CN101424686A - ELISA reagent for detecting malachite green and method - Google Patents
ELISA reagent for detecting malachite green and method Download PDFInfo
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- CN101424686A CN101424686A CNA2008102027338A CN200810202733A CN101424686A CN 101424686 A CN101424686 A CN 101424686A CN A2008102027338 A CNA2008102027338 A CN A2008102027338A CN 200810202733 A CN200810202733 A CN 200810202733A CN 101424686 A CN101424686 A CN 101424686A
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Abstract
The invention discloses an Elisa agent for detecting malachite green and a method thereof. The Elisa agent comprises a malachite green antigen or antibody coated Elisa plate, an enzyme label, a malachite green specific antibody, a malachite green standard solution, a substrate developing solution, a stop solution, a concentrated cleaning solution, enzyme label diluents and a concentrated combined solution. The Elisa agent is used for the quantitative detection of the content of the malachite green in fishes, shrimps and water samples and has the advantages of high specificity and sensitivity, easy sample pretreatment, short detection time, large sample detection amount, and the like.
Description
Technical field
The present invention relates to a kind of ELISA reagent and the method that detects malachite green in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
(Malachite Green, MG) as antifungal agent, anti-exophytic parasite medicine and sanitizer are widely used in the fish culture industry malachite green.But, recent studies have shown that malachite green has high carcinogenic.So many countries all classify malachite green as the aquaculture forbidden drugs.China is also listed malachite green in " veterinary drug and the compound inventory thereof of food animal forbidding " in May, 2002, forbids being used for all food animals.Yet because malachite green is cheap, it is remarkable to kill mould effect, therefore still prohibits in aquaculture and exhausted.
In order to prevent that malachite green vestigial from entering food chain, culturist and supervision department of government need a kind of quick, accurate and responsive technology to detect it.At present, malachite green vestigial detects two kinds of conventional method: HPLC and LC/MS, but they all compare costliness and sample preparation get up very complicated, during check fee.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of enzyme linked immunological kit that detects malachite green in aquatic products and the water body, it contains:
(1) bag by the elisa plate of malachite green antigen or the bag by the elisa plate of antiantibody;
(2) enzyme labeling thing;
(3) malachite green specific antibody;
(4) malachite green standard solution;
(5) substrate colour developing liquid;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) enzyme labeling thing dilution;
(9) concentrate redissolution liquid.
The malachite green envelope antigen adopts active ester method that malachite green haptens and bovine gamma globulin(BGG) are carried out coupling to obtain in the elisa plate of the present invention, antiantibody can be sheep anti mouse antiantibody or goat-anti rabbit antiantibody, the sheep anti mouse antiantibody is to be that immunogene is carried out immunity to the pathogen-free domestic goat and obtained with mouse source antibody, and goat-anti rabbit antiantibody is to be that immunogene is carried out immunity to the pathogen-free domestic goat and obtained with rabbit source antibody.Used bag is cushioned the carbonate buffer solution that liquid is pH value 9.6,0.05mol/L in the preparation elisa plate process; Bag be can be polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc. by the carrier mass of malachite green antigen or antiantibody; The form of carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.; Used cleansing solution is for containing 0.1%～0.5% Tween 80, the phosphate buffer of 0.5% sodium azide; Used confining liquid is to contain 8%～15% the horse serum and the solution of 1% inert protein.
The enzyme labeling thing is enzyme labeling antiantibody or enzyme labeling malachite green antigen in the kit of the present invention, and used enzyme can be peroxidase or the sweet enzyme of galactose, the preferred peroxidase of the present invention; Enzyme labeling thing form can be freeze-dried powder, concentrate and working fluid; The used dilution of enzyme labeling thing working fluid is for containing the solution of 50% glycerine (can prevent that the enzyme labeling thing of putting into-20 ℃ of environment from freezing, also can keep the biologically active of enzyme labeling thing for a long time), 1% sodium azide antiseptic (being convenient to preserve).
The preparation process of enzyme labeling antiantibody is in the kit of the present invention:
(1) preparation of antiantibody: with the mouse endogenous antibody is that immunogene is carried out immunity to the pathogen-free domestic goat, obtains the sheep anti mouse antiantibody; Or be that immunogene is carried out immunity to the pathogen-free domestic goat with the rabbit endogenous antibody, obtain goat-anti rabbit antiantibody.
(2) preparation of peroxidase labelling antiantibody: antiantibody and peroxidase (HRP) are carried out coupling, the method that adopts is a glutaraldehyde method, adopt glutaraldehyde method to make the combination rate of antiantibody and horseradish peroxidase raise, tradition GA single stage method coupling reaction is wayward, spontaneous polymerization easily takes place in the fast molecule of reaction velocity, and coupling efficiency is not high.In order to address these problems, we improve single stage method, have overcome the shortcoming of single stage method.At first in by two kinds of molecules of coupling, the molecule more weak with the coupling agent reflection activates with excessive coupling agent earlier, then the unnecessary coupling agent in place to go; Second step was connected an end with certain molecule coupling agent couples together with another kind of molecule by changing reaction conditions.Though the two step method operation is more numerous, coupling efficiency improves, and the same Molecularly Imprinted Polymer that forms reduces.
Enzyme-labelled antigen is to adopt active ester method that marker enzyme and malachite green haptens are carried out coupling to obtain in the kit of the present invention.
The malachite green specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody in the kit of the present invention, and immunogene adopts active ester method that malachite green haptens and key hole maple hemocyanin are carried out coupling and obtains; Antibody formation can be freeze-dried powder, concentrate, working fluid; Antibody diluent is pH value 8.2,0.05mol/L, contain the phosphate buffer of 3% horse serum and 5 ‰ gelatin.
In the kit of the present invention when marker enzyme is peroxidase substrate colour developing liquid be that hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, stop buffer are sulfuric acid or the hydrochloride buffer of 0.1～0.5mol/L; When marker enzyme was the sweet enzyme of galactose, substrate colour developing liquid was the 0.5mol/L kaliumphosphate buffer, and stop buffer is the citrate buffer solution of 2mol/L; Concentrated cleaning solution is for containing 0.1%～0.5% Tween 80, the phosphate buffer of 0.5% sodium azide; Concentrate and redissolve liquid for containing 20% methyl alcohol, 1% calf serum (BSA) phosphate buffer.
Selected single substrate solution among the present invention first for use,, operated more easy compared to two substrate solutions (need add 2 kinds of solution during use) of other similar kits.
The malachite green standard solution is the malachite green solution of six concentration gradients in the kit of the present invention, and the malachite green dilution is a phosphate buffer.
The preparation of reagent is specially in the kit of the present invention:
A. malachite green standard solution: 6 bottles of malachite green series standard solution, concentration are 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 1～3ml/ bottle.
B. bag is cushioned liquid: the pH value is 9.6, the carbonate buffer solution of 0.05mol/L.
C. confining liquid: the solution of 8%～15% horse serum and 1% inert protein.
D. concentrated cleaning solution: contain 0.1%～0.5% Tween 80, the phosphate buffer of 0.5% sodium azide, 1 bottle.
E. enzyme labeling thing: enzyme labeling antiantibody working fluid or enzyme labeling malachite green antigen working fluid, 7～12ml/ bottle, 1 bottle.
F. substrate colour developing liquid is the mixed liquor of hydrogen peroxide or urea peroxide and o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
G. stop buffer: 1～2mol/L sulfuric acid, hydrochloric acid or 2mol/L sodium hydrate buffer solution, 5～8ml/ bottle, 1 bottle.
H. antibody work dilution: for pH value 8.2,0.05mol/L, contain the phosphate buffer of 3% horse serum and 5 ‰ gelatin.
I. concentrate redissolution liquid: contain 20% methyl alcohol, 1% calf serum (BSA) phosphate buffer, 30～50ml/ bottle, 1 bottle.
The method of malachite green in detection aquatic products of the present invention and the water body has comprised following steps:
1. sample pre-treatments
Take by weighing 5g behind the sample homogeneous in clean centrifuge tube; Add 10ml acetonitrile (100%), fully mix 30min; The centrifugal 10min of room temperature 4000g pipettes supernatant in centrifuge tube, with the neutral post purifying of JTBaker Alumina1000/6ml 3ml filtrate (flow velocity 1-2 drips/second) (detailed process saw below for 4 steps); Add 3ml acetonitrile (100%) and wash post; Add the 3ml sample extracting solution and cross post; Collect in the new pipe of sample extracting solution to.Add 3ml acetonitrile (100%) and wash post, collect in the supreme pipe of extract; To mix 10 times of eluate dilutions (50ul acetonitrile extract+450ul concentrates the redissolution liquid) with concentrating redissolution liquid; Pipetting 75ul analyzes.Sample extension rate: 20
2. detect with kit
In 96 hole ELISA Plate micropores of malachite green coupled antigen bag quilt, add series standard product or sample solution (each 2 hole) 75 μ l, add malachite green antibody working fluid 75 μ l again,, react 30min in 25 ℃ of constant temperature ovens with cover plate film shrouding.Pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole after 10 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds enzyme labeling thing 150 μ l, with cover plate film shrouding, reacts 30min in 25 ℃ of constant temperature ovens, repeated washing work.Add substrate colour developing liquid 150 μ l, the mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuge colour developing 30min.Every hole adds stop buffer 100 μ l, and the mixing that vibrates is gently measured the every hole of 450nm absorbance with microplate reader.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained, the absorbance (Bo) divided by first standard solution (0 standard) multiply by 100% again, obtains the percentage absorbance.Semilog with malachite green concentration is the x axle, and the percentage absorbance is a Y-axis, the drawing standard curve map.Percentage absorbance with same way calculation sample solution, in the percentage absorbance substitution typical curve with sample solution, read the pairing concentration of malachite green the sample solution from typical curve, multiply by the actual concentrations that its corresponding extension rate is malachite green in the sample solution.
Detection principle of the present invention is:
Be that malachite green haptens and bovine gamma globulin(BGG) conjugate (CAP-BGG) are adsorbed on the solid phase carrier when coating antigen is the malachite green coupled antigen, add sample or malachite green standard items, add the malachite green specific antibody then, the malachite green antigenic competition malachite green specific antibody of bag quilt on residual malachite green and the ELISA Plate in the testing sample.Add the enzyme labeling antiantibody again and carry out the amplification of enzymatic activity, the colour developing back stops, the absorbance of working sample, and the malachite green residual quantity is negative correlation in this value and the sample, relatively can draw the concentration of malachite green with typical curve.
Coating antigen is that antiantibody is adsorbed on the solid phase carrier during for antiantibody, add the malachite green specific antibody, add malachite green enzyme-labelled antigen and sample or malachite green standard solution again, residual malachite green and enzyme labeling malachite green antigenic competition malachite green specific antibody in the sample to be tested, the colour developing back stops, the absorbance of working sample, the malachite green residual quantity is negative correlation in this value and the sample, relatively can draw the concentration of malachite green with typical curve, but with the standard solution color concentration range of malachite green in the judgement sample more then.
Enzyme linked immunoassay testing cassete of the present invention, utilize emulative integrated enzyme reaction principle, the content of quantitative detecting analysis malachite green in fish, shrimp and water sample has high specificity, highly sensitive, advantages such as sample pretreatment is simple, and detection time is short, the detection sample size is big.
Description of drawings
Fig. 1 is the examination criteria curve map of malachite green.
Embodiment
Below described the specific embodiment of the present invention, but it can not be interpreted as limitation of the invention, its alternative, modifications and changes of making have all been dropped in protection scope of the present invention with this area routine techniques means.
1. antigen is synthetic
A. coating antigen is synthetic
Adopt derivative method to synthesize the malachite green haptens malachite green, again haptens is carried out coupling by diazo-reaction and bovine gamma globulin(BGG) carrier protein with active ester method and obtain.
B. immunogenic synthetic
The malachite green haptens is carried out coupling by diazo-reaction and key hole maple hemocyanin carrier protein with active ester method to be obtained.
2. the preparation of malachite green mouse monoclonal antibody
A. animal immune
Adopt the Balb/c mouse as immune animal, with malachite green haptens and key hole maple hemocyanin conjugate is immunogene, immunizing dose is 300 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 2 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole in 5:1 ratio and SP2/0 myeloma cell.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
C. cell cryopreservation and recovery
Get the hybridoma that is in exponential phase and make 5 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt in the body and induce method, the Balb/c mouse peritoneal injection in 8 ages in week is only sterilized paraffin oil 0.5ml/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
6Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, bottle packing, 20 ℃ of preservations.
3. the preparation of malachite green rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with malachite green haptens and key hole maple hemocyanin conjugate is immunogene, immunizing dose is 3mg/kg, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 7 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
4. the preparation of ELISA Plate
Bag by the elisa plate of malachite green antigen or bag by the preparation process of the elisa plate of antiantibody is in the kit of the present invention:
(1) is cushioned liquid with bag the malachite green haptens is become antigenic dilution or antiantibody dilution with bovine gamma globulin(BGG) (BGG) conjugate or antiantibody with 0.02～0.08 μ g/ml concentration dilution;
(2) in every hole of elisa plate, add 100 μ l and diluted good antigenic dilution or antiantibody dilution, 37 ℃ of incubation 2h, the coating buffer that inclines, with cleansing solution washing 1 time, each 15～30s pats dry;
(3) in every hole of elisa plate, add 150～200 μ l confining liquids, 37 ℃ of incubation 1～2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
The elisa plate of above method preparation has good stability, and through cold and hot stability test, the correlation technique parameter of elisa plate is all in normal range, and coating antigen has good specificity.
Embodiment 2 detects the establishment of malachite green monoclonal enzyme linked immunological kit
Set up the enzyme linked immunological kit that detects malachite green, make it comprise following component:
(1) bag is by the elisa plate of malachite green antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) malachite green mouse monoclonal antibody;
(4) the malachite green standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L;
(5) substrate colour developing liquid is the mixed liquor of hydrogen peroxide or urea peroxide and o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
(6) stop buffer is the phosphate buffer of 2mol/L;
(7) concentrated cleaning solution: contain 0.1%～0.5% Tween 80, the phosphate buffer of 0.5% sodium azide;
(8) antibody diluent is pH value 8.2,0.05mol/L, contains the phosphate buffer of 3% horse serum and 5 ‰ gelatin.
(9) concentrate redissolution liquid for containing the phosphate buffer of 20% methyl alcohol, 1% calf serum (BSA).
Embodiment 3 detect malachite green polyclone enzyme linked immunoassay reagent kits establishment
Set up the enzyme linked immunological kit that detects malachite green, make it comprise following component:
(1) bag is by the elisa plate of goat-anti rabbit antiantibody;
(2) the malachite green antigen of usefulness alkaline phosphate ester enzyme labeling;
(3) malachite green rabbit polyclonal antibody;
(4) the malachite green standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L;
(5) substrate colour developing liquid is to the nitro phosphate buffer;
(6) stop buffer is the sodium hydrate buffer solution of 2mol/L;
(7) concentrated cleaning solution contains 0.1%～0.5% Tween 80, the phosphate buffer of 0.5% sodium azide, 1 bottle;
(8) concentrate redissolution liquid for containing the phosphate buffer of 20% methyl alcohol, 1% calf serum (BSA).
The detection of malachite green vestigial in the embodiment 4 aquatic products samples
(1) sample pre-treatments;
(2) detect with kit of the present invention;
(3) analyzing and testing result.
Sample-pretreating method is among the present invention:
The sample pre-treatment need be prepared:
The 1mL acetonitrile is mixed with the concentrated redissolution liquid that 9mL provides.
The enzyme labeling thing that kit provides be 25 times concentrated.Before each the detection, the enzyme labeling thing that concentrates dilutes with enzyme labeling thing dilution.For example, the doubly concentrated enzyme labeling thing of 40ul25 mixes with 1ml enzyme labeling thing dilution to obtain enzyme labeling thing working fluid.
The preparation of standard specimen
Storing standard specimen need dilute with 10% second eyeball/diluted sample damping fluid (V/V), allots 0,0.01,0.05,0.1,0.5 and the malachite green standard specimen of 1ug/L (ppb).
1ppb standard specimen: 40ul 100ppb storage liquid and 4mL standard sample dilution liquid
0.5ppb standard specimen: 1mL 1ppb standard specimen and 1mL standard sample dilution liquid
0.1ppb standard specimen: 200ul 1ppb standard specimen and 1.8mL standard sample dilution liquid
0.05ppb standard specimen: 1mL 0.1ppb standard specimen and 1mL standard sample dilution liquid
0.01ppb standard specimen: 200ul 0.05ppb standard specimen and 800ul standard sample dilution liquid
(a) aquatic products (shrimp, fish etc.) pre-treating method
1. take by weighing 5g behind the sample homogeneous in clean centrifuge tube;
2. add 10ml acetonitrile (100%), fully mix 30min;
3. the centrifugal 10min of room temperature 4000g;
4. pipette supernatant in centrifuge tube, with the neutral post purifying of JTBaker Alumina1000/6ml 3ml filtrate (flow velocity 1-2 drips/second) (detailed process saw below for 4 steps);
5. add 3ml acetonitrile (100%) and wash post;
6. add the 3ml sample extracting solution and cross post;
7. collect in the new pipe of sample extracting solution to.
8. add 3ml acetonitrile (100%) and wash post. collect in extract to 7 pipe.
9. will mix 10 times of eluate dilutions (the concentrated liquid that redissolve of 50ul acetonitrile extract+450ul) with concentrating redissolution liquid;
10. pipetting 75ul analyzes.
Sample extension rate: 20
(b) water sample
Getting 50 μ l clarification water sample is used for analyzing.
Detect with kit of the present invention:
1, from 4 ℃ of cold storage environment, takes out required reagent, put room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses.
2, take out micropore and the framework that needs quantity, no micropore is put into valve bag, be stored in 2-8 ℃.
3, numbering: the corresponding micropore of sample and standard items is numbered according to the order of sequence, and it is parallel that each sample and standard items are done 2 holes, and the position at record standard hole and sample aperture place.
4, add standard items/sample 75 μ l/ holes in micropore separately, add antibody working fluid 75 μ l/ holes then, with cover plate film shrouding, light shaking mixing.React 30min in 25 ℃ of environment.
5, take out liquid drying in the hole, wash plate 4-5 time, each 10 seconds at interval, pat dry (bubble that is not eliminated after patting dry can puncture with clean rifle head) with thieving paper with washing lotion 250 μ l/ holes.
6, every hole adds enzyme labeling thing 150 μ l, reacts 30min in the rearmounted 25 ℃ of environment of cover plate membrane cover plate.Liquid in the hole is dried, fully wash with cleansing solution, 4-5 pats dry (bubble that is not eliminated after patting dry can puncture with clean rifle head) all over (the same) with thieving paper.
7, colour developing: every hole adds substrate 150 μ l, the mixing that vibrates gently, lucifuge colour developing 30min in 25 ℃ of environment.
8, measure: every hole adds stop buffer 100 μ l, and the mixing that vibrates is gently set microplate reader and (run through data in 5min) in the 450nm place, measures every hole OD value.
The result judges
The result judges two kinds of methods, judges available the 1st kind of method roughly, quantitatively judges with the 2nd kind of method.Notice that sample light absorption value malachite green contained with it is inversely proportional to.
1, rough judgement:
Mean light absorbency value and standard value with sample relatively can draw its concentration range (ppb).The absorbance of supposing sample 1 is 0.250, and the absorbance of sample 2 is 0.720, and the titer absorbance is respectively: 0ppb is 1.610; 0.01ppb be 1.380; 0.05ppb be 1.100; 0.1ppb be 0.620; 0.5ppb be 0.289; 1ppb is 0.108.Then the concentration range of sample 1 is 0.5ppb-1ppb; The concentration range of sample 2 is 0.05ppb-0.1ppb.
2, quantitative test
(1) calculating of percentage absorptance, the percentage absorptance of standard items or sample equal the mean value (diplopore) of percentage absorbance of standard items or sample divided by the absorbance of first standard (0 standard), multiply by 100% again, promptly
The mean light absorbency value of B-standard solution or sample solution
B
0The mean light absorbency value of-0ng/ml standard solution
(2) drafting of typical curve and calculating
With standard items percentage absorptance is ordinate, is horizontal ordinate with the semilog of malachite green standard items concentration (ng/ml), the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be malachite green actual concentrations in the sample from typical curve.If utilize kit specialty analysis software to calculate, accurate, the express-analysis of the great amount of samples of being more convenient for.
The detection of malachite green vestigial in embodiment 5 water body examples
1. sample pre-treatments
Directly getting 75ul clarification water sample analyzes.
2. detect with kit
Method is with embodiment 4
3. testing result analysis
Method is with embodiment 4
The test of experimental example 1 standard items precision
From every batch of elisa plate according to the preparation of the method the embodiment 1 (4), each extracts 10 micropores out, measures 0.5 μ g/L.The absorbance of standard solution (OD value) repeats 3 times, calculates coefficient of variation CV%, the results are shown in Table 1.。
The repeatable test of table 1 standard (CV%)
The result shows coefficient of variation scope between 4.3%～9.7%, has met the coefficient of variation less than 20% regulation, illustrates that this kit standard items precision has reached standard.
The repeatable test of experimental example 2 samples
With the malachite green of 0.5 μ g/L concentration, add in fishes and shrimps and the water body example, get each five of the kits of three different batches respectively, each concentration repeats 5 times, calculates the coefficient of variation respectively, the results are shown in Table 2, table 3.
The repeatable test of table 2 fishes and shrimps sample
But table 3 water body example repeated experiments
The result shows that the fishes and shrimps sample coefficient of variation all is lower than 20%, the Variation Lines number average of water body sample is lower than 20%, has met the coefficient of variation less than 25% regulation, illustrates that the precision of this kit measurement sample has reached standard.
The specificity of experimental example 3 antibody:
Specificity is meant the recognition capability of antibody to determinand, emphasizes the selectivity of association reaction between antibody and determinand and the separating capacity of or related substances close to structure.Specificity depends on the cross reaction of determinand and other materials.In competition analysis, the cross reacting rate of different material can calculate with following formula:
Cross reacting rate (%)=IC50 (competition thing)/IC50 (determinand) * 100
| Compound | Cross reacting rate (%) |
| Malachite green | 100 |
| Concealed malachite green | <1 |
| Crystal violet | 120 |
| Recessive crystal violet | <1 |
The accuracy test of experimental example 4 kits
Get the malachite green standard solution of two concentration, be respectively 0.5 μ g/kg (L) and 1 μ g/kg (L), respectively sample is added recovery test, each concentration do 4 parallel, calculate recovery rate respectively.
The accuracy of table 4 kit
The result shows the interpolation recovery of fishes and shrimps between 78%～98%, and water sample adds the recovery between 80%～98%.
Experimental example 5
The kit preservation condition is 2～8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, malachite green added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under the condition of 37 ℃ of preservations, 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 ℃.
Experimental example 6
Get the malachite green standard solution of two concentration, be respectively 50 μ g/kg (L) and 100 μ g/kg (L), respectively sample is added recovery test, each concentration do 4 parallel, run a curve according to experimental data and to calculate its recovery respectively.
The titer absorbance is respectively: 0ppb is 2.045; 0.01ppb be 1.800; 0.05ppb be 1.235; 0.1ppb be 0.850; 0.5ppb be 0.350; 1ppb is 0.133
The shading value of sample liquid clingfish sample: blank pipe is 2.015; 0.5ppb be 0.36; 0.381; 0.343; 0.359; 1ppb is 0.135; 0.143; 0.142; 0.150; Water sample: blank pipe is 1.995; 0.5ppb be 0.361; 0.354; 0.367; 0.384; 1ppb is 0.142; 0.139; 0.152; 0.148; Formulate typical curve (Fig. 1) according to standard items OD value
The accuracy of table 5 kit
The result shows the recovery of adding between 84%～99% in fish, add the recovery in the water sample between 82%～96%.
Claims (10)
1. enzyme linked immunological kit that detects malachite green in aquatic products and the water body is characterized in that it contains:
(1) bag by the elisa plate of malachite green antigen or the bag by the elisa plate of antiantibody;
(2) enzyme labeling thing;
(3) malachite green specific antibody;
(4) malachite green standard solution;
(5) substrate colour developing liquid;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) enzyme labeling thing dilution;
(9) concentrate redissolution liquid.
The malachite green envelope antigen adopts active ester method that malachite green haptens and bovine gamma globulin(BGG) are carried out coupling to obtain in the kit, antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody, the sheep anti mouse antiantibody is to be that immunogene is carried out immunity to the pathogen-free domestic goat and obtained with mouse source antibody, and goat-anti rabbit antiantibody is to be that immunogene is carried out immunity to the pathogen-free domestic goat and obtained with rabbit source antibody.
2. the enzyme linked immunological kit of malachite green is characterized in that in detection aquatic products as claimed in claim 1 and the water body, and the enzyme labeling thing is enzyme labeling antiantibody or enzyme labeling malachite green antigen in the kit, and used enzyme is peroxidase or the sweet enzyme of galactose; Enzyme labeling thing form is freeze-dried powder, concentrate or working fluid.
3. the enzyme linked immunological kit of malachite green is characterized in that in detection aquatic products as claimed in claim 1 and the water body, and used enzyme is a peroxidase.
4. as the enzyme linked immunological kit of malachite green in claim 2 or 3 described detection aquatic products and the water body, it is characterized in that enzyme labeling malachite green antigen is to adopt active ester method that marker enzyme and malachite green haptens are carried out coupling to obtain in the kit.
5. the enzyme linked immunological kit of malachite green in detection aquatic products as claimed in claim 4 and the water body, it is characterized in that, the malachite green specific antibody is mouse resource monoclonal antibody or rabbit source polyclonal antibody in the kit, and immunogene adopts active ester method that malachite green haptens and key hole maple hemocyanin are carried out coupling and obtains; Antibody formation is freeze-dried powder, concentrate or working fluid; Antibody diluent is pH value 8.2,0.05mol/L, contain the phosphate buffer of 3 volume % horse serums and 5 volumes, ‰ gelatin.
6. the enzyme linked immunological kit of malachite green in detection aquatic products as claimed in claim 5 and the water body, it is characterized in that, in the kit when marker enzyme is peroxidase substrate colour developing liquid be that hydrogen peroxide, urea peroxide or tetramethyl benzidine sulfate mixed solution, stop buffer are sulfuric acid or the hydrochloride buffer of 0.1～0.5mol/L; When marker enzyme was the sweet enzyme of galactose, substrate colour developing liquid was the 0.5mol/L kaliumphosphate buffer, and stop buffer is the citrate buffer solution of 2mol/L; Concentrated cleaning solution is for containing 0.1 volume %～0.5 volume % Tween 80, the phosphate buffer of 0.5 quality % sodium azide; Concentrating redissolution liquid is the phosphate buffer that contains 20 volume % methyl alcohol, 1 volume % calf serum.
7. the enzyme linked immunological kit of malachite green is characterized in that in detection aquatic products as claimed in claim 6 and the water body, and used bag is cushioned the carbonate buffer solution that liquid is pH value 9.6,0.05mol/L in the preparation elisa plate process; Bag is polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber or Ago-Gel by the carrier mass of malachite green antigen or antiantibody; The form of carrier is test tube, micro-reaction plate shrinkage pool, globule or sequin; Cleansing solution is for containing 0.1 volume %～0.5 volume % Tween 80, the phosphate buffer of 0.5% sodium azide; Confining liquid is to contain the horse serum of 15 volume %～30 volume % and the solution of 1% inert protein.
8. as the enzyme linked immunological kit of malachite green in claim 1-3 and arbitrary described detection aquatic products of 5-7 and the water body, it is characterized in that, the malachite green standard solution is the malachite green solution of six concentration gradients in the kit, the malachite green dilution is a phosphate buffer, the malachite green concentration of standard solution is: 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 1～3ml/ bottle.
9. the enzyme linked immunological kit of malachite green in detection aquatic products as claimed in claim 4 and the water body, it is characterized in that, the malachite green standard solution is the malachite green solution of six concentration gradients in the kit, the malachite green dilution is a phosphate buffer, the malachite green concentration of standard solution is: 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 1～3ml/ bottle.
10. method that detects malachite green in aquatic products and the water body has comprised following steps:
(1) sample pre-treatments;
(2) detect with each described kit of claim 1-9;
(3) analyzing and testing result.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102027338A CN101424686A (en) | 2008-11-14 | 2008-11-14 | ELISA reagent for detecting malachite green and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102027338A CN101424686A (en) | 2008-11-14 | 2008-11-14 | ELISA reagent for detecting malachite green and method |
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| CN101424686A true CN101424686A (en) | 2009-05-06 |
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| CNA2008102027338A Pending CN101424686A (en) | 2008-11-14 | 2008-11-14 | ELISA reagent for detecting malachite green and method |
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