CN101813697A - Broad-spectrum pesticide residue immunity test strip and preparation method and application thereof - Google Patents
Broad-spectrum pesticide residue immunity test strip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a broad-spectrum pesticide residue immunity test strip, which adopts a nitrocellulose membrane containing monoclonal or polyclonal antibodies for identifying the specificity of different organic phosphorus and different synthetic pyrethroid insecticides. The invention also discloses a preparation method and an application of the test strip. The test strip has the advantages that the measurement speed is fast and no particular device is required.
Description
Technical field
The present invention relates to a kind of detection test paper that is used for residues of pesticides and its production and application, especially a kind ofly can be used for detecting multiple organophosphorus and multiple pyrethroid pesticide remained immunity test strip and its production and application.
Background technology
Agricultural chemicals is that current agricultural production is used for anti-curing the disease, worm, weeds be to the indispensable material of murrain, to promoting that agricultural produce has epochmaking effect, but bring serious environmental problem simultaneously: disruption of ecological balance, quality of agricultural product descends, and human body health is compromised.At present, organophosphorus, pyrethroid pesticide have insecticidal effect efficiently, are the main forces of chemical protection of plant.Along with the cry of environment in recent years protection is more and more higher, organophosphorus, pyrethroid pesticide use the environmental problem paid more and more attention of bringing.
But with regard to present situation, still hunger stares at sb. in the face for the mankind, poor puzzlement, and the use of completely forbidding agricultural chemicals is unpractical, in order to reach highly efficient and productive target, the status of agricultural chemicals can't replace in a short time.Given this, be to ensure people health, rationally the using and its residual quantity is monitored of agricultural chemicals during effectively control is produced developed a kind of fast, reliable, sensitive, and the trace analysis method that is suitable for the residues of pesticides on-site supervision has important practical significance.The residue analysis method of organophosphorus, pyrethrin pesticide mainly is complicated pre-treatment process at present, is equipped with efficient HPLC or GC (band ECD) again, and this detection method expense is relatively costly, and the operating personnel that must be skilled in technique.Immunoassay is a kind of quick, sensitive method that is used for trace detection, but agricultural chemicals mostly is micromolecular compound, the synthetic difficulty of immunogene is bigger, again because antibody has specificity, many of the immune analysis methods of setting up are applicable to the check and analysis of single persticide residue, have limited the application of immune analysis method, and existing in addition immunoassay is many to be solid phase carrier with the polystyrene ELISA Plate, testing result generally needs 3-8h, the testing process complexity.
Summary of the invention
The object of the present invention is to provide a kind of immunity test strip that contains the broad spectrum activity labelled antibody and its production and application.Have the advantages that finding speed soon, does not need special instruments and equipment.
For reaching above-mentioned purpose, the present invention adopts following technical scheme:
A kind of broad-spectrum pesticide residue immunity test strip, this test paper adopts to contain has the monoclonal of specific recognition or the nitrocellulose filter test paper of polyclonal antibody to multiple organophosphorus and multiple pyrethroid insecticides.
It is to be haptens with organophosphorus or pyrethrin pesticide universal architecture that described multiple organophosphorus or multiple pyrethroid insecticides have the monoclonal antibody of specific recognition, by the active ester method synthetic immunogen, immune balb/c mice, every mouse 100 μ g immunogenes, even with equal-volume Fu Shi Freund's complete adjuvant mixing and emulsifying, inject in the abdominal cavity film along groin; After 4 weeks, booster immunization, dosage is constant, and adjuvant changes the Fu Shi Freund into; Behind the booster immunization three times, blood sampling is surveyed mouse and is tired, when treating that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10: 1 ratio is mixed in the 50ml centrifuge tube and merges, cell after the fusion treats that after cultivating the hybrid cell quantity in the hole reaches 300 when above, get the cell culture supernatant enzyme linked immunosorbent detection, the each detection is the detection of multiple hole, second day duplicate detection after each the detection is with confirmed results; Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the cell in all positive hole is the hybridoma cell strain of secrete monoclonal antibody; Be used for preparing ascites through after the enlarged culture; Carry the last week with 0.5ml paraffin oil injection mouse peritoneal, with 10
6Individual hybridoma is suspended in the 1ml serum free medium, injects in the mouse peritoneal; After 10 days, mouse web portion obviously expands, and collects ascites; Come monoclonal antibody purification with caprylic acid-ammonium sulfate precipitation method.
It is to be haptens with organophosphorus or pyrethrin pesticide universal architecture that described multiple organophosphorus and multiple pyrethroid insecticides have the polyclonal antibody of specific recognition, by the active ester method synthetic immunogen, the immunity new zealand white rabbit, when reaching the best when tiring, the heart blood sampling, with behind the ammonium sulfate step-by-step precipitation method preliminary purification, be further purified high-quality broad spectrum activity organophosphorus antibody or the pyrethroid antibody that obtains through Sephadex G-200 more earlier.
A kind of preparation method of broad-spectrum pesticide residue immunity test strip, its preparation method comprises the steps:
1) at first adopts nitrocellulose filter (NCM), and be named as A1 respectively, A2, B1 and B2 its every by 2 * 2 0.5cm * 0.5cm size;
2), then with 1 μ L organophosphorus coating buffer drop in A1, A2 central authorities, 1 μ L pyrethroid coating buffer drop is in B1, B2 central authorities fill up thieving paper below, 4 ℃ are spent the night, and in PBST rinsing 3 times, dry;
3) being will be through step 2, again) 4 nitrocellulose filters after handling are immersed in respectively and soak 30min in the confining liquid, and rinsing is 3 times in PBST, dries;
4) secondly be that A1, B1 after handling through step 3) are immersed in the premix of organophosphorus antibody, pyrethroid antibody and testing sample extract; A2, B2 are immersed in the premix of organophosphorus antibody, pyrethroid antibody and negative sample extract, sample in contrast, and rinsing 3 times in PBST is dried behind the 10min;
5) and then 4 nitrocellulose filters after will handling through step 4) be immersed in 10min in the ELIAS secondary antibody dilution respectively, rinsing is 3 times in PBST, dries;
6) 4 nitrocellulose filters after will handling through step 5), again are immersed in substrate reactions liquid dark place reaction 5min respectively, and rinsing is 3 times in PBST, dries;
7), last visual observation, the negative sample of A2, B2 forms specificity brown precipitation on nitrocellulose filter, and the positive sample of A1, B1 does not develop the color or shows light brown at nitrocellulose filter, A1, B1 is with regard to the test paper of preparation cost invention detection usefulness.
Described organophosphorus coating buffer is meant the PBS dilution of diethyl phosphonic acids acetate and ovalbumin conjugate, and the pyrethroid coating buffer is meant the PBS dilution of m-phenoxybenzoic acid and ovalbumin conjugate.
Described confining liquid is the PBS dilution of 1% ovalbumin.
Described sample extracting solution is meant and adds acetonitrile 30mL homogenate 1min in 20g dish sample, and add 5-7g sodium chloride behind the suction filtration and saltout, getting its supernatant is sample extracting solution, and with organophosphorus antibody and pyrethroid antibody premixed 10min after to form premix to be measured.
A kind of application of broad-spectrum pesticide residue immunity test strip, take by weighing the crops sample of 10g chopping, add acetonitrile 30ml homogenate 1min, adding 5-7g sodium chloride behind the suction filtration saltouts, getting its supernatant is sample extracting solution, keep flat after adopting the thieving paper end of pesticide residue immunity test strip bar of the broad spectrum activity of method of the present invention preparation to immerse to take out behind the sample liquid 10s, observations behind the 10min, as have only the check plot to develop the color, the expression sample is negative, if sample area band colour developing, show in the crops sample that organophosphorus or pyrethrin pesticide are residual.
The invention has the beneficial effects as follows: the present invention is directed to the practical problems that exists in the present Pesticides Testing, a kind of immunity test strip that contains the broad spectrum activity labelled antibody and its production and application is provided.Immunity test strip of the present invention is a kind of effective tool of fast detecting pesticide residue, can carry out qualitative and half-quantitative detection to the total residual of a class agricultural chemicals, compare with immunologic detection method on the present general plate, have finding speed and soon, do not need special instruments and equipment, and easy and simple to handle, quick, economic advantage is the emphasis of present technical development.Be highly suitable for on-the-spot detection of agricultural product agricultural chemicals and testing laboratory to the scalping of batch sample.
Description of drawings
Fig. 1 is a ultraviolet spectrogram of the present invention.
Embodiment
Embodiment 1: the preparation method of multiple organophosphorus insecticides immune detection antibody
1), the preparation method 1 of artificial antigen
Immunogen preparing: with diethyl phosphonic acids acetate is haptens, get the diethyl phosphonic acids acetate and the N-hydroxy-succinamide (NHS) of equimolar amounts, N, N '-dicyclohexyl carbimide (DCC), with dioxane potpourri is dissolved, lucifuge reaction is spent the night under the room temperature, and after removing post precipitation and getting the supernatant drying, residue is suspended in 3 milliliters of borate buffer solutions that are dissolved with bovine serum albumin (BSA).Potpourri is magnetic agitation 1 hour at room temperature, 4 ℃ of phosphate buffers to 1 liter of pH7.4 (PBS) dialysis.
Envelope antigen: be the conjugate of haptens and ovalbumin (OVA), the preparation method is identical with immunogene.
Dialyzed sample is carried out full wavelength scanner with ultraviolet scanner and is identified the coupling situation, and diethyl phosphonic acids acetate has maximum absorption band at the 232nm place after measured, according to OD separately
232Value calculations incorporated ratio.
2), the preparation method 2 of artificial antigen
Immunogen preparing: take by weighing diethyl phosphonic acids acetate earlier and be dissolved in the dimethyl formamide (DMF), low temperature stirs.Water intaking dissolubility carbodiimide (EDC) is dissolved among the 1mlDMF, slowly joins among the above-mentioned DMF, and other gets BSA20mg and is dissolved among the 2mlDMF, and 4 ℃ of stirrings slowly join in the above-mentioned mixed liquor.4 ℃ of stirring reaction 8h spend the night in 14 ℃ of reactions again, and the centrifugal 10min of 2000g next day gets precipitation, and dialysis, authentication step is the same.
Envelope antigen: be the conjugate of haptens and ovalbumin (OVA), the preparation method is identical with immunogene.
Artificial antigen sample by above-mentioned 2 kinds of methods preparation carries out drawing after the ultra-violet analysis: as shown in Figure 1, a and b are 2 artificial antigen samples by method for preparing artificial antigen 1 (active ester method) preparation among the figure; C and d are 2 artificial antigen samples by method for preparing artificial antigen 2 (carbodiimide method) preparation among the figure; 3 kinds of materials are respectively carrier protein BSA, carrier protein BSA and hapten conjugation thing, haptens from top to bottom among the figure.At 254nm place haptens one trough is arranged as can be seen, carrier-antigen conjugates also has trough to occur, and the stack of absorption peak takes place herein for the two.At 210nm, 238nm place tangible absorption superposition phenomenon is arranged also in addition.Illustrate reaction has taken place between the two, can infer haptens and carrier protein BSA success coupling.According to above-mentioned estimation method, can determine 3 kinds of also successful couplings of material among Fig. 2-b, 2-c, the 2-d.
3), Polyclonal Antibody Preparation and purifying
Immune programme for children is: at first, dosage is every kilogram of rabbit body weight 1mg immunogene, and the Jia Fushi Freund's complete adjuvant is made water in oil emulsion with antigen, the subcutaneous multi-point injection in back; Every the immunity for the second time of 2 weeks, Jia Fushi Freund; After this, every 3 week immunity 1 time, method is with immunity for the second time; The 4th beginning, each immunity back one all rabbit ear edge veins are adopted a small amount of blood, and separation of serum adopts the indirect competitive ELISA method to measure antibody titer.Tire qualified after, carry out last 1 immunity, with the physiological saline dilution auricular vein injection of doubled amount antigen, the back heart blood sampling of 1 week causes death, and separates antiserum, and adopts sad-two step of ammonium sulfate precipitation method antibody purification, make freeze-dried powder, standby in-20 ℃ of preservations.Utilize the square formation titrimetry that two kinds of antiserums are detected respectively with two kinds of coating antigens, determine best Ag-Ab combination and best effort concentration thereof.
Multiple organophosphorus antibody titer measurement result sees Table 1, as can be seen from Table 1, compares relatively poor with the effect of EDC method (method for preparing artificial antigen 2) synthetic antigen immune animal with active ester method (method for preparing artificial antigen 1).The antibody that active ester type antigen produces has faint recognition capability to the antigen of EDC method preparation; The antibody that EDC type antigen produces to the coating antigen of active ester method preparation without any effect.Main cause may be that the antigen of EDC method preparation can not make antigenic determinant fully expose, so do not obtain the antibody of high affinity.Secretory antibody is tired, and to be that 25600 rabbit is tired the highest, with this antibody as further research.Utilize this antibody to carry out the square formation titration, the gained result shows that antigen, antibody best effort concentration are 2000 times.
4), antibody is to the cross reaction of different organophosphorus medicaments,
Table 2 is an antibody to the cross reaction of different agricultural chemicals indicator gauge as a result, and as can be seen from Table 2, gained antibody has produced recognition reaction to Multiple Pesticides.Antibody resists former guide's thing diethyl phosphonic acids acetate stronger affinity, I
50Be 0.0143 μ g/mL.The selective difference of this method between Rogor and omethoate is bigger, and the two structural difference is that P=O is different with the P=S key, and all the other groups are all identical.Antibody has very strong affinity to omethoate, but very weak to the affinity of Rogor, this shows that in the process of antigenic activation immuning system generating antibody, the P=O key has played conclusive effect.But antibody is to the IC of chlopyrifos, basudin, ethyl parathion
50All less than 1 μ g/mL, these 3 kinds of agricultural chemicals all contain common structure (C
2H
5O-), C is described
2H
5O-also exists as an antigenic determinant, plays important effect.Profenofos is because contain a C
2H
5O--and a P=O, and make antibody stronger recognition reaction be arranged to it, I
50Be 0.97 μ g/mL.It is relatively poor that isocarbophos, malathion, parathion-methyl etc. suppress ability to antigen-antibody reaction, illustrates that antibody does not have or have only faint recognition capability to these medicaments.From above result as can be seen, at diethyl phosphonic acids acetate c-terminus coupling carrier albumen, can make the C on the phosphonyl group
2H
5O--and a P=O fully expose as antigenic determinant, stimulate animal to produce antibody, and the organophosphorus insecticides that contains this universal architecture is had affinity widely.
Certainly present embodiment also can adopt monoclonal antibody, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying are to be haptens with the organophosphorus insecticide universal architecture, by the active ester method synthetic immunogen, immune balb/c mice, every mouse 100 μ g immunogenes, even with equal-volume Fu Shi Freund's complete adjuvant mixing and emulsifying, inject in the abdominal cavity film along groin; After 4 weeks, booster immunization, dosage is constant, and adjuvant changes the Fu Shi Freund into; Behind the booster immunization three times, blood sampling is surveyed mouse and is tired, when treating that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10: 1 ratio is mixed in the 50ml centrifuge tube and merges, cell after the fusion treats that after cultivating the hybrid cell quantity in the hole reaches 300 when above, get the cell culture supernatant enzyme linked immunosorbent detection, the each detection is the detection of multiple hole, second day duplicate detection after each the detection is with confirmed results; Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the cell in all positive hole is the hybridoma cell strain of secrete monoclonal antibody; Be used for preparing ascites through after the enlarged culture; Carry the last week with 0.5ml paraffin oil injection mouse peritoneal, with 10
6Individual hybridoma is suspended in the 1ml serum free medium, injects in the mouse peritoneal; After 10 days, mouse web portion obviously expands, and collects ascites; Come monoclonal antibody purification with caprylic acid-ammonium sulfate precipitation method.
Embodiment 2: the preparation method of multiple pyrethroid insecticides immune detection antibody
1, Antibody Preparation: with the m-phenoxybenzoic acid is haptens, is equipped with artificial immunity antigen and envelope antigen by active ester method and 6-aminocaprolc acid legal system.By the male new zealand white rabbit of subcutaneous multi-point injection immune health, prepare polyclonal antibody after 6 months with artificial immunogen.
2, purifying antibody: get 5ML antibody and equivalent physiological saline in stirring the saturated ammonium sulfate that dropwise adds equivalent down, place down for 4 ℃ and spend the night, it is fully precipitated, centrifugal 3000 change 20min, abandon supernatant, be precipitated to 12ml with physiological saline solution, dropwise adding saturated sulfuric acid, put 4 ℃ of preservations and spend the night to 18ml, repeat the above-mentioned second step process 1 time, the centrifugal back of last gained sediment is dissolved to 5ml with 0.02mol/l PBS, and the dialysis of packing into is with dialysing among the 0.02mol/lPBS.
3, polyclonal antibody detects pyrethroid pesticide
Antibody can be discerned Permethrin, decis, cypermethrin, decis, Cyhalothrin, the result shows that PBA-antibody and CPA-antibody can not only discern haptens, and identification contains the haptens structure at interior macromolecular compound, and decis, cypermethrin, decis cross reaction also have the identification of certain intersection.
Certainly present embodiment also can adopt monoclonal antibody, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying are to be haptens with the pyrethrin pesticide universal architecture, by the active ester method synthetic immunogen, immune balb/c mice, every mouse 100 μ g immunogenes, even with equal-volume Fu Shi Freund's complete adjuvant mixing and emulsifying, inject in the abdominal cavity film along groin; After 4 weeks, booster immunization, dosage is constant, and adjuvant changes the Fu Shi Freund into; Behind the booster immunization three times, blood sampling is surveyed mouse and is tired, when treating that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10: 1 ratio is mixed in the 50ml centrifuge tube and merges, cell after the fusion treats that after cultivating the hybrid cell quantity in the hole reaches 300 when above, get the cell culture supernatant enzyme linked immunosorbent detection, the each detection is the detection of multiple hole, second day duplicate detection after each the detection is with confirmed results; Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the cell in all positive hole is the hybridoma cell strain of secrete monoclonal antibody; Be used for preparing ascites through after the enlarged culture; Carry the last week with 0.5ml paraffin oil injection mouse peritoneal, with 10
6Individual hybridoma is suspended in the 1ml serum free medium, injects in the mouse peritoneal; After 10 days, mouse web portion obviously expands, and collects ascites; Come monoclonal antibody purification with caprylic acid-ammonium sulfate precipitation method.
Embodiment 3: the preparation method of immunity test strip
Set up enzyme-linked immunosorbent assay earlier, optimize the condition of work of antigen, antibody, and further study the quick detection test paper best operating condition on this basis.
This test paper is by nitrocellulose filter (the NCM) (A1 of 2 * 2 0.5cm * 0.5cm size, A2, B1 B2) forms, its mensuration program is: 1, with 1 μ L organophosphorus coating buffer drop in A1, A2 central authorities, 1 μ L pyrethroid coating buffer drop is in B1, B2 central authorities, fill up thieving paper below, 4 ℃ are spent the night, and in PBST rinsing 3 times, dry.2, NCM is immersed in soaks 30min in the confining liquid, rinsing is 3 times in PBST, dries.3, A1, B1 are immersed in the premix of organophosphorus antibody, pyrethroid antibody and testing sample extract, A2, B2 are immersed in the premix of organophosphorus antibody, pyrethroid antibody and negative sample extract (comparing), rinsing 3 times in PBST is dried behind the 10min; 4, NCM is immersed in 10min in the ELIAS secondary antibody dilution, rinsing is 3 times in PBST, dries; 5, NCM is immersed in substrate reactions liquid dark place reaction 5min, rinsing is 3 times in PBST, dries; 6, visual observation, the negative sample of A2, B2 form specificity brown precipitation on nitrocellulose filter, and the positive sample of A1, B1 does not develop the color or shows light brown at nitrocellulose filter, and invention detects the test paper of usefulness with regard to preparation cost for A1, B1.
Embodiment 4: organophosphorus and pyrethroid pesticide are residual in the application immunity test strip detection cucumber
Take by weighing the cucumber sample of 10g chopping, add acetonitrile 30ml homogenate 1min, adding 5-7g sodium chloride behind the suction filtration saltouts, getting its supernatant is sample extracting solution, the thieving paper end of the test strips of embodiment 3 preparation immersed keep flat after taking out behind the sample liquid 10s, observations behind the 10min, as have only the check plot colour developing, the expression sample is negative.If sample area band colour developing, show in the cucumber sample that organophosphorus or pyrethrin pesticide are residual.The colour developing of the sample sample area band of present embodiment shows in the cucumber sample that organophosphorus or pyrethrin pesticide are residual.
Embodiment 5: using immunity test strip, to detect in the little green vegetables organophosphorus and pyrethroid pesticide residual
Take by weighing 20g dish sample, add acetonitrile 50ml homogenate 1min, adding 5-7g sodium chloride behind the suction filtration saltouts, getting its supernatant is sample extracting solution, the thieving paper end of the test strips of embodiment 3 preparation immersed to take out behind the sample liquid 10s keep flat, the 10min observations has only the check plot to develop the color, the expression sample is negative, shows that not have organophosphorus or pyrethrin pesticide in the dish sample residual.
Many kinds of organophosphorus antibody titers of table 1 measurement result
Table 2 antibody is to the cross reaction of different agricultural chemicals
Claims (7)
1. broad-spectrum pesticide residue immunity test strip is characterized in that: this test paper adopts to contain has the monoclonal of specific recognition or the nitrocellulose filter test paper of polyclonal antibody to multiple organophosphorus and multiple pyrethroid insecticides.
2. a kind of broad-spectrum pesticide residue immunity test strip as claimed in claim 1, it is characterized in that: it is to be haptens with organophosphorus or pyrethrin pesticide universal architecture that described multiple organophosphorus or multiple pyrethroid insecticides have the polyclonal antibody of specific recognition, by the active ester method synthetic immunogen, the immunity new zealand white rabbit, when reaching the best when tiring, the heart blood sampling, with behind the ammonium sulfate step-by-step precipitation method preliminary purification, be further purified high-quality broad spectrum activity organophosphorus antibody or the pyrethroid antibody that obtains through Sephadex G-200 more earlier.
3. the preparation method of a broad-spectrum pesticide residue immunity test strip as claimed in claim 1, it is characterized in that: its preparation method comprises the steps:
1) at first adopts nitrocellulose filter, and be named as A1 respectively, A2, B1 and B2 its every by 2 * 2 0.5cm * 0.5cm size;
2), then with 1 μ L organophosphorus coating buffer drop in A1, A2 central authorities, 1 μ L pyrethroid coating buffer drop is in B1, B2 central authorities fill up thieving paper below, 4 ℃ are spent the night, and in PBST rinsing 3 times, dry;
3) being will be through step 2, again) 4 nitrocellulose filters after handling are immersed in respectively and soak 30min in the confining liquid, and rinsing is 3 times in PBST, dries;
4) secondly be that A1, B1 after handling through step 3) are immersed in the premix of organophosphorus antibody, pyrethroid antibody and testing sample extract; A2, B2 are immersed in the premix of organophosphorus antibody, pyrethroid antibody and negative sample extract, sample in contrast, and rinsing 3 times in PBST is dried behind the 10min;
5) and then 4 nitrocellulose filters after will handling through step 4) be immersed in 10min in the ELIAS secondary antibody dilution respectively, rinsing is 3 times in PBST, dries;
6) 4 nitrocellulose filters after will handling through step 5), again are immersed in substrate reactions liquid dark place reaction 5min respectively, and rinsing is 3 times in PBST, dries;
7), last visual observation, the negative sample of A2, B2 forms specificity brown precipitation on nitrocellulose filter, and the positive sample of A1, B1 does not develop the color or shows light brown at nitrocellulose filter, A1, B1 is with regard to the test paper of preparation cost invention detection usefulness.
4. the preparation method of a kind of broad-spectrum pesticide residue immunity test strip as claimed in claim 3, it is characterized in that: described organophosphorus coating buffer is meant the PBS dilution of diethyl phosphonic acids acetate and ovalbumin conjugate, and the pyrethroid coating buffer is meant the PBS dilution of m-phenoxybenzoic acid and ovalbumin conjugate.
5. the preparation method of a kind of broad-spectrum pesticide residue immunity test strip as claimed in claim 3, it is characterized in that: described confining liquid is the PBS dilution of 1% ovalbumin.
6. the preparation method of a kind of broad-spectrum pesticide residue immunity test strip as claimed in claim 3, it is characterized in that: described sample extracting solution is meant and adds acetonitrile 30mL homogenate 1min in 20g dish sample, adding 5-7g sodium chloride behind the suction filtration saltouts, getting its supernatant is sample extracting solution, and with organophosphorus antibody and pyrethroid antibody premixed 10min after to form premix to be measured.
7. the application of a broad-spectrum pesticide residue immunity test strip, it is characterized in that: the crops sample that takes by weighing the 10g chopping, add acetonitrile 30ml homogenate 1min, adding 5-7g sodium chloride behind the suction filtration saltouts, getting its supernatant is sample extracting solution, keep flat after adopting the thieving paper end of pesticide residue immunity test strip bar of the broad spectrum activity of method of the present invention preparation to immerse to take out behind the sample liquid 10s, observations behind the 10min, as have only the check plot to develop the color, the expression sample is negative, if sample area band colour developing, show in the crops sample that organophosphorus or pyrethrin pesticide are residual.
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