CN102539746A - Reagent kit for fast detecting domoic acid and application method thereof - Google Patents
Reagent kit for fast detecting domoic acid and application method thereof Download PDFInfo
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- CN102539746A CN102539746A CN2011104424767A CN201110442476A CN102539746A CN 102539746 A CN102539746 A CN 102539746A CN 2011104424767 A CN2011104424767 A CN 2011104424767A CN 201110442476 A CN201110442476 A CN 201110442476A CN 102539746 A CN102539746 A CN 102539746A
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- domoic acid
- fast detecting
- domoic
- enzyme
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Abstract
The invention relates to a reagent kit for fast detecting domoic acid and an application method thereof. The reagent kit for fast detecting the domoic acid comprises an enzyme-labeled plate, an enzyme-labeled antigen, diluent, cleaning solution, color developing solution and stop solution. The enzyme-labeled antigen is a conjugate of the domoic acid and horse radish peroxidase, and the enzyme-labeled plate is already coated through an antibody of anti-domoic acid and is sealed through seal liquid. The invention further relates to a method for applying the reagent kit to fast detect the domoic acid. The reagent kit for fast detecting the domoic acid and the application method of the reagent kit adopt a method for directly competing enzyme-linked immuno sorbent assay (ELISA), avoid using secondary antibodies, and are simple in step, convenient to operate, capable of enabling detection time to be greatly shortened and reducing cost and work load of detecting personnel and suitable for fast detection.
Description
Technical field
The present invention relates to a kind of kit and method of fast detecting biotoxin, specifically relate to a kind of kit and method of application thereof of fast detecting domoic acid.
Background technology
According to statistics, the whole world is because of eating by about more than 300 of the mussel poisoning incident of red-tide toxin pollution, dead people more than 300.And China, in recent years, in Fujian, all there is the generation of red-tide toxin poisoning on ground such as Taiwan, Hong Kong, causes personnel's death.Amnesia property shellfish poison domoic acid is to plant generation by plan Nitzschia (Pseudo-nitzschia) in Bacillariophyta, plumage line Diatomacae (Pennatae), shell seam order (Aulonoraphidinals), the rhombus algae section (Nitzschiaceae) and some of the middle diatom of Nitzschia (Nitzschia); Can occur behind human the poisoning feeling sick, vomiting, gastritis, can cause symptoms such as dizzy, dysopia, the loss of memory, stupor when serious.
The method of detection domoic acid (DA) commonly used comprises mouse bioassay detection method, high performance liquid chromatography (HPLC) detection, enzyme linked immunosorbent assay etc. at present.Small white mouse detection method sense cycle is long, detects limit for height.The high performance liquid chromatography accuracy is high, and detection limit is low, but instrument is expensive, heaviness should not be carried out field monitoring, and enzyme linked immunosorbent assay is highly sensitive, is easy to carry, the suitable on-the-spot fast monitored that is applied to domoic acid.
The at present domestic kit of having developed detects principle and is mostly indirect competitive ELISA, earlier solid phase antigen is fixed on the ELISA Plate, adds the confining liquid sealing to reduce the non-specific binding of antibody antigen; Washing adds antibody and sample generation competitive reaction, washs behind the incubation certain hour; Add two and resist, inculation washing adds colour developing liquid normal temperature colour developing 20min; Add stop buffer; Cessation reaction, the working sample OD value (OD value) under 450nm and 630nm comes the domoic acid in the sample is carried out quantitatively through OD value.
The kit of indirect competitive ELISA method exploitation detects sensitivity, and specificity is high; But needing to add two resists; Two anti-and antibody need to combine the regular hour, increased the operating process and the time of detection, commercially available domoic acid kit major part is the indirect competitive ELISA kit; Have certain limitation at 2～3h detection time.
Summary of the invention
The objective of the invention is to defective to prior art; The kit and the method for domoic acid in a kind of test sample quickly and accurately be provided, avoided two anti-uses, step is simple; Easy to operate; Shortened detection time greatly, reduced cost and testing staff's workload, be applicable to fast detecting.
The present invention realizes through following technical scheme,
First invention; The present invention relates to a kind of kit of fast detecting domoic acid; This kit comprises: ELISA Plate, enzyme-labelled antigen, dilution, cleansing solution, colour developing liquid, stop buffer; Said enzyme-labelled antigen is the conjugate of domoic acid and horseradish peroxidase, and said ELISA Plate seals with the antibody sandwich of anti-domoic acid and with confining liquid.
Second aspect the invention still further relates to the method for application of aforementioned kit fast detecting domoic acid, may further comprise the steps:
Step 1; Get with the antibody sandwich of anti-domoic acid and with the ELISA Plate of confining liquid sealing, every hole adds the enzyme-labelled antigen of domoic acid and horseradish peroxidase formation, and adds the domoic acid standard solution of known variable concentrations respectively; Simultaneously in all the other holes, add testing sample; Incubation, the cleansing solution washing is clapped and is done;
Step 3, the logarithm value and corresponding combination rate (OD of drawing the domoic acid standard solution of said known variable concentrations
Mark/ OD
0) typical curve;
Step 4 is brought the OD value of said testing sample into said typical curve, can try to achieve the concentration of domoic acid in the sample.
Preferably, the coupling of said domoic acid and horseradish peroxidase is accomplished through carbodiimide method.
Preferably, carry out pre-service before testing sample is measured, this pre-treatment step comprises: sample thief adds homogenate; After the homogenate, vortex oscillation, ultrasonic Extraction, centrifugal; Take out supernatant to volumetric flask, residual residue adds homogenate again, repeats to extract; Supernatant all moves in the volumetric flask, distilled water constant volume, membrane filtration.
Preferably, said centrifugal be the centrifugal 20min of 4000rpm.
Preferably, the said number of times that repeats to extract is 2 times.
Preferably, the aperture of said filter membrane is 0.22 μ m.
Preferably, the temperature of said incubation is 37 ℃, and the time is 60min.
Preferably, the used antibody of said coated elisa plate is anti-domoic acid monoclone antibody, and 4 ℃ encapsulate and spend the night.
Preferably, described confining liquid is that massfraction is 1% gelatin, and 37 ℃ of sealing 60min process the good ELISA Plate of sealing.
The present invention has following beneficial effect: the present invention has adopted direct competitive ELISA method, has avoided two anti-uses, and step is simple, and is easy to operate, and shortened detection time greatly, reduced cost and testing staff's workload, is applicable to fast detecting.
Description of drawings
Fig. 1 is an ELISA EUSA typical curve among the embodiment 1.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated.Following examples will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.Should be pointed out that to those skilled in the art, under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement.These all belong to protection scope of the present invention.
Embodiment 1
Present embodiment relates to a kind of method that detects domoic acid, comprises the steps:
(1) shellfishes such as mussel, clam, scallop carry out pre-service by following mode: get fresh band shell or freezing after the sample that at room temperature thaws; Cut closed shell flesh with cutter and take out shellfish meat; The edible part is separated with mid intestinal gland, be placed on draining 5min on the tiny wire netting of mesh; Get this sample of 5g, add 10ml homogenate (1: 1 methanol in water of volume ratio), after the homogenate, vortex oscillation 1min, ultrasonic Extraction 5min, the centrifugal 20min of 4000rpm; Take out supernatant to the 25ml volumetric flask, residual residue adds homogenate 5min again, repeats to extract 2 times, and supernatant all moves in the 25ml volumetric flask, the distilled water constant volume; 0.22 μ m membrane filtration;
(2) get with the antibody sandwich of anti-domoic acid and with the ELISA Plate of confining liquid sealing, every hole adds the enzyme-labelled antigen 50 μ L of domoic acid and horseradish peroxidase formation, and adds a series of 0ngmL respectively
-1, 10ngmL
-1, 50ngmL
-1, 100ngmL
-1, 250ngmL
-1, 500ngmL
-1, 1000ngmL
-1Domoic acid standard solution 50 μ L, each concentration is done three repetitions, in all the other holes, adds simultaneously testing sample 50 μ L, each sample is done three repetitions, at 37 ℃ of following incubation 60min, cleansing solution washing three times each three minutes, is clapped and is done;
(3) every hole adds colour developing liquid 100 μ L, and normal temperature is colour developing 20min down, adds 50 μ L stop buffers, measures OD450 and OD630, makes typical curve, brings the OD value of testing sample into typical curve, can calculate the concentration of domoic acid in the sample; Testing result is following: the logarithm value with antigen DA concentration of standard solution is a horizontal ordinate, with combination rate (OD
Mark/ OD
0) be ordinate, draw the typical curve of this direct competitive ELISA experiment, as shown in Figure 1, coefficient R
2=0.97235, linear equation is: y=-0.1169x+1.06312.Get inhibiting rate and be 90% o'clock DA concentration, be the detection limit of this method, must detect and be limited to 24.85ngmL
-1Fig. 1 is an ELISA EUSA typical curve; Obtain the DA content of different types of shellfish through the mensuration of direct competitive ELISA method, it is as shown in table 1 to measure the result, confirms that detecting of this method is limited to 24.85ngmL
-1, the concentration that obtains is all less than 24.85ngmL
-1, can learn not contain DA in the shellfish sample.
The mensuration result of DA in the table 1 shellfish sample
| Sample | The OD value | B/B0 | Concentration (ngmL -1) |
| The red heart shellfish | 1.475 | 1.128 | 0.341 |
| Scallop | 1.495 | 1.133 | 0.252 |
| Clam | 1.409 | 1.068 | 0.913 |
| Mussel | 1.437 | 1.081 | 0.700 |
Obviously; Present embodiment also provides a kind of kit of fast detecting domoic acid; This kit comprises: ELISA Plate, enzyme-labelled antigen, dilution (0.01MPBS solution), cleansing solution (1L concentration is to add the 0.5ml Tween-20 in the PBS solution of 0.01M), colour developing liquid (are dissolved in 4mL ethanol with 10mg tetramethyl benzidine (TMB) earlier; Be prepared into the TMB storage liquid, 4 ℃ of preservations.During use, get 0.4mL TMB storage liquid, substrate buffer solution 10mL and 3,0%H,2O2 10 μ l mix; Face with join at present), stop buffer (2MH
2SO
4Solution), said enzyme-labelled antigen is the conjugate of domoic acid and horseradish peroxidase, said ELISA Plate with the antibody sandwich of anti-domoic acid, and seal with confining liquid (massfraction is 1% gelatin, the PBS solution dilution).
Present embodiment relates to a kind of method that detects domoic acid, comprises the steps:
The shellfish such as mussel, clam, scallop that (1) will not contain domoic acid respectively carries out pre-service by the method for embodiment 1, and mark-on is to 1000ngmL in sample
-1
(2) get with the antibody sandwich of anti-domoic acid and with the ELISA Plate of confining liquid sealing, every hole adds the enzyme-labelled antigen 50 μ L of domoic acid and horseradish peroxidase formation, and adds a series of 0ngmL
-1, 10ngmL
-1, 50ngmL
-1, 100ngmL
-1, 250ngmL
-1, 500ngmL
-1, 1000ngmL
-1Domoic acid standard solution 50 μ L, each concentration is done three repetitions, in all the other holes, adds simultaneously testing sample 50 μ L, each sample is done three repetitions, at 37 ℃ of following incubation 60min, cleansing solution washing three times each three minutes, is clapped and is done;
(3) every hole adds colour developing liquid 100 μ L, and normal temperature is colour developing 20min down, adds 50 μ L stop buffers, measures OD
450And OD
630, make typical curve.Obtain the concentration of domoic acid in the sample according to typical curve, calculate recovery of standard addition.Typical curve such as embodiment 1.According to typical curve and OD value, the concentration and the recovery that obtain the mark-on sample are as shown in table 2, and different shellfish matrix can obtain different recovery of standard addition, on the whole, change comparatively stable.
The mensuration result of table 2 sample recovery of standard addition
In sum, the present invention has adopted direct competitive ELISA method, has avoided two anti-uses, and step is simple, and is easy to operate, and shortened detection time greatly, reduced cost and testing staff's workload, is applicable to fast detecting.
Claims (10)
1. the kit of a fast detecting domoic acid; It is characterized in that; This kit comprises: ELISA Plate, enzyme-labelled antigen, dilution, cleansing solution, colour developing liquid, stop buffer; Said enzyme-labelled antigen is the conjugate of domoic acid and horseradish peroxidase, and said ELISA Plate seals with the antibody sandwich of anti-domoic acid and with confining liquid.
2. an application rights requires the method for 1 described kit fast detecting domoic acid, it is characterized in that, may further comprise the steps:
Step 1; Get with the antibody sandwich of anti-domoic acid and with the ELISA Plate of confining liquid sealing, every hole adds the enzyme-labelled antigen of domoic acid and horseradish peroxidase formation, and adds the domoic acid standard solution of known variable concentrations respectively; Simultaneously in all the other holes, add testing sample; Incubation, the cleansing solution washing is clapped and is done;
Step 2, every hole add colour developing liquid, and colour developing adds the stop buffer cessation reaction, measures the OD value;
Step 3 is drawn logarithm value and the typical curve of corresponding combination rate of the domoic acid standard solution of said known variable concentrations;
Step 4 is brought the OD value of said testing sample into said typical curve, can try to achieve the concentration of domoic acid in the sample.
3. the method for fast detecting domoic acid as claimed in claim 2 is characterized in that, the coupling of said domoic acid and horseradish peroxidase is accomplished through carbodiimide method.
4. the method for fast detecting domoic acid as claimed in claim 2 is characterized in that, carries out pre-service before testing sample is measured, and this pre-treatment step comprises: sample thief; Add homogenate, after the homogenate, vortex oscillation, ultrasonic Extraction; Centrifugal, take out supernatant to volumetric flask, residual residue adds homogenate again, repeats to extract; Supernatant all moves in the volumetric flask, distilled water constant volume, membrane filtration.
5. the method for fast detecting domoic acid as claimed in claim 4 is characterized in that, said centrifugal be the centrifugal 20min of 4000rpm.
6. the method for fast detecting domoic acid as claimed in claim 4 is characterized in that, the said number of times that repeats to extract is 2 times.
7. the method for fast detecting domoic acid as claimed in claim 4 is characterized in that, the aperture of said filter membrane is 0.22 μ m.
8. the method for fast detecting domoic acid as claimed in claim 2 is characterized in that, the temperature of said incubation is 37 ℃, and the time is 60min.
9. the method for fast detecting domoic acid as claimed in claim 2 is characterized in that, the said antibody that encapsulates use is anti-domoic acid monoclone antibody, and 4 ℃ encapsulate and spend the night.
10. the method for fast detecting domoic acid as claimed in claim 2 is characterized in that, said confining liquid is that massfraction is 1% gelatin, and 37 ℃ of sealing 60min process the good ELISA Plate of sealing.
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Citations (6)
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|---|---|---|---|---|
| CN1588073A (en) * | 2004-07-15 | 2005-03-02 | 上海交通大学 | Indirect enzyme-linked immunosorbent test method for quantitative detecting red tide hinge algae |
| CN1892224A (en) * | 2005-07-06 | 2007-01-10 | 国家海洋环境监测中心 | Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison |
| CN1979170A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Paralytic shellfish-poison competitive enzyme-lined immue quantitative detecting reagent box, its preparing and use |
| CN1979171A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Amnestic shellfish-poison competitive immue quantitative detection reagent box, its preparation and use |
| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN101949923A (en) * | 2010-09-16 | 2011-01-19 | 上海交通大学 | Enzyme-linked immunization kit of domoic acid and detection method thereof |
-
2011
- 2011-12-26 CN CN2011104424767A patent/CN102539746A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588073A (en) * | 2004-07-15 | 2005-03-02 | 上海交通大学 | Indirect enzyme-linked immunosorbent test method for quantitative detecting red tide hinge algae |
| CN1892224A (en) * | 2005-07-06 | 2007-01-10 | 国家海洋环境监测中心 | Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison |
| CN1979170A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Paralytic shellfish-poison competitive enzyme-lined immue quantitative detecting reagent box, its preparing and use |
| CN1979171A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Amnestic shellfish-poison competitive immue quantitative detection reagent box, its preparation and use |
| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN101949923A (en) * | 2010-09-16 | 2011-01-19 | 上海交通大学 | Enzyme-linked immunization kit of domoic acid and detection method thereof |
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Application publication date: 20120704 |