiluminescence preexciting liquid A is by H2O2And HNO3Mixed liquor composition.Wherein, H2O2Mass fraction be
0.05-5%, HNO3Concentration is 0.05-2.5 mol/L；Chemiluminescence exciting liquid B by Triton X-100 and NaOH mixed liquor
Composition.Wherein, Triton X-100 concentration is 0.05-2.0 mol/L, and NaOH concentration is 0.05-1.0 mol/L.
The Tris-HCl solution that described cleaning fluid is pH 7.0-9.0, concentration is 5-50 mmol/L, wherein being containing concentration
0.05-0.50 mol/L NaCl and 0.01-0.25% Tween-20.
The principle that detection kit of the present invention uses is competition law, particular by anti-in the antigen and system in sample
Former competition binding specific antibody, using acridinium ester react caused by photon to detect production concentration.
The present invention has advantages below：
1. kit of the present invention is carried out using Magneto separate chemiluminescence as detection means using acridinium ester label technology
Detect to establish a kind of direct chemical luminescence reagent kit for detecting melamine.Acridinium ester have molecular weight is small, background luminescence is low,
Signal to noise ratio is high, luminous efficiency is high, mark is stable, can improve the clear superiority of system overall sensitivity.
2. the present invention requires low to Sample pretreatment, sample can be used to detect after the processing such as extraction, dilution, and can
Realize high-volume automatic detection.
To sum up, kit of the present invention is simple to operate, has luminous value stabilization, high sensitivity, detection quick and convenient, accurate
The advantages that true property is high, reproducible, high specificity.
Embodiment
The present invention provides a kind of chemical luminescence reagent kit and its composition of melamine, to make the purpose of the present invention, technology
Scheme and effect definitely, it is clear, the present invention is described in more detail below.
The present invention provides a kind of chemical luminescence reagent kit of melamine, and the kit includes：Acridinium ester label, coupling
There are magnetic particle, calibration object solution, cleaning fluid, chemiluminescence preexciting liquid A, the chemiluminescence exciting liquid B of antigen or antibody.
Kit of the present invention is entered using Magneto separate chemiluminescence as detection means in combination with acridinium ester label technology
Row detection.
Embodiment 1：The establishment and its preparation of kit 1
1. kit 1 is set up
A kind of chemiluminescence detection kit of melamine is set up, it is contained following component：
The melamine antibody of acridinium ester label；
The melamine antigen of carboxyl magnetic bead coupling；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Melamine series calibration object solution, concentration are respectively：0 ng/mL、0.01 ng/mL、0.1 ng/mL、1 ng/mL、10
ng/mL、100 ng/mL；
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. coupling has the preparation of the magnetic particle suspension of melamine antigen
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds 200 μ L 0.1 mol/L MES（pH 5.0）Buffering
Liquid, it is vortexed and mixes, be placed on magnetic frame, standing 5 min makes magnetic particle be separated with liquid, supernatant discarding, washs 3 times, adds
200 μ L MES buffer solutions（pH 5.0）, it is vortexed.
（2）Add 20 μ L（20 μg）Melamine antigen, be vortexed, on rotatable reactor, be incubated at room temperature 30 minutes.
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL（1- (3- dimethylamino-propyls) -3- ethyl carbon
Diimine）, it is vortexed, on rotatable reactor, is incubated at room temperature 2 hours.
（4）Supernatant is removed, adds 200 μ L cleaning buffer solutions（TBS+0.05% Tween-20）, wash 3 times.
（5）Closed with containing 1% BSA buffer solutions, repeatedly closing 4 times, every time 10 min.The magnetic particle suspension is put
In 2-8 DEG C of preservation.
3. the preparation of the melamine antibody of acridinium ester label
（1）A certain amount of melamine antibody is placed in bag filter, and bag filter is placed in the mark buffer solution not less than 1 L
Middle dialysis, during which buffer solution at least change 3 times, last time dialysed overnight, mark buffer solution be pH 10.1, concentration 0.1
Mol/L Na2CO3-NaHCO3Buffer solution.
（2）A certain amount of acridinium ester is weighed, is NSP-DMAE-NHS from chemiluminescent labels, is dissolved in anhydrous dimethyl base
In formamide DMF, the acridine ester solution that concentration is 6.5 mmol/L is made into.
（3）Melamine antibody after dialysis is placed in a centrifuge tube, adds 6.5 mmol/L's of certain volume
The molar ratio of NSP-DMAE-NHS DMF solutions, acridinium ester and antibody is 10:1,200 μ L mark buffer solutions are added, at room temperature
React 1 h.The μ L of 10 g/L lysines 100 are added, 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（1×25
cm）Separation, with the mol/L of purification buffer 0.1 PBS（pH 6.3）Balance and elute chromatographic column.Chromatograph is used in separation process
Protein peak is detected, measures the chemiluminescence intensity and 280 nm absorbances of efflux respectively.Collect shading value height and absorbance
Big eluent, add 1% BSA（Volume）After dispense stored frozen.
4. the preparation of melamine calibration object
Melamine sterling is configured into sign concentration with the Tris-NaCl buffer solutions containing 2% BSA and 0.25% PC300 is
0 ng/mL, 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, a series of 100 ng/mL calibration objects.
5. the preparation of chemiluminescence exciting agent
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Mixed liquor composition.Wherein, H2O2Mass fraction be 1.5%, HNO3
Concentration is 0.1 mol/L, is distributed into 20 mL/ branch with 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 and its preparation of kit 2
1. the establishment of kit 2
A kind of chemiluminescence detection kit of melamine is set up, it is contained following component：
The melamine antigen of acridinium ester label；
Carboxyl magnetic bead is coupled melamine antibody；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Melamine series calibration object solution, concentration are respectively：0 ng/mL、0.01 ng/mL、0.1 ng/mL、1 ng/mL、10
ng/mL、100 ng/mL；
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. coupling has the preparation of the magnetic particle suspension of melamine antibody
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds 200 μ L 0.1 mol/L MES（pH 6.0）Buffering
Liquid, it is vortexed and mixes, be placed on magnetic frame, standing 5 min makes magnetic particle be separated with liquid, supernatant discarding, washs 3 times, adds
200 μ L MES（pH 6.0）Buffer solution, it is vortexed.
（2）Add 15 μ L（15 μg）Melamine antibody, it is vortexed, on rotatable reactor, is incubated at room temperature 30 minutes.
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL（1- (3- dimethylamino-propyls) -3- ethyl carbon
Diimine）, it is vortexed, on rotatable reactor, is incubated at room temperature 2 hours.
（4）Supernatant is removed, adds 200 μ L cleaning buffer solutions（TBS+0.05% Tween-20）, wash 3 times.
（5）Closed with containing 1% BSA buffer solutions, repeatedly closing 4 times, every time 10 min.The magnetic particle suspension is put
In 2-8 DEG C of preservation.
3. the preparation of the melamine antigen of acridinium ester label
（1）A certain amount of melamine antigen is placed in bag filter, and bag filter is placed in the mark buffer solution not less than 1 L
Middle dialysed overnight, the Na that mark buffer solution is pH 9.8, concentration is 0.1 mol/L2CO3-NaHCO3Buffer solution.
（2）A certain amount of acridinium ester is weighed, is NSP-DMAE-NHS from chemiluminescent labels, is dissolved in anhydrous dimethyl base
In formamide DMF, the acridine ester solution that concentration is 6.5 mmol/L is made into.
（3）Melamine antigen after dialysis is placed in a centrifuge tube, adds 6.5 mmol/L's of certain volume
The molar ratio of NSP-DMAE-NHS DMF solutions, acridinium ester and antigen is 20:1,200 μ L mark buffer solutions are added, at room temperature
React 1 h.Add the μ L of 10 g/L lysines 100 to 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（1×25
cm）Separation, with the mol/L of purification buffer 0.1 PBS（pH 6.3）Balance and elute chromatographic column.Chromatograph is used in separation process
Protein peak is detected, measures the chemiluminescence intensity and 280 nm absorbances of efflux respectively.Collect shading value height and absorbance
Big eluent, add 1% BSA（Volume）After dispense stored frozen.
4. the preparation of melamine calibration object
It is 0 to be configured to indicate concentration by melamine sterling with the Tris-HCl buffer solutions containing 2% BSA and 0.25% PC300
Ng/mL, 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, a series of 100 ng/mL calibration objects.
5. the preparation of chemiluminescence exciting agent：
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Mixed liquor composition.Wherein, H2O2 Mass fraction be 1.5%,
HNO3Concentration is 0.1 mol/L, is distributed into 20 mL/ branch with 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 3：The detection of melamine in actual sample
The use operation sequence of melamine quantitative detection reagent box of the present invention is as follows：
1. Sample pretreatment
（1）Milk sample process
The acquisition of milk test sample solution：150 μ L fresh milks are taken in 500 μ L centrifuge tubes, 4 DEG C of 10 min of centrifugation
（3000 r/min）, discard upper-layer fat.Pipette the μ L of milk sample 25 after centrifugation to be placed in clean teat glass, add
475 μ L concentration are that 0.02 mol/L phosphate buffers are diluted.
（2）Eggs sample process
The sample after 2 g homogeneous is taken in 10 mL centrifuge tubes, the NaCl of 5 mL 2% is added and 0.2 mol/L HCl- methanol mixes
Liquid is closed, vibrates 1 min.5000 r/min centrifuge 8 min, take 0.5 mL supernatants, add 0.05 mL, 0.5 mol/L NaOH
Solution, the phosphate buffer that 9.45 mL concentration are 0.02 mol/L is added, is mixed.
2. the detection of kit
（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, clean 5 times.
（3）Reaction vessel after washing, which is fully vibrated, makes magnetic particle scatter.
（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 melamine luminous intensity proportion relation corresponding thereto in sample.
Embodiment 4：Performance indications testing result
（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 melamine is 0.01 ng/mL.
（2）The specificity of kit
The competition medicine similar to melamine structure or function：Cyanuric acid, triazine, triazinediamine.Grasped by kit step
Make, be separately added into melamine, cyanuric acid, triazine, triazinediamine, make suppression curve, calculated according to linear equation each competing
Strive 50% inhibition concentration of thing.Cross reacting rate is IC of the antibody to melamine50With antibody to melamine competitor
IC50The ratio between percentage.As a result show：This kit has higher specificity to melamine, pair with melamine structure
Or intimate competition 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 (10)
1. the chemiluminescence detection kit and its composition of a kind of melamine, it is characterised in that including：The three of acridinium ester label
Poly cyanamid antibody, it is coupled the magnetic particle for having melamine antigen, calibration object solution, cleaning fluid, chemiluminescence preexciting liquid A, change
Learn luminous exciting liquid B.
A kind of 2. chemiluminescence detection kit of melamine according to claim 1, it is characterised in that described change
Luminous marker is acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
A kind of 3. chemiluminescence detection kit of melamine according to claim 1, it is characterised in that described a word used for translation
Pyridine ester mark is melamine antibody.
A kind of 4. chemiluminescence detection kit of melamine according to claim 1, it is characterised in that described magnetic
Particulate directly can be coupled with melamine antigen, or magnetic particle and Streptavidin are coupled, while use biotin labeling three
Poly cyanamid antigen.
A kind of 5. chemiluminescence detection kit of melamine according to claim 1, it is characterised in that described school
Quasi- product are using the Tris-HCl buffer solutions containing 0.5-5.0% BSA and 0.1-0.5% PC300 as matrix, and it is pure to add melamine
The calibration object solution for the series concentration gradient that product configuration forms.
A kind of 6. chemiluminescence detection kit of melamine according to claim 1, it is characterised in that described change
Luminous preexciting liquid A is learned by H2O2And HNO3Mixed liquor composition, chemiluminescence exciting liquid B is by Triton X-100 and NaOH
Mixed liquor forms.
7. the chemiluminescence detection kit and its composition of a kind of melamine, it is characterised in that including：The three of acridinium ester label
Poly cyanamid antigen, it is coupled the magnetic particle for having melamine antibody, calibration object solution, cleaning fluid, chemiluminescence preexciting liquid A, change
Learn luminous exciting liquid B.
A kind of 8. chemiluminescence detection kit of melamine according to claim 7, it is characterised in that described change
Luminous marker is acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
A kind of 9. chemiluminescence detection kit of melamine according to claim 7, it is characterised in that described a word used for translation
Pyridine ester mark is melamine antigen.
10. the chemiluminescence detection kit of a kind of melamine according to claim 7, it is characterised in that described
Magnetic particle directly can be coupled with melamine antibody, or magnetic particle and Streptavidin are coupled, while use biotin labeling
Melamine antibody.
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| CN113109558A (en) * | 2021-04-21 | 2021-07-13 | 山东康华生物医疗科技股份有限公司 | Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof |
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