CN101949923A - Enzyme-linked immunization kit of domoic acid and detection method thereof - Google Patents
Enzyme-linked immunization kit of domoic acid and detection method thereof Download PDFInfo
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- CN101949923A CN101949923A CN2010102834546A CN201010283454A CN101949923A CN 101949923 A CN101949923 A CN 101949923A CN 2010102834546 A CN2010102834546 A CN 2010102834546A CN 201010283454 A CN201010283454 A CN 201010283454A CN 101949923 A CN101949923 A CN 101949923A
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Abstract
The invention discloses an enzyme-linked immunization kit of domoic acid, belonging to the technical field of biological detection. The kit comprises a specific antibody containing the domoic acid, an enzyme labeled plate coated with a domoic acid antigen, an enzyme labeled secondary antibody, a domoic acid standard solution, a coating buffer solution, a confining liquid, a cleaning solution, antibody diluent, a color developing agent and a stopping solution. The detection method comprises the following steps: detecting a preprocessed sample by the kit; adding a standard substance or sample solution and the antibody diluent into the enzyme labeled plate holes coated with the domoic acid antigen; incubating, washing and drying by patting; adding the enzyme-labeled secondary antibody; further incubating, washing and drying by patting; developing, stopping, determining the absorbance value by an elisa reader; and analyzing the detection result. The enzyme-linked immunization kit of the invention has the characteristics of high specificity, high sensitivity, high accuracy and the like, and has the advantages of low instrument and equipment requirement, long reagent storage time, high automatic degree and no pollution and the like.
Description
Technical field
What the present invention relates to is a kind of detection kit and detection method thereof of technical field of biological, specifically is the enzyme linked immunological kit and the detection method thereof of domoic acid in a kind of shellfish sample.
Background technology
(Domoic Acid DA) is a kind of of red-tide toxin to domoic acid, because the characteristic symptoms after its poisoning is a loss of memory, so claim losing the memory of property shellfish poison again.DA is from a kind of large-scale red algae---branch cartilage algae is separating obtained the earliest.Molecular formula is C
15H
21NO
6, molecular weight is 311.34, and pure product are the white solid powder, and fusing point is 223～224 ℃, and water soluble (8mg/mL) is slightly soluble in methyl alcohol (0.6mg/mL), and the maximum absorption wavelength in the ultraviolet spectrum district is 242nm.DA is mainly produced by marine diatom, by the enrichment of sea life institute, not only has suitable ecological risk by food chain, has threatened human health especially.There are some researches show that dysgenesias such as premature labor, miscarriage can appear in the edible fish that polluted by DA of sea lion.There are some researches show also that under the exposure of low dosage DA study and the memory capability of newborn mouse have produced permanent damage.The PI incident that ate the Mytilus galloprovincialis that is polluted by DA in Canadian Prince Edward Island generation in 1987 has caused people's common concern, the poisoner produces vomiting, diarrhoea, memory confusion, the loss of memory, direction identification obstacle have appearred in some of the staff, even dead.Therefore various countries have formulated the aquatic products standard of 20 μ g/g shellfish meat in succession.China does not have relevant examination criteria at present.
Set up several different methods for the analysis of DA so far, what be used for conventional sense mainly is mouse bioassay detection method, high performance liquid chromatography (HPLC) detection method and immunodetection.The mouse bioassay method is progressively eliminated because detectability does not reach requirement, but because its method of operating is simple, equipment requirements is low still to be used by some countries, and high performance liquid chromatography is with its low detectability, and high sensitivity is subjected to the favor of various countries, has become the standard method of many countries.But high performance liquid chromatography is to instrument and technician's requirement height, and is not suitable for the detection of a large amount of samples.And immunological method just in time combines the advantage of above-mentioned two kinds of methods, and instrumentation is simple, detectability is lower, is applicable to the conventional sense of department of basic unit.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of enzyme linked immunological kit and detection method thereof of domoic acid is provided, it is low to make that the satisfied pre-treatment to sample of the present invention requires, and can also reduce the operation steps of kit; To instrument and equipment require low, the reagent holding time is long, automaticity is high, advantage such as pollution-free.
The present invention relates to a kind of enzyme linked immunological kit of domoic acid, comprising: comprised the domoic acid specific antibody, bag is cushioned liquid, confining liquid, cleansing solution, antibody diluent, developer and stop buffer by the ELISA Plate of domoic acid antigen, ELIAS secondary antibody, domoic acid standard solution, bag;
Described ELISA Plate adopts 96 or 40 hole ELISA Plate, be coated with can with the domoic acid antigen of anti-domoic acid antibody specific bond, and the site of domoic acid antigen is not adsorbed on the closed porosity surface.
Described domoic acid specific antibody is the anti-domoic acid polyclonal antibody of rabbit.
Described domoic acid antigen is to adopt carbodlimide method, active ester method or mixed anhydride method that domoic acid micromolecule haptens and carrier protein are carried out coupling to obtain.
It is the horseradish peroxidase-labeled goat anti-rabbit igg antibody that described enzyme labeling two resists.
It is 0.05-0.1mol/L that described bag is cushioned liquid, the carbonate buffer solution of pH9.6.
Described confining liquid is the poly-vinyl alcohol solution of 1-3%.
Described cleansing solution is the phosphate buffer that contains the 0.05-5% Tween-20.
Described antibody diluent is the polyglycol solution that contains 4-10%.
Described colour developing liquid is to be made up of developer A and developer B, and developer A is a hydrogen peroxide, and developer B is a tetramethyl benzidine.
Described stop buffer is the sulfuric acid solution of 1-5mol/L.
The present invention relates to the detection method that the mentioned reagent box is used for shellfish sample domoic acid, may further comprise the steps:
1. sample pre-treatments
2. with above-mentioned kit sample is detected, in the ELISA Plate hole that is coated with domoic acid antigen, add standard items or sample solution and antibody diluent;
3. washing pats dry behind the incubation, adds ELIAS secondary antibody again;
4. further wash behind the incubation and pat dry, colour developing, termination are measured absorbance with microplate reader;
5. analyzing and testing result.
The detection principle of kit of the present invention is for to be adsorbed in the domoic acid envelope antigen on the solid phase carrier, add series standard product or sample solution and domoic acid specific polyclonal antibody dilution, the antigenic competition binding specificity antibody of domoic acid in the testing sample and pre-bag quilt, add ELIAS secondary antibody and carry out the enzymatic activity amplification, the colour developing back stops.The content of sample absorbance and its residue domoic acid is negative correlation, relatively can draw the concentration range of domoic acid in the sample with typical curve.
The enzyme linked immunological kit of detection domoic acid of the present invention mainly adopts the content of domoic acid in the qualitative or detection by quantitative shellfish sample of indirect competitive ELISA method; Pre-treatment to sample requires low.Adopt the domoic acid polyclonal antibody of high specific, main agents provides with the form of working fluid, can reduce the operation steps of kit, for the user saves time and reduces the error that causes because of operation steps is miscellaneous, the present invention have the specificity height, highly sensitive, degree of accuracy is high and characteristics such as accuracy height, to instrument and equipment require low, the reagent holding time is long, automaticity is high, advantage such as pollution-free, be applicable to the qualitative and quantitative analysis of batch samples screening, can in the fast detecting of shellfish food, play a significant role.
Description of drawings
Fig. 1 domoic acid typical curve.
Embodiment
Following example will the invention will be further described in conjunction with the accompanying drawings.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
The preparation of reagent constituents
1.1 the domoic acid artificial antigen is synthetic
Get 1mg domoic acid (DA) and be dissolved in a small amount of solvent (dimethyl sulfoxide (DMSO): water=1: 9), add 0.8mg N-hydroxy-succinamide (20mg/mL, face with now joining), 1.2mg water-soluble carbodiimide (10mg/mL, face with now joining) and a small amount of 0.1mol/L phosphate buffer (pH 7.0), mix and be placed on 25 ℃ of incubator internal reaction 2h.Be transferred to subsequently in 4 ℃ of refrigerators, placement is spent the night.Added 2mg keyhole limpet hemocyanin (KLH) or bovine serum albumin(BSA) (BSA) in second day, and added a small amount of 0.1mol/L phosphate buffer, mix and be placed on 25 ℃ of incubator internal reaction 3h.Mixed liquor inserted in the ultrafiltration pipe manage, behind the centrifugal 15min of 4500rpm, an amount of dilution is stored in-20 ℃ after the packing.
1.2 the preparation of domoic acid specific polyclonal antibody
Above-mentioned domoic acid-KLH conjugate is diluted to the 1mg/ml solution for standby with physiological saline.
Select 4 body weight 1.8-2kg Healthy female new zealand white rabbits for use.Conjugate and equivalent Freund's complete adjuvant are mixed into water in oil emulsion by syringe, press the amount first immunisation of 1mg/kg body weight, take the subcutaneous multi-point injection of nape portion.Every two all booster immunizations once, replace Freund's complete adjuvant, the same first immunisation of dosage and method with incomplete Freund.From immunity for the third time, back 7 days of each immunity, auricular vein is got blood 1ml, carry out antibody titer and detect, when antibody titer no longer raises, do not add adjuvant and carry out for the last time (the 9th time) immunity, heart bloodletting after 7 days, after room temperature leaves standstill 30min, 37 ℃ of incubation 1h, make blood clotting, back 4 ℃ are spent the night, centrifugal 15 minutes of 4000r/min, remove clot, the serum part is sad-and the ammonium sulfate method carries out the purifying dialysis, promptly gets polyclonal antibody, after the packing, stored frozen.
1.3 the preparation of ELISA Plate
Be cushioned liquid (0.05-0.1mol/L, the carbonate buffer solution of pH9.6) with bag the conjugate DA-BSA of domoic acid and bovine serum albumin(BSA) (BSA) is diluted to 1ug/ml, the every hole of 96 orifice plates bag is by 100 μ l, and 4 ℃ are spent the night.Pour out liquid in the hole, with cleansing solution vibration washing 3 times, each 3min pats dry.Every hole adds 200 μ l confining liquids (1% poly-vinyl alcohol solution), 37 ℃ of sealing 2h.Pour out liquid in the hole, preserve with the vacuum seal of aluminium film dry back.
Detect the establishment of domoic acid enzyme linked immunological kit
Set up the enzyme linked immunological kit that detects domoic acid, make it comprise following component:
(1) bag is by the ELISA Plate of domoic acid and bovine serum albumin(BSA) (BSA) conjugate;
(2) concentration is the domoic acid specific polyclonal antibody working fluid of 8.4ug/L;
(3) goat anti-rabbit igg antibody of horseradish peroxidase-labeled;
(4) domoic acid standard solution, concentration are respectively 0ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1 μ g/mL, 10 μ g/mL;
(5) to be cushioned liquid be 0.05mol/L to bag, the carbonate buffer solution of pH9.6;
(6) confining liquid is 1% poly-vinyl alcohol solution;
(7) cleansing solution is the phosphate buffer that contains 0.05% Tween-20;
(8) antibody diluent is to contain 4% polyglycol solution;
(9) substrate colour developing liquid is made up of developer A and developer B, and developer A is a hydrogen peroxide, and developer B is a tetramethyl benzidine;
(10) reaction terminating liquid is the sulfuric acid solution of 1mol/L.
The detection of domoic acid in the embodiment 3 shellfish samples
3.1 sample pre-treatments
Get fresh band shell or freezing after the sample that at room temperature partly thaws, cut closed shell flesh with cutter and take out shellfish meat, edible part is separated with mid intestinal gland, be placed on draining 5min on the tiny wire netting of mesh.Get the 5g sample, add 10mL homogenate (50% methanol aqueous solution), after the homogenate, vortex oscillation 1min, ultrasonic Extraction 5min; The centrifugal 20min of 4000rpm.Shift out supernatant to the 25mL volumetric flask, residual residue adds homogenate 5mL again, repeats to extract twice, and supernatant all moves in the 25mL volumetric flask, the distilled water constant volume.0.22 μ m membrane filtration.
3.2 detect with kit
In the ELISA Plate micropore of domoic acid haptens and ovoserum albumen (BSA) conjugate bag quilt, add the polyclonal antibody of 50 μ l with the antibody diluent dilution of 4% polyglycol, simultaneously, every hole adds testing sample 50 μ l again, and every block of plate is done graticule simultaneously, 37 ℃ of incubation 1h.Wash 3 times, pat dry with thieving paper.Add the goat-anti rabbit two anti-working fluid 100 μ l of horseradish peroxidase-labeled, 37 ℃ of incubation 1h wash 5 times, pat dry.Every hole adds freshly prepared substrate colour developing liquid 100 μ l, and every hole adds stop buffer 50 μ l behind the colour developing 10min.The light shaking mixing is measured the absorbance in every hole with microplate reader.
3.3 interpretation of result
Calculate the percentage absorbance, the drawing standard curve;
Each the concentration standard solution that is obtained or the mean value (A) of sample absorbance are divided by the absorbance (A of first standard (0 standard)
0) multiply by 100% again, i.e. inhibiting rate.
Inhibiting rate (%)=(A/A
0* 100%)
A is the mean light absorbency value of standard solution or sample solution in the formula, A
0Mean light absorbency value for the 0ng/mL standard solution.Logarithm with domoic acid standard series concentration is an ordinate as horizontal ordinate, with the inhibiting rate, the drawing standard curve, calibration curve has favorable linearity at 0.01～10 μ g/mL, and the concentration of domoic acid can be read from typical curve in corresponding each sample.
The kit sensitivity determination
Utilize the domoic acid standard solution to react, according to experimental result drawing standard curve (Fig. 1), can be got by Fig. 1: straight-line equation is Y=109.40-15.45X, inhibiting rate (A/A0) concerns that with logarithm value significant linear in concentration is 0.01～10 μ g/mL scope of domoic acid related coefficient is R
2=0.9682, with inhibiting rate be 90% o'clock concentration as lowest detectable limit, can get lowest detectable limit and be about 18ng/mL.
Recovery experiment
Get the domoic acid standard specimen of 3 concentration, add in the sample, each concentration is established 6 repetitions, measures.
The result of the kit recovery is as follows, and the red heart shellfish is 95.2～101.3%, and scallop is 49.6～84.7%, and clam is 47.2～65%
The experiment of kit storage life
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value of kit (zero adds), 50% inhibition concentration, domoic acid added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed for two weeks under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 12 months at 2-8 ℃.
Claims (10)
1. enzyme linked immunological kit that detects domoic acid in the shellfish sample, it is characterized in that, comprising: comprised the domoic acid specific antibody, bag is cushioned liquid, confining liquid, cleansing solution, antibody diluent, developer and stop buffer by the ELISA Plate of domoic acid antigen, ELIAS secondary antibody, domoic acid standard solution, bag;
Described ELISA Plate adopts 96 or 40 hole ELISA Plate, be coated with can with the domoic acid antigen of anti-domoic acid antibody specific bond, and the site of domoic acid antigen is not adsorbed on the closed porosity surface.
2. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described domoic acid specific antibody is the anti-domoic acid polyclonal antibody of rabbit.
3. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described domoic acid antigen is to adopt carbodlimide method, active ester method or mixed anhydride method that domoic acid micromolecule haptens and carrier protein are carried out coupling to obtain.
4. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, it is the horseradish peroxidase-labeled goat anti-rabbit igg antibody that described enzyme labeling two resists.
5. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, it is 0.05-0.1mol/L that described bag is cushioned liquid, the carbonate buffer solution of pH9.6.
6. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described confining liquid is the poly-vinyl alcohol solution of 1-3%.
7. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described cleansing solution is the phosphate buffer that contains the 0.05-5% Tween-20.
8. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described antibody diluent is the polyglycol solution that contains 4-10%.
9. the enzyme linked immunological kit of detection domoic acid according to claim 1 is characterized in that, described colour developing liquid is to be made up of developer A and developer B, and developer A is a hydrogen peroxide, and developer B is a tetramethyl benzidine.
10. a kit according to claim 1 detects the method for domoic acid in the shellfish sample, it is characterized in that, may further comprise the steps:
1. sample pre-treatments;
2. with above-mentioned kit sample is detected, in the ELISA Plate hole that is coated with domoic acid antigen, add standard items or sample solution and antibody diluent;
3. washing pats dry behind the incubation, adds ELIAS secondary antibody again;
4. further wash behind the incubation and pat dry, colour developing, termination are measured absorbance with microplate reader;
5. analyzing and testing result.
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Cited By (4)
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| CN102680693A (en) * | 2012-05-18 | 2012-09-19 | 上海交通大学 | Domoic acid collaurum immunochromatographic test strip and preparation method thereof |
| CN104345143A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Technology for improving activity of enzyme-labeled working fluid anti-human IgA alkaline phosphatase |
| WO2019169798A1 (en) * | 2018-03-07 | 2019-09-12 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody repertoire detection kit and preparation method therefor |
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