CN103792357A - Spectinomycin fast time-resolved fluoroimmunoassay quantitative detection test strip - Google Patents
Spectinomycin fast time-resolved fluoroimmunoassay quantitative detection test strip Download PDFInfo
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- CN103792357A CN103792357A CN201210434957.8A CN201210434957A CN103792357A CN 103792357 A CN103792357 A CN 103792357A CN 201210434957 A CN201210434957 A CN 201210434957A CN 103792357 A CN103792357 A CN 103792357A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention relates to a preparation method and an application of a spectinomycin fast time-resolved fluoroimmunoassay quantitative detection test strip. The test strip consists of a Fusion5 membrane, a nitrocellulose membrane and water absorption paper; quick and fast quantitative detection is performed on the antigen to be detected by taking a time-resolved fluorescent microsphere as an immune marker based on the principle of competition law.
Description
Technical field
The invention belongs to food safety detection field, be specifically related to quantitative time-resolved fluoroimmunoassay chromatograph test strip detecting of the detection method of the Harmful Residue in food, particularly spectinomycin and its preparation method and application.
Background technology
Spectinomycin (English name Spectinomycin) is a kind of aminocyclitol compounds, by strepto-Streptomyces
Spectabilis is isolated, and is mainly present in the tissue and liver of animal product, can damage the 8th pair of cranial nerve, has Toxicity of Kidney and to nervimuscular blocking effect.
European Union has promulgated about spectinomycin the maximum residue limit in tissue (residual marker is original shape) for 2002.In the muscle of all food animals, fat, liver, kidney, the maximum residue limit of spectinomycin is respectively 300 μ g/kg, 500 μ g/kg, 1000 μ g/kg and 1500 Ministry of Agriculture of μ g/kg China and has also promulgated the maximum residue limit in tissue about spectinomycin in 2003, and spectinomycin maximum residue limit in the muscle of ox/sheep/pig/chicken, fat, liver, kidney is respectively 500 μ g/kg, 2000 μ g/kg, 2000 μ g/kg and 5000 μ g/kg.Therefore strengthening the research to spectinomycin residue detection technology, is the key measure of effectively controlling animal tissue's drug residue, is also to set up and improve one of task of medicament residue monitoring system.
Current immunochromatography (lateral flow immunoassay, LFIA) Rapid detection test strip is mainly with collaurum, color latex microballoon or the fluorescein thing that serves as a mark.The problems such as the fast detecting product of developing based on colloidal gold-labeled method, exists qualitative or sxemiquantitative, and differences between batches are larger; Although color latex microballoon difference between batch improves to some extent, sensitivity is still lower, also can only qualitative or sxemiquantitative; Immunochromatography sensitivity based on fluorescein-labelled technology is greatly improved, also can quantitatively detect, but owing to containing higher fluorescence background signal in sample, and stock displacement is less, can produce larger impact to detecting.
Time-resolved fluorescence (Time-resolved Fluorescence, TRF) be a kind of heterotope fluorescent marker, compared with common fluorescence, there is stock displacement large, the features such as fluorescence lifetime is long, can effectively avoid the background fluorescence in sample, and the impact of the parasitic light such as exciting light, therefore compare common fluorescence and there is higher sensitivity and antijamming capability.
The present invention utilizes Fusion5 to substitute traditional sample pad, hemofiltration film, pad, by original four layers of five tunic system simplification even, develop and only need the fast quantification of trilamellar membrane structure immunity chromatography detection test paper, not only make production technology be simplified, and effectively reduce batch interpolation and the difference between batch of product, improve detection sensitivity, had practical value.
Summary of the invention
The object of the invention is for time-resolved fluoroimmunoassay chromatography detecting test paper strip novel, energy Quantitative detection spectinomycin and its preparation method and application is provided.
One aspect of the present invention discloses spectinomycin time-resolved fluoroimmunoassay chromatography quantitative testing test paper bar, and this test strips comprises Fusion5 film, nitrocellulose filter and thieving paper three parts.In the middle of nitrocellulose filter is positioned at, Fusion5 film and thieving paper are overlapped in respectively two ends, nitrocellulose filter left and right.Wherein, Fusion5 film is provided with sample application zone and microballoon district, and microballoon district uploads free resolution fluorescent microsphere; Nitrocellulose filter is provided with detection line (T line) and nature controlling line (C line), and T line connects coated spectinomycin antigen, and C line is coated with anti-rabbit antibody.
Advantage of the present invention is the content that energy fast detecting goes out spectinomycin in muscle, tissue.
Described spectinomycin time-resolved fluorescence microballoon, comprises and detects microballoon and Quality Control microballoon, detecting microballoon is the fluorescent microsphere that pan coating has spectinomycin monoclonal antibody, and Quality Control microballoon is the fluorescent microsphere that pan coating has the anti-labelled protein of rabbit.
In described fluorescent microsphere, fill bright-coloured series elements compound; Preferably, this bright-coloured is that first rope huge legendary turtle compound is europium huge legendary turtle compound; Optimum, this europium huge legendary turtle compound can be Eu(TTA)
3/ TOPO or Eu(TTA)
3/ Phen.
Described albumen can be cow's serum Y-globulin (BGG) or bSA (BSA).
Described time-resolved fluorescence microspherulite diameter scope is 100-1000 nm.
On described nitrocellulose filter, on T line, coated antibody is spectinomycin monoclonal antibody, the anti-rabbit antibody being coated with on C line.
Described spectinomycin time-resolved fluoroimmunoassay quantitative testing test paper bar bottom is provided with plastic bottom board.
Second aspect present invention discloses the preparation method of spectinomycin time-resolved fluoroimmunoassay chromatography quantitative testing test paper bar, comprises the following steps:
(1) Quality Control microballoon preparation:
1. use biotinylated protein;
2. adopt above-mentioned labelled protein to be coated with aldehyde group modified fluorescent microsphere;
(2) detect microballoon preparation: the coated aldehyde group modified fluorescent microsphere of monoclonal antibody that adopts spectinomycin;
(3) blank kilocalorie is pasted: nitrocellulose filter is sticked in the middle of plastic bottom board, and Fusion5 film and thieving paper are overlapped in respectively two ends, nitrocellulose filter left and right;
(4) spray film: adopt buffer release liquid mixed diluting to finite concentration the fluorescent microsphere of the fluorescent microsphere of the anti-labelled protein of rabbit and spectinomycin monoclonal antibody, be sprayed onto the microballoon district of Fusion5 film; After spectinomycin antigen and anti-rabbit antibody dilution, be sprayed onto respectively T line and the C line position of nitrocellulose filter;
(5) dry and slitting: by above-mentioned kilocalorie oven dry, the slitting that has sprayed reagent.
The buffer release liquid of using in step (4) contains 10-20% sucrose, 3-10% trehaloses, 0. 5-1%N, 0-bis-trimethylsilyl acetamides (BSA), 0.2 one 0.5% gentamicins.
After step (4) dilution, detecting microballoon final concentration is 0.5-2 mg/mL, Quality Control microballoon final concentration 0.05-0.2 mg/mL; T line antigen final concentration 0.5-2 mg/mL, C line antigen final concentration 0.5-2 mg/mL; C, T line spray film liquid measure is 0.5-1.5 μ L/cm, microballoon spray film amount is 2-8 μ L/cm.
Oven dry described in step (5) can be in constant temperature oven or drying room, dries 8-12 hours for 37 ℃.
Third aspect present invention discloses the application of spectinomycin time-resolved fluoroimmunoassay chromatography quantitative testing test paper bar.
Test strips of the present invention can be measured biomolecule by competition law principle, and the immunochromatography that is applicable to all employing competition law patterns detects.
Spectinomycin time-resolved fluoroimmunoassay chromatograph test strip of the present invention is highly sensitive, withinrun precision can reach 10% left and right, betweenrun precision can reach 15%, can detect whole blood, serum, blood plasma, urine specimen simultaneously, 250 mg/dL haemoglobins, 500 mg/dL triglycerides, 10 mg/dL cholerythrin without impact, far exceed most of immunochromatography quick diagnosis products on market on the detection of this test strips.
Accompanying drawing explanation
Fig. 1: test strips structural representation of the present invention (1. plastic bottom board, 2. Fusion5 film, 3. nitrocellulose filter, 4. thieving paper, 5. sample application zone, 6. microballoon district, 7. T line district, 8. C line district).
Fig. 2: SPE ELISA test strip typical curve.
embodiment
1. the preparation of the each composition of test strips
The preparation of preparing the coated Quality Control microballoon of biotin labeling γ-globulin (BGG) of 1.1 Quality Control microballoons.
(1) preparation of biotin labeled BGG
With 0.1 M NaCNBH
3by BGG(purchased from Pel-Freez Biological) be mixed with l0 mg/mL solution, adopt DMSO(dimethyl formamide) configuration Biotin-X-X-NHS(N-N-Hydroxysuccinimide modified biological element, manufacturer: SIGMA, production number: B3295) solution to 16.172 mg/mL, add the amount of 5.4 μ L Biotin-X-X-NHS that Biotin-X-X-NHS liquid is joined in BGG solution according to 1 mg BBG albumen, mix and place and spend the night at 4 ℃.Adopt dialysis to remove free unreacted biotin, dislysate is biotinylated protein dialysis buffer liquid (0.1 M Tris, 0.3 M NaCl, 0.005 M EDTA-Na-2H
20, pH8.0).Dialyse complete, BCA method is measured protein concentration.
(2) adopt above-mentioned labelled protein to be coated with aldehyde group modified fluorescent microsphere
To the Tween-20 solution, 2 mg that add 6.4 μ L 20% in the aldehyde group modified fluorescent microsphere of 10 mL above-mentioned (1) in the biotinylated protein that obtains of dialysis and the NaCNBH of 16 μ L
3, (25 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0, now with the current), adding 0.1 M pH6.0 MES is 400 μ L to cumulative volume, 37 ℃ of lucifuges are hatched 48 h.Add the Gly solution (75 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0) of 40 μ L, 37 ℃ of lucifuge revolving reaction 2 h.Add 250 μ L N, the two trimethylsilyl acetamides of O-(200 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0) solution.37 ℃ of lucifuge revolving reaction 16 h.Centrifugal 30 minutes of 4 ℃ of 13000 rpm.Abandon supernatant, wash again twice with the MES damping fluid of 1 mL pH6.0.With HEPES damping fluid (0.05 M HEPES, 0.3 M NaCl, the 0.025 M EDTA-Na-2H of 1 mL 0.05 M pH8.0
20,1.6% N, O-bis-trimethylsilyl acetamides (BSA), 0.1% Dextran, 0.1% Tween-20,0.3745% Triton X-405,0.0l% gentamicin, 0.05 % Proclin) (its final concentration is 10 mg/mL) suspends.
(3) Quality Control microballoon working fluid preparation
According to need of work, the HEPES damping fluid of employing 0.05M pH 8.0 (2) middle suspension is diluted to respective concentration, and packing is preserved.
1.2 detect the preparation of microballoon
To the NaCNBH that adds Tween-20 solution, 2 mg spectinomycin monoclonal antibodies and the 16 μ L of 6.4 μ L 20% in the aldehyde group modified fluorescent microsphere of 10 mg
3(25 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0, now with the current), adding 0.1 M pH6.0 MES is 400 μ L to cumulative volume, 37 ℃ of lucifuges are hatched 48 h.Add the Gly solution (75 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0) of 40 μ L, 37 ℃ of lucifuge revolving reaction 2 h.Add 250 μ L BSA(200 mg/mL, the MES damping fluid preparation of 0.05 M pH6.0) solution.37 ℃ of lucifuge revolving reaction 16 h.Centrifugal 30 minutes of 4 ℃ of 13000 rpm.Abandon supernatant, wash again twice with the MES damping fluid of 1 mL pH6.0.With HEPES damping fluid (0.05 M HEPES, 0.3 M NaC1, the 0.25 M EDTA-Na-2H of 1 mL 0.05 M pH8.0
20,1.6% BSA, 0.1% Dextran, 0.1% Tween-20,0.3745% Triton X-405,0.01% gentamicin, 0.05% Proclin) (its final concentration is 10 mg/mL) suspends.
The configuration of 1.3 microspheres solution
(contain 20% sucrose, 5% trehalose, 0.5% BSA with buffer release liquid, 0.2% gentamicin) Quality Control microballoon and detection microballoon are mixed with to time-resolved fluorescence microballoon mixed liquor, Quality Control microballoon final concentration is 0.2 mg/mL, and detecting microballoon final concentration is 1 mg/mL.
The preparation of 1.4 detection lines (T line) solution
Spectinomycin antigenic compound is diluted to 0.5 mg/mL with 0.01 M PB solution.
The preparation of 1.5 nature controlling lines (C line) solution
Streptavidin is diluted to 0.5 mg/mL with 0.01 M PB solution.
2. the preparation of test strips.
2.1 blank kilocalories are pasted
According to the film array mode of accompanying drawing 1, in the middle of nitrocellulose filter 3 is positioned at, Fusion5 film 2 is overlapped in respectively two ends, nitrocellulose filter left and right with thieving paper 4, sticks on the plastic bottom board with gum.
2.2 spray films
T line 7 in Fig. 1 respectively, T is sprayed in C line 8 positions, C line solution, C, T line spray film liquid measure is 0.8 μ L/cm, and in Fig. 1, microspheres solution is sprayed in 6 positions, and microspheres solution spray film amount is 4 μ L/cm.
2.3 dry
The spectinomycin that has sprayed reagent in step (2.2) is stuck in greatly in constant temperature oven to 37 ℃ dries 12 hours.
2.4 slitting
The spectinomycin kilocalorie of oven dry is cut into the paper slip of 4 mm width, obtain spectinomycin test strips.
3. detecting instrument Principle and application
3.1 measuring instrument principles: condenser lens is positioned at exciting light and becomes 90
.direction, the fluorescence signal line focus lens focus that sample in sample detection pond is produced, then arrive photomultiplier after grating beam splitting, photomultiplier changes light signal into electric signal, is finally sent to A/D converter and processes.Described LASER Light Source adopts diode pumped solid state laser, and output power is 20 mW, and emission wavelength is 375 ± 5 nm.The fiber power of exciting light after conduction coupling fiber is more than 5 mW.Ultraviolet quartz glass is selected in sample detection pond.It can light splitting acquisition wavelength be the light of 440 ± 5 nm that grating is set to.
3.1 detector application: by the edible animal muscle that contains spectinomycin, organize extract after pretreatment to pack in sample detection pond, LASER Light Source is sent the laser that wavelength is 375 ± 5 nm, after conduction coupling fiber, conduct to sample detection pond, and through testing sample, after absorption of sample exciting light in sample detection pond, produce fluorescence, the fluorescence signal line focus lens focus producing, again through grating beam splitting, obtain the light that wavelength is 440 ± 5 nm, arrive photomultiplier, photomultiplier changes light signal into electric signal, input a/d converter after simultaneously electric signal being amplified, finally export to reading device processing, can directly read result by reading device.
4. the quantitative detection of test strips
4.1 drawing standard curves
At the spectinomycin test strips preparing (lot number: 20120620) add the spectinomycin antigen standard items of variable concentrations (to get five different concentration, be respectively 0,125 in sample application zone, 250,500,750 ng/mL, each concentration is established 5 repetitions), (PBS, contains 1.6% BSA to drip sample-loading buffer, 0.1% Tween-20, antiseptic), after rete is analysed 10 minutes, detecting instrument reads C, T line signal, experimental result and analysis in table 1:
Table 1 spectinomycin standard items testing result
| SPE standard items (ng/mL) | 0 | 125 | 250 | 500 | 750 |
| 1 | 18.25 | 12.29 | 8.52 | 3.72 | 1.35 |
| 2 | 18.92 | 13.07 | 9.06 | 3.66 | 1.58 |
| 3 | 19.6 | 12.55 | 8.2 | 4.01 | 1.22 |
| 4 | 19.18 | 11.81 | 8.47 | 3.47 | 1.46 |
| 5 | 18.64 | 12.63 | 8.38 | 3.63 | 1.54 |
| mean | 18.918 | 12.47 | 8.526 | 3.698 | 1.43 |
| SD | 0.507 | 0.445 | 0.397 | 0.231 | 0.094 |
With the signal averaging drawing standard curve of antigen standard items concentration and mensuration, adopt four parameter fitting modes, typical curve data are in table 2, and typical curve is as shown in Figure 2.
Table 2 spectinomycin quantitative measurement standard curve data
| SPE standard items (ng/mL) | 0 | 125 | 250 | 500 | 750 |
| Reading | 18.918 | 12.470 | 8.526 | 3.698 | 1.430 |
4.2 sample detection
In spectinomycin test strips, (lot number: sample application zone 20120620) adds testing sample successively, drips sample-loading buffer, and after rete is analysed 10 minutes, instrument reads C, T line signal.Typical curve according to step in (1) calculates spectinomycin antigen concentration in testing sample.
5. test strips performance test
Table 3 test strips is criticized interpolation
| Concentration of specimens (ng/mL) | mean | SD | CV% |
| 250 | 5.37 | 0.336 | 10.70 |
| 500 | 2.88 | 0.175 | 7.21 |
The spectinomycin quantitative testing test paper bar sensing range that adopts the method to prepare can reach 0-1000 ng/mL, sensitivity is below 125 ng/mL, withinrun precision can reach 11% left and right, betweenrun precision can reach 15%, can detect whole blood, serum, plasma sample simultaneously, 250 mg/dL haemoglobins, 500 mg/dL triglyceride, 10 mg/dL cholerythrin without impact, far exceed most of immunochromatography quick diagnosis products on market on this detection.
Claims (10)
1. the quick time-resolved fluoroimmunoassay chromatography of spectinomycin quantitative testing test paper bar, it is characterized in that: comprise Fusion5 film, nitrocellulose filter and thieving paper three parts, in the middle of nitrocellulose filter is positioned at, Fusion5 film and thieving paper are overlapped in respectively two ends, nitrocellulose filter left and right, on Fusion5 film, there are sample application zone and microballoon district, in microballoon district, are loaded with spectinomycin monoclonal antibody time-resolved fluorescence microballoon; On nitrocellulose filter, there are detection line and nature controlling line, coated spectinomycin antigen on detection line, coated anti-rabbit antibody on nature controlling line.
2. test strips as claimed in claim 1, it is characterized in that: described time-resolved fluorescence microballoon comprises detection microballoon and Quality Control microballoon, detecting microballoon is the fluorescent microsphere that pan coating has spectinomycin monoclonal antibody, and Quality Control microballoon is the fluorescent microsphere that pan coating has the anti-labelled protein of rabbit.
3. test strips as claimed in claim 1 or 2, is characterized in that: the particle size range of described time-resolved fluorescence microballoon is 100-1000 nm.
4. test strips as claimed in claim 1, is characterized in that: on described detection line, coated spectinomycin detection is the conjugates that spectinomycin and carrier mass form with antigen; Coated two anti-anti-rabbit antibody on detection line.
5. the preparation method of test strips as claimed in claim 1, its preparation process is as follows:
(1) Quality Control microballoon preparation:
1. use biotinylated protein;
2. adopt above-mentioned labelled protein to be coated with aldehyde group modified fluorescent microsphere;
(2) detect microballoon preparation: the coated aldehyde group modified fluorescent microsphere of monoclonal antibody that adopts spectinomycin;
(3) blank kilocalorie is pasted: nitrocellulose filter is sticked in the middle of plastic bottom board, and Fusion5 film and thieving paper are overlapped in respectively two ends, nitrocellulose filter left and right;
(4) spray film: adopt buffer release liquid mixed diluting to finite concentration the fluorescent microsphere of the fluorescent microsphere of the anti-labelled protein of rabbit and spectinomycin monoclonal antibody, be sprayed onto the microballoon district of Fusion5 film; After spectinomycin antigen and anti-rabbit antibody dilution, be sprayed onto respectively T line and the C line position of nitrocellulose filter;
(6) dry and slitting: by above-mentioned kilocalorie oven dry, the slitting that has sprayed reagent.
6. preparation method as claimed in claim 5, is characterized in that: step (4) middle buffer release liquid consists of 10-20% sucrose, 3-10% trehaloses, 0. 5-1% N, the two trimethylsilyl acetamides (BSA) of O-, 0.2-0.5% gentamicin.
7. preparation method as claimed in claim 5, is characterized in that: (4) step dilutes rear detection microballoon final concentration is 0.5-2 mg/mL, Quality Control microballoon final concentration 0.05-0.2 mg/mL; T line antigen final concentration 0.5-2 mg/mL, C line antigen final concentration 0. 5-2 mg/mL; C, T line spray film liquid measure is 0.5-1.5 μ L/cm, microballoon spray film amount is 2-8 μ L/cm.
8. preparation method as claimed in claim 5, is characterized in that: step (5) middle drying condition is 37 ℃ of oven dry 8-12 hours.
9. the quick time-resolved fluoroimmunoassay chromatography of the spectinomycin quantitative testing test paper bar as described in claim 5 claim, is characterized in that: test strips bottom is provided with plastic bottom board.
10. the application of test strips as claimed in claim 1, is characterized in that: this test strips is used for the quantitative detection of the immunochromatography that adopts competition law pattern
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