CN104133063B - A kind of tetraodotoxin detection kit - Google Patents
A kind of tetraodotoxin detection kit Download PDFInfo
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- CN104133063B CN104133063B CN201410251894.1A CN201410251894A CN104133063B CN 104133063 B CN104133063 B CN 104133063B CN 201410251894 A CN201410251894 A CN 201410251894A CN 104133063 B CN104133063 B CN 104133063B
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- tetraodotoxin
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a kind of tetraodotoxin detection kit, described tetraodotoxin detection kit comprises following composition: the standard items of A, concentration known; B, buffer system L; C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.01-0.03mol/L of pH7.4, Sodium azide (antiseptic) 0.1-0.3g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.1-0.2g/L of 30-50nm, particle diameter is the latex particle 0.2-0.4g/L of 60-90nm, and particle diameter is the latex particle 0.6-0.8g/L of 120-200nm; D, tetraodotoxin concentrating sample extract 10 × each component and concentration thereof are: methyl alcohol 10-15g/L, acetic acid 2-5g/L, ammonium acetate 1-3g/L.The present invention has quick, easy, accurate and sensitivity high, can carry out sample Fast Measurement in enormous quantities simultaneously, can reduce operate miss and working strength to greatest extent.
Description
Technical field
The present invention relates to a kind of kit, particularly a kind of tetraodotoxin detection kit.
Background technology
Tetraodotoxin (Tetrodotoxin, TTX) is a kind of alkaloid (amino perhydro quinazoline type compound) contained in Puffer fish (being commonly called as globe fish) and other biosome, is one of maximum neurotoxin of toxicity that occurring in nature finds; Taller more than 1250 times of the sodium cyanide of its toxicity ratio severe toxicity, 0.5mg can be lethal.
The patent of invention of application number 200410036380.0 discloses a kind of simple and rapid detection method of tetraodotoxin.The method highly basic process tetraodotoxin, obtains and the 2-amino-6-methylol-oxine of tetraodotoxin equimolar amounts and oxalates.Utilize oxalate oxidase that oxalates Catalytic Oxygen is changed into hydrogen peroxide.Developed the color by developer and hydroperoxidation afterwards, by getting final product the content of quantitative tetraodotoxin in 520nm colorimetric.The defect that the method exists is: complicated operation, detection time is long, cannot carry out the Simultaneously test of batch samples.
Summary of the invention
The object of the present invention is to provide a kind of tetraodotoxin detection kit, there is quick, easy, accurate and sensitivity high, sample Fast Measurement in enormous quantities can be carried out simultaneously, operate miss and working strength can be reduced to greatest extent.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of tetraodotoxin detection kit, described tetraodotoxin detection kit comprises following composition:
The standard items of A, concentration known;
B, buffer system, the each component of described buffer system and concentration thereof are: the PBS damping fluid 0.01-0.03mol/L of pH7.4, hexadimethrine bromide (reaction promoter) 1-3g/L, Sodium azide (antiseptic) 0.1-0.5g/L, polysorbas20 (surfactant) 0.5-1.5mol/L, citric acid 0.8-1.6g/L, bovine serum albumin(BSA) 1-3g/L;
C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.01-0.03mol/L of pH7.4, Sodium azide (antiseptic) 0.1-0.3g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.1-0.2g/L of 30-50nm, particle diameter is the latex particle 0.2-0.4g/L of 60-90nm, and particle diameter is the latex particle 0.6-0.8g/L of 120-200nm;
D, tetraodotoxin concentrating sample extract 10 × each component and concentration thereof are: methyl alcohol 10-15g/L, acetic acid 2-5g/L, ammonium acetate 1-3g/L.
Cleaning Principle of the present invention is latex enhancing immune turbidimetry, be specially: tetraodotoxin concentrating sample extract is diluted to after normal concentration extracts sample and obtains sample extracting solution, sample extracting solution is added in buffer system, then detection liquid is added, detect tetraodotoxin specific binding in tetraodotoxin monoclonal antibody on the latex particle that is cross-linked with tetraodotoxin monoclonal antibody in liquid and sample extracting solution and form soluble antigen antibody complex, the turbidity of formation becomes positive correlation with the content of tetraodotoxin in sample.
Small size particle (30-50nm), middle particles (60-90nm) and large-size particles (120-200nm) three part is divided into the latex particle that tetraodotoxin monoclonal antibody is crosslinked, small size particle specific surface area is large, crosslinked tetraodotoxin monoclonal antibody is more, adds the range of linearity of detection.Large-size particles is comparatively large, easily manifests turbidity, improve the sensitivity of detection in conjunction with after tetraodotoxin.While middle particles can ensure to detect the range of linearity, turbidity display can also be strengthened, ensure that the stability of detection.The content controlling the latex particle be cross-linked with tetraodotoxin monoclonal antibody is abnormal important, serves central role for detection.The concentration of large-size particles (120-200nm), middle particles (60-90nm) concentration easily manifest turbidity comparatively greatly, small size particle concentration 0.1-0.2g/L, ensure that the range of linearity of detection, and such Detection results is best.
As preferably, standard concentration is respectively 0 μ g/L, 2 μ g/L, 5 μ g/L, 15 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L.
As preferably, the preparation method of the latex particle be cross-linked with tetraodotoxin monoclonal antibody is as follows:
(1) in centrifuge tube, add 150-180 μ L nano rubber latex particle respectively, 1200-1400 μ LpH7.40.01-0.03mol/LPBS damping fluid, tetraodotoxin monoclonal antibody 3 strain, 300-400 μ LEDAC aqueous solution, magnetic agitation 3-5 hour, 4 DEG C, the centrifugal 60-80min of 12000g, abandons supernatant;
(2), precipitation is placed in ice bath, sonic oscillation 1-2min;
(3) quencher 0.2mol/L glycine solution 300-400 μ L is added, stirring 20-30min after the precipitation equal-volume PBS damping fluid, after step (2) process is resuspended;
(4), 4 DEG C, the centrifugal 60-80min of 10000g, removes supernatant, after precipitation equal-volume PBS damping fluid is resuspended the crosslinked latex particle of tetraodotoxin monoclonal antibody.
Nano rubber latex particle is commercially available prod, and product is emulsion form, and in product, nano rubber latex granule content is 20%(w/v); EDAC concentration of aqueous solution is 0.2g/ml.Tetraodotoxin monoclonal antibody has three strains, and three strain tetraodotoxin monoclonal antibodies have different binding site from fugutoxin, and sensitivity, the specificity of detection are like this better.
Carry out chemical crosslinking with the nano rubber latex particle of tetraodotoxin monoclonal antibody and band amino, optimize the concentration, pH, parameter of noncentricity, ultrasound condition control etc. of damping fluid, obtain the latex particle crosslinked with tetraodotoxin monoclonal antibody that detection perform is good.
As preferably, during the sonic oscillation described in step (2), ultrasonic 1 second, interval 3-4 second.
As preferably, the particle diameter of described nano rubber latex particle is 30-50nm, 60-90nm or 120-200nm.
The invention has the beneficial effects as follows: there is quick, easy, accurate and sensitivity high, sample Fast Measurement in enormous quantities can be carried out simultaneously, operate miss and working strength can be reduced to greatest extent.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, the raw material adopted and equipment etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the conventional method of this area.
Three strain tetraodotoxin monoclonal antibodies of the present invention conventionally record: Wang Jianwei etc., the anti-preparation of tetraodotoxin monoclonal antibody and the Primary Study of characteristic thereof, health research, 1996, volume the 5th phase in September the 25th, the method that 308-311 page is recorded obtains (1G3,4G3,6D9 tri-strain).
Embodiment 1:
A kind of tetraodotoxin detection kit, described tetraodotoxin detection kit comprises following composition:
The standard items of A, concentration known: 0 μ g/L, 2 μ g/L, 5 μ g/L, 15 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L.
The PBS damping fluid 0.01mol/L of B, buffer system: each component of described buffer system and concentration thereof are: pH7.4, hexadimethrine bromide 1g/L, Sodium azide 0.1g/L, polysorbas20 0.5mol/L, citric acid 1.6g/L, bovine serum albumin(BSA) 1g/L.
C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.01mol/L of pH7.4, Sodium azide 0.1g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.1g/L of 30nm, particle diameter is the latex particle 0.2g/L of 60nm, and particle diameter is the latex particle 0.6g/L of 120nm.
The preparation method of the latex particle be cross-linked with tetraodotoxin monoclonal antibody is as follows:
(1) in centrifuge tube, 150 μ l nano rubber latex particles, are added respectively (commercially available, in product, nano rubber latex granule content is 20%(w/v), particle diameter is the latex particle crosslinked with tetraodotoxin monoclonal antibody that 30nm, 60nm or 120nm obtain three kinds of specifications respectively), 1200 μ lpH7.40.01mol/LPBS damping fluids, tetraodotoxin monoclonal antibody 3 strain (1G3,4G3,6D9 tri-strain), 300 μ lEDAC aqueous solution (concentration is 0.2g/ml), magnetic agitation 3 hours, 4 DEG C, the centrifugal 60min of 12000g, abandons supernatant;
(2), precipitation be placed in ice bath, sonic oscillation 1min, during sonic oscillation, ultrasonic 1 second, 3 seconds, interval;
(3) quencher 0.2mol/L glycine solution 300 μ l is added, stirring 20min after the precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.01mol/L), after step (2) process is resuspended;
(4), 4 DEG C, the centrifugal 60min of 10000g, removes supernatant, after precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.01mol/L) is resuspended the crosslinked latex particle of tetraodotoxin monoclonal antibody.
D, tetraodotoxin concentrating sample extract 10 × each component and concentration thereof are: methyl alcohol 10g/L, acetic acid 2g/L, ammonium acetate 3g/L.
Embodiment 2:
A kind of tetraodotoxin detection kit, described tetraodotoxin detection kit comprises following composition:
The standard items of A, concentration known: 0 μ g/L, 2 μ g/L, 5 μ g/L, 15 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L.
The PBS damping fluid 0.03mol/L of B, buffer system: each component of described buffer system and concentration thereof are: pH7.4, hexadimethrine bromide 3g/L, Sodium azide 0.5g/L, polysorbas20 1.5mol/L, citric acid 0.8g/L, bovine serum albumin(BSA) 3g/L.
C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.03mol/L of pH7.4, Sodium azide 0.3g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.2g/L of 50nm, particle diameter is the latex particle 0.4g/L of 90nm, and particle diameter is the latex particle 0.8g/L of 200nm.
The preparation method of the latex particle be cross-linked with tetraodotoxin monoclonal antibody is as follows:
(1) in centrifuge tube, 180 μ l nano rubber latex particles, are added respectively (commercially available, in product, nano rubber latex granule content is 20%(w/v), particle diameter is the latex particle crosslinked with tetraodotoxin monoclonal antibody that 50nm, 90nm or 200nm obtain three kinds of specifications respectively), 1400 μ lpH7.40.03mol/LPBS damping fluids, tetraodotoxin monoclonal antibody 3 strain, 400 μ lEDAC aqueous solution (concentration is 0.2g/ml), magnetic agitation 5 hours, 4 DEG C, the centrifugal 80min of 12000g, abandons supernatant;
(2), precipitation be placed in ice bath, sonic oscillation 2min, during sonic oscillation, ultrasonic 1 second, 4 seconds, interval;
(3) quencher 0.2mol/L glycine solution 400 μ l is added, stirring 30min after the precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.03mol/L), after step (2) process is resuspended;
(4), 4 DEG C, the centrifugal 80min of 10000g, removes supernatant, after precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.03mol/L) is resuspended the crosslinked latex particle of tetraodotoxin monoclonal antibody.
D, tetraodotoxin concentrating sample extract 10 × each component and concentration thereof are: methyl alcohol 15g/L, acetic acid 5g/L, ammonium acetate 1g/L.
Embodiment 3:
A kind of tetraodotoxin detection kit, described tetraodotoxin detection kit comprises following composition:
The standard items of A, concentration known: 0 μ g/L, 2 μ g/L, 5 μ g/L, 15 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L.
The PBS damping fluid 0.02mol/L of B, buffer system: each component of described buffer system and concentration thereof are: pH7.4, hexadimethrine bromide 2g/L, Sodium azide 0.3g/L, polysorbas20 1mol/L, citric acid 1g/L, bovine serum albumin(BSA) 2g/L.
C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.02mol/L of pH7.4, Sodium azide 0.2g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.15g/L of 40nm, particle diameter is the latex particle 0.3g/L of 70nm, and particle diameter is the latex particle 0.7g/L of 160nm.
The preparation method of the latex particle be cross-linked with tetraodotoxin monoclonal antibody is as follows:
(1) in centrifuge tube, 170 μ l nano rubber latex particles, are added respectively (commercially available, in product, nano rubber latex granule content is 20%(w/v), particle diameter is the latex particle crosslinked with tetraodotoxin monoclonal antibody that 40nm, 70nm or 160nm obtain three kinds of specifications respectively), 1300 μ lpH7.40.02mol/LPBS damping fluids, tetraodotoxin monoclonal antibody 3 strain, 350 μ lEDAC aqueous solution (concentration is 0.2g/ml), magnetic agitation 4 hours, 4 DEG C, the centrifugal 70min of 12000g, abandons supernatant;
(2), precipitation be placed in ice bath, sonic oscillation 1.5min, during sonic oscillation, ultrasonic 1 second, 3 seconds, interval;
(3) quencher 0.2mol/L glycine solution 350 μ l is added, stirring 25min after the precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.02mol/L), after step (2) process is resuspended;
(4), 4 DEG C, the centrifugal 70min of 10000g, removes supernatant, after precipitation equal-volume PBS damping fluid (the PBS damping fluid of pH7.4,0.02mol/L) is resuspended the crosslinked latex particle of tetraodotoxin monoclonal antibody.
D, tetraodotoxin concentrating sample extract 10 × each component and concentration thereof are: methyl alcohol 12g/L, acetic acid 3g/L, ammonium acetate 2g/L.
The concrete specification of reagent kit product of the present invention is as follows:
| Component title | Dosage |
| Standard items × 7 bottle * | 1ml/ bottle |
| Buffer system | 50ml |
| Detect liquid | 15ml |
| Tetraodotoxin concentrating sample extract 10 × | 50ml |
Reagent kit product of the present invention: holding conditions: preserve kit in 2 ~ 8 DEG C; Storage life: this keeping life is 12 months.
Tetraodotoxin concentrating sample extract 10 × when using: with deionized water by tetraodotoxin concentrating sample extract 10 × dilute by 1:9 volume ratio, that is: 1 part of tetraodotoxin concentrating sample extract+9 parts of deionized waters, the sample extracting solution prepared can preserve one month at 4 DEG C of environment.
Sample extraction method is:
The freezing sample of globe fish, be loaded in sealed plastic bag and thaw rapidly under flowing water, overall fish is with after distilled water cleaning, blot with filter paper, then fish is resolved into the parts such as muscle, liver, enteron aisle, skin, ovary (male is essence bundle), wash away blood stains with distilled water, then blot with filter paper, respectively the sample obtained after dissection is shredded, abundant homogeneous.Take above-mentioned homogeneous samples 1.0g ± 0.02g in 50mL polystyrene centrifuge tube, add sample extracting solution 1mL, eddy current mixing 3min; In 4000rpm, centrifugal 10min, gets 50 μ L and analyzes.
Tetraodotoxin in sample is combined with the antibody on latex particle surface, and adjacent latex particle is cross-linked to each other, and measurement solution turbidity is increased, according to the increase of 500nm place absorbance, compares with typical curve, can calculate the content of tetraodotoxin in sample.
performance evaluation of the present invention:
Performance characteristic: the range of linearity: 2-100 μ g/L; Sensitivity for analysis: △ A >=0.005/20 μ g/L; Precision of measurement: CV≤7.0%; Difference between batch≤10.0%; Stability: 12 months.Can Simultaneously test in enormous quantities.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
Claims (4)
1. a tetraodotoxin detection kit, is characterized in that: described tetraodotoxin detection kit comprises following composition:
The standard items of A, concentration known;
B, buffer system, each component of described buffer system and concentration thereof are: the PBS damping fluid 0.01-0.03mol/L of pH7.4, hexadimethrine bromide 1-3g/L, Sodium azide 0.1-0.5g/L, polysorbas20 0.5-1.5mol/L, citric acid 0.8-1.6g/L, bovine serum albumin(BSA) 1-3g/L;
C, detection liquid, the each component of described detection liquid and concentration thereof are: the PBS damping fluid 0.01-0.03mol/L of pH7.4, Sodium azide 0.1-0.3g/L, the latex particle be cross-linked with tetraodotoxin monoclonal antibody: wherein particle diameter is the latex particle 0.1-0.2g/L of 30-50nm, particle diameter is the latex particle 0.2-0.4g/L of 60-90nm, and particle diameter is the latex particle 0.6-0.8g/L of 120-200nm;
D, 10 × tetraodotoxin concentrating sample extract, each component of described 10 × tetraodotoxin concentrating sample extract and concentration thereof are: methyl alcohol 10-15g/L, acetic acid 2-5g/L, ammonium acetate 1-3g/L.
2. a kind of tetraodotoxin detection kit according to claim 1, is characterized in that: standard concentration is respectively 0 μ g/L, 2 μ g/L, 5 μ g/L, 15 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L.
3. a kind of tetraodotoxin detection kit according to claim 1, is characterized in that: the preparation method of the latex particle be cross-linked with tetraodotoxin monoclonal antibody is as follows:
(1) in centrifuge tube, add 150-180 μ L nano rubber latex particle respectively, 1200-1400 μ LpH7.40.01-0.03mol/LPBS damping fluid, tetraodotoxin monoclonal antibody 3 strain, 300-400 μ LEDAC aqueous solution, magnetic agitation 3-5 hour, 4 DEG C, the centrifugal 60-80min of 12000g, abandons supernatant; The particle diameter of described nano rubber latex particle is 30-50nm, 60-90nm or 120-200nm;
(2), precipitation is placed in ice bath, sonic oscillation 1-2min;
(3) quencher 0.2mol/L glycine solution 300-400 μ L is added, stirring 20-30min after the precipitation equal-volume PBS damping fluid, after step (2) process is resuspended;
(4), 4 DEG C, the centrifugal 60-80min of 10000g, removes supernatant, after precipitation equal-volume PBS damping fluid is resuspended the crosslinked latex particle of tetraodotoxin monoclonal antibody.
4. a kind of tetraodotoxin detection kit according to claim 3, is characterized in that: during the sonic oscillation described in step (2), ultrasonic 1 second, interval 3-4 second.
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| WO2007013157A1 (en) * | 2005-07-28 | 2007-02-01 | Kabushiki Kaisha Yakult Honsha | Antibody-sensitized latex |
| WO2008055995A2 (en) * | 2006-11-10 | 2008-05-15 | Alphaptose Gmbh | Methods and compositions for detecting receptor ligand mimetics |
| CN101781365A (en) * | 2010-01-29 | 2010-07-21 | 华南农业大学 | Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof |
| CN102175847A (en) * | 2011-01-26 | 2011-09-07 | 福建农林大学 | Kit and method for detecting tetrodotoxin with gene engineering single-chain antibody |
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