CN1672049A - Rapid detection of bt-cry toxins - Google Patents
Rapid detection of bt-cry toxins Download PDFInfo
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- CN1672049A CN1672049A CNA038176416A CN03817641A CN1672049A CN 1672049 A CN1672049 A CN 1672049A CN A038176416 A CNA038176416 A CN A038176416A CN 03817641 A CN03817641 A CN 03817641A CN 1672049 A CN1672049 A CN 1672049A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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- G01N2333/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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Abstract
The present invention is based on the use of 1) two polyclonal IgGs against two Cry toxins, 2) Cry toxin receptors isolated from lepidopteran larvae and 3) manual striping methods to manufacture immunochromatographic strips that are useful in detecting a variety of analytes. Immunochromatographic strips, which facilitate rapid detection of analytes are expensive if made using monoclonal antibodies and cannot be affordable for Indian farmers and extension workers. The main objective of the current method was to simplify the development of the immunochromatographic detection method for Cry (Bt) toxin detection using affinity purified polyclonal antibodies specific to the analyte and also to provide a robust and easy method suitable for manufacture of 'Cry1Ac/Cry1Ab/Cry2Aa/Cry2Ab toxin' detection strips at affordable cost, under Indian conditions. Anti-Bt (anti-Cry1 Ac or anti-Cry2Aa/Ab or both) antibody or Cry receptor proteins are immobilized on a cellulose nitrate membrane by manual striping. The membrane is blocked using casein or BSA and preservatives. Anti-Bt (anti-Cry1 Ac or anti-Cry2Aa/Ab or both) antibody is labeled with gold and adsorbed on glass-fibre membrane which is placed at the bottom the membrane so as to overlap 2mm. Specificity and accuracy of the assay are greatly enhanced due to the use of antigen-affinity and Protein-A affinity purified polyclonal IgG raised in two different animals (goat and rabbit). The schematic diagram of the Cry toxin detection lateral flow strip assembly is shown in a diagram appended herewith. The strips thus made represent rapid, sensitive devices and methods for detecting the presence of Cry1Ac/Cry1Ab/Cry2Aa/Cry2Ab and crystal toxins either from transgenic plants or in Bt-fermented products.
Description
Technical field
The present invention relates to use the fast immune chromatographic of polyclonal antibody to measure, be used for the crystalline toxin Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab of detection side seed or plant tissue on fluxion strap.The crystalline toxin that its particular attention given is separated from natural bacillus thuringiensis (Bacillus thuringiensis) and with the method for polyclonal antibody is cultivated antibody and is resisted described crystalline toxin.Invent the mensuration that more specific simplification development that relates to stripping means by hand and specificity and accuracy greatly improve, raising is by using the affine and albumin A affinity purification polyclone IgG of the antigen of cultivating in two kinds of different animals-goats and the rabbit.Invention comprise use from the crystalline toxin acceptor of lepidopterous insects as catching aglucon to detect crystalline toxin.Invention also promote to detect simultaneously single with on two kinds of crystalline toxins.
Background technology
The soil bacteria bacillus thuringiensis generates some albumin crystals, and is poisonous but to host plant and environmentally friendly to insect.Transform host plant and make them can synthesize described toxin and resist insect with separating gene genetic from bacterium.Separation is used at insect pest resistance program hybridization converting cotton (Gossypium hirsutum) and some other crop plants from the gene (being also referred to as the Bt-gene usually) of bacillus thuringiensis, and gene such as Cry1 Ac, cry 1Ab, Cry2 Aa and Cry2 Ab are the most frequently used genes of this genetic transformation.
The method that the genetic transformation of affirmation plant must and have some to do like this to the hybridization program.In addition, the expression key of toxin in transforming plant is because this contratoxin is meaningful to the effect of insect target kind.If insect pest control must be effectively, it is most important characteristic that the Cry1 Ac in the genetically modified plants expresses, and it is important too therefore to detect its expression.Generally express, in addition,, test the possibility that becomes rapidly along with the appearance of the immune chromatography method that detects transgene expression with the quantitative toxin of standard ELISA method.Yet, when production process comprises use monoclonal antibody and some instruments, immunochromatography band costliness.
Summary of the invention
Purity and efficient (expression of quality toxin) that must the commercial Bt-genetically modified crops of assessment, assessment be in the commercial share of different phase and level-research, technical development and affirmation, environmental impact, seed quality control, cultivation field, production etc.The method of any rapid detection Bt-transgenic technology is useful, and in addition, cheap method is valuable.Another kind of selection can be to use polyclonal antibody to replace monoclonal antibody not influence with development immunochromatography band and detect the efficient that cry expresses in seed or the plant tissue, so it is a target of this research.Another target is to develop described band with minimum instrument, comprises that expense and price are lower.The another one target is to develop the method that detects described expression with polyclonal antibody, cultivates the anti-crystalline toxin that separates from natural bacillus thuringiensis bacterial strain of antibody.Method aims to provide strong and easy method, and method is applicable to that the development immunochromatographic measurement is to detect Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab.
Quantitatively tachysynthesis diagnostic test ((1988) one step device for immunochromatography U.S. Patent number such as Huang 5,712,172) generally is used for detection side and contrasts as plus or minus to the antigen of flow assay.The example of this mensuration comprises that conceived detection kit, AIDS detect and many urinalysis thing types.Developed based on the Cry1 Ac detection kit of immunochromatography detection method and respectively by two u s companys ' Agdia, USA ' and ' StrategicDiagnostic Inc, USA ' is in July calendar year 2001 and commercialization in October calendar year 2001.Two companies claim in the promotional document that all kit is the basis that is combined as with monoclonal antibody.Therefore, another target of development the inventive method is to simplify the immunochromatography detection method that detects Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab, and the polyclonal antibody of use is specific to analyte and complete crystalline toxin.
Principle: caught Cry1Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab and carry it along film by the polyclonal immunoglobulin of gold (IgG) by capillarity from animal such as rabbit or goat, bag, in conjunction with capture antibody line (from the polyclonal immunoglobulin (IgG) of animal such as rabbit or goat) or N-aminopeptidase or cadherin line, stride stripper wire in the middle of the film up to the free-end of Cry1 Ac/Cry1Ab/Cry2 Aa/Cry2 Ab.The IgG of bag quilt accumulates in and catches the IgG line and produce the visible signal that indication Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab toxin exists in the gold.When lacking Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab in the sample, the IgG of Jin Bao quilt advances along film, in conjunction with the anti-rabbit/goat antibody of goat/rabbit, gathers and produce visible signal.
Preparation N-aminopeptidase or cadherin: according to an aspect of invention, preparation BBM bubble uses difference precipitation and centrifugal method from the lepidopterous larvae internal organ, with sweet mellow wine, ethylene glycol-two (beta-amino ether)-N, N, N ', N '-tetraacethyl (EGTA) and magnesium chloride (MgCl
2), according to (1987) such as Wolfersberger " transhipment is determined from the amino acid preparation and the Partial Feature of the BBM bubble of cabbage butterfly larva (Pierris brassicae) midgut " (Preparation and partial characterization of amino acidtransporting brush border memebrane vescicles from the larval midgut ofthe cabbage butterfly (Pierris brassicae)) .Comp.Biochem.Physiol.86A, 301-308.The bead that contains Cry albumen N-aminopeptidase/cadherin acceptor is used to peel off as antigen capture albumen.
Antigen: according to second aspect of invention, (theInstitute of Microbial Technology (IMTECH), Chandigarh determine characteristic and determine peculiar property by antibiotic resistance distribution, colonial morphology and growth characteristics to isolate natural bacillus thuringiensis bacterial strain ' x '.The gene cry1Ac/cry2A that generates crystalline protein, is cloned in pUC18 and the pRK223-3 expression vector with this gene of special primer pcr amplification from the bacterial strain plasmid., chromatography centrifugal by difference and sds polyacrylamide gel electrophoresis be the purified crystals toxin from the clone.Purified toxins and the anti-particular toxin of cultivation antiserum.
Cultivate antibody: the crystalline toxin mixing Freund's complete adjuvant of purifying also injects rabbit/goat.Strengthened dosage at interval, the dissolving toxin in the use incomplete Freund, collection serum with every month.With ammonium sulfate and IgG precipitation serum, IgG purification is with DEAE cellulose, albumin A/Protein G post and/or finally use Cry1Ac/Cry2A antigen affinity column chromatography in case of necessity.
The conjugate of preparation aurosol and IgG.The preparation aurosol is to reduce golden chloride by citric acid in the coloured particle conjugate is synthetic, standard method and engineering philosophy were explained in the past in Horisberger, (1979) " assessment is as the aurosol of the cytochrome mark of transmission and scanning electron microscope " (Evaluation of Colloidal Gold asa Cytochromic Marker for Transmission and Scanning Electron Microscopy), Biol.Cellulaire, 36,253-258; Leuvering etc., (1980) " immunoassays of solution particle " (SolParticle Immunoassay), J Immunoassay, 1 (1): 77-91 and Frens (1973) " regulating single control nucleation of disperseing grain size in the golden suspending liquid " (Controlled Nucleation for the Regulationof the Particle Size in Monodisperse Gold Suspensions), Nature, PhysicalScience, 241:20-22.To put together gold and be applied to 30cm * 1cm conjugate release fiberglass packing from the affinity purification IgG antibody of a kind of animal according to the method for delivering in the described operation of above-mentioned these publications.
The set of preparation lateral flow.(30 * 6cm) bags are by acryloid cement and 2.5 * 30cm nylon/nitrocellulose/nitrocellulose filter band (S﹠amp for the polyester plastics sheet; S/Whatman/Millipore/Pall-Gelman) be bonded at the plastic lining back.Detect band and special IgG of crystalline toxin or crystalline toxin acceptor for 2 toxin, peel manually, detects for single toxin and to be with, only from film two ends 1.25cm as 1.25cm in the middle of film with from the line of 30cm * 0.1cm of film bottom 1cm from the special IgG of crystalline toxin.Peel manually is from goat anti-rabbit igg or the anti-goat IgG of rabbit, and it is the line from 30cm * 0.1cm of film top 0.5cm.Film seals with damping fluid, the phosphate-buffered saline washed twice, and damping fluid contains 2% casein/BSA and 3% skimmed milk power, and skimmed milk power contains sugar and antiseptic.Behind the bone dry film, do the combination bag and placed the film lower end with overlapping 2mm in the above by fiberglass packing.The thick fiber pad of 30cm * 1 * 0.1cm places conjugate to discharge the pad lower end and is bonded on the plastics backing effective coverage with overlapping 2mm and with acryloid cement.The fiber mat of another 30cm * 1 * 0.1cm places film upper end with overlapping 2mm and be bonded at acryloid cement on the film effective coverage of plastics backing.Make set stacked and be cut into 6cm * 0.5cm band with cold stacked (cold-lamination sheet).
Symptom: (be used for single toxin and detect band) in positive, 2 line-index strips occurring the control line of function and the sample wire of the indication sample positive.And in the sample that lacks Cry1 Ac/Cry 1Ab/Cry2 Aa/Cry2 Ab, the line that 1 index strip has function only appears; Sample detection is the toxin feminine gender.Design identical with on detect two kinds of toxin (Cry1A and Cry2A) simultaneously band in the sample all positive, 3 lines are arranged to two kinds of toxin, or 2 lines are only arranged in the sample positive to arbitrary toxin, if or sample all negative then 1 line (control line) is only arranged to two kinds of toxin.
Embodiment (1): preparation fast immune chromatographic mensuration/band is to detect Cry1Ac
1. separation of C ry1Ac from the clone, this is by ultrasonic degradation bacterial clone culture, precipitation is removed cell fragment and lysotoxin not.Toxin is dissolved in alkaline buffer and centrifugal extraction.Finish the toxin purifying by 25% saturated ammonium sulfate precipitation and polyacrylamide gel electrophoresis.
2. by injecting purified toxins is cultivated anti-Cry1Ac toxin in rabbit or goat antiserum respectively.
3. cultivate sero-fast method and be initial injection and Freund's complete adjuvant antigens mixed (Cry1Ac) and mix with incomplete Freund repeat to strengthen dosage, collect serum then, this ordinary skill can be available from immunology teaching material (" antibody-laboratory manual " (Antibodies-A laboratory Manual)-Harlow Ed and DavidLane; Cold Spring Harbor Laboratory, USA, 1988) and here do not describe in detail.
4. purifying Immunoglobulin IgG from antiserum, this is by ammonium sulfate precipitation, sediment is dissolved in damping fluid and makes it continuously through DEAE cellulose, albumin A and antigen (Cry1Ac toxin) affinity column.Method used herein is described in detail in " antibody-laboratory manual " (Harlow Ed and David Lane; Cold Spring HarborLaboratory, USA, 1988).
5. available from the IgG purification of antigen affinity purification 0.01M sodium phosphate buffer, pH7.2 dialyses.
6. peel off the anti-Cry1Ac-IgG that cultivates in rabbit/goat, as 2.5 * 30cm nylon/nitrocellulose/nitrocellulose filter band (S﹠amp; S/Whatman/Millipore/Pall-Gelman) on the side from the center line of top and bottom end 1.25cm distance, the long 30cm of thick 1mm uses hand-held sable hairbrush (numbering 0,00,000,1,2,3,4 or 5), supports along the 30cm chi.
7. anti-rabbit igg/anti-goat IgG (can be commercial available from some companies, comprise Sigma Chemical Company, USA) be dissolved in the 0.01M sodium phosphate buffer, pH7.2 and peel manually from, it is the line from 30cm * 0.1cm of film top 0.5cm.
Film under dry-air blower 50 ℃ dry 10-15 minute.
9. use the damping fluid closing membrane subsequently, damping fluid contains 2% casein/BSA and 3% skimmed milk power, and skimmed milk power contains 1-5% sucrose and sodium azide antiseptic, uses the 0.01M sodium phosphate buffer, the pH7.2 washed twice.
Film under dry-air blower 50 ℃ dry 10-15 minute.
11. (30 * 6cm) bags are sticked at by acryloid cement and film in the middle of the thin slice polyester plastics sheet, and are equidistant from the top and bottom end.
12. aurosol can be commercial available from Sigma Chemical Company, USA.The anti-Cry1Ac-IgG of affinity purification is diluted to 2mg/ml in the borate buffer of pH8.5 to 9.0, stir to add aurosol (pH transfers to 9.0).Stablize conjugate by adding 10%BSA (bovine serum albumin(BSA)) to potpourri.
13. conjugate is with 12, centrifugal 30 minutes of 4 ℃ of 000g are to obtain the pine precipitation.Precipitation is dissolved in 100ml solution, and solution contains 0.01M Tris, 5%BSA, 2% sucrose, 0.87%NaCl and 0.1M sodium azide.
14. aurosol puts together that solution is applied to that 30cm * 1cm conjugate discharges fiberglass packing and under dry-air blower dry 10-15 minute.
15. the fiberglass packing of dried conjugate bag quilt places the described film of step 10 lower end with overlapping 2mm in the above.
16.30 the thick fiber pad of * 1 * 0.1cm places conjugate to discharge the pad lower end and is bonded on the plastics backing effective coverage with overlapping 2mm and with acryloid cement.Here the indication end is the bottom.
17. the fiber mat of another 30 * 1 * 0.1cm places film upper end with overlapping 2mm and be bonded at acryloid cement on the film effective coverage of plastics backing.Here the indication end is the top.
18. make set stacked and be cut into 6cm * 0.5cm band with cold stacked.
19. plant tissue (ca.50mg) pulverizes among the pH7.2 as 0.5ml 0.01M sodium phosphate buffers in 1.5ml microcentrifugation plastic bottle such as leaf, stem, root, flower, seeds, uses the Teflon pestle.
20. the band bottom is immersed and is contained the bottle that pulverizes leaf/seed.Solution upwards flow in the fiberglass packing and by capillarity and rises along film.
21. in about 10 to 15 minutes, solution arrives the band top.A clearly purple line appears in the upper part of 1cm under the diaphragm area top, and the indication film has function.This is called control line.If specimen to the Cry1Ac positive, another purple line occurs at the film middle body.
22. 2 line-index strips therefore in the Cry1Ac positive, occur the control line of function and the sample wire of the indication sample positive are arranged.And in the sample that lacks Cry1Ac, the line that 1 index strip has function only appears; Sample detection is the toxin feminine gender.
Embodiment (2): preparation fast immune chromatographic mensuration/band uses the Cry-toxin receptor proteins as catching aglucon to detect Cry1Ac/Cry2Ab.
1. separation of C ry1Ac and Cry2Aa/Cry2Ab from the clone, this is by ultrasonic degradation bacterial clone culture, precipitation is removed cell fragment and lysotoxin not.Toxin is dissolved in alkaline buffer and centrifugal extraction.Finish the toxin purifying by 25% saturated ammonium sulfate precipitation and polyacrylamide gel electrophoresis.
2. by injecting purified toxins is cultivated anti-Cry1Ac/Cry2Aa/Cry2Ab toxin in rabbit or goat antiserum respectively.
3. cultivate sero-fast method and be initial injection and Freund's complete adjuvant antigens mixed (Cry1Ac/Cry2Aa/Cry2Ab) and mix with incomplete Freund repeat to strengthen dosage, collect serum then, this ordinary skill can be available from immunology teaching material (" antibody-laboratory manual "-Harlow Ed and David Lane; Cold Spring Harbor Laboratory, USA, 1988) and here do not describe in detail.
4. purifying Immunoglobulin IgG from antiserum, this is by ammonium sulfate precipitation, sediment is dissolved in damping fluid and makes it continuously through DEAE cellulose, albumin A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity column.Method used herein is described in detail in " antibody-laboratory manual " (Harlow Ed and David Lane; ColdSpring Harbor Laboratory, USA, 1988).
5. available from the IgG purification of antigen affinity purification 0.01M sodium phosphate buffer, pH7.2 dialyses.
6. preparation BBM bubble (BBMVs) from the lepidopterous larvae internal organ uses difference precipitation and centrifugal method, with sweet mellow wine, ethylene glycol-two (beta-amino ether)-N, N, N ', N '-tetraacethyl (EGTA) and magnesium chloride (MgCl
2), according to (1987) described operations such as Wolfersberger.Purifying Cry protein receptor from BBMVs uses ammonium sulfate and standard spin-column chromatography method.
7. peel off the cry-toxin receptor proteins, as 2.5 * 30cm nylon/nitrocellulose/nitrocellulose filter band (S﹠amp; S/Whatman/Millipore/Pall-Gelman) on the side from the center line of top and bottom end 1.25cm distance, the long 30cm of thick 1mm uses hand-held sable hairbrush (number 0,00,000,1,2,3,4 or 5), supports along the 30cm chi.
8. anti-rabbit igg/anti-goat IgG (can be commercial available from some companies, comprise Sigma Chemical Company, USA) be dissolved in the 0.01M sodium phosphate buffer, pH7.2 and peel manually from, as line from 30cm * 0.1cm of film top 0.5cm.
Film under dry-air blower 50 ℃ dry 10-15 minute.
10. use the damping fluid closing membrane subsequently, damping fluid contains 2% casein/BSA and 3% skimmed milk power, and skimmed milk power contains 1-5% sucrose and sodium azide antiseptic, uses the 0.01M sodium phosphate buffer, the pH7.2 washed twice.
11. film under dry-air blower 50 ℃ dry 10-15 minute.
12. (30 * 6cm) bags are sticked at by acryloid cement and film in the middle of the thin slice polyester plastics sheet, and are equidistant from the top and bottom end.
13. aurosol can be commercial available from Sigma Chemical Company, USA.The anti-Cry1Ac-IgG or the anti-Cry2Ab-IgG of affinity purification are diluted to 2mg/ml in the borate buffer of pH8.5 to 9.0, stir to add aurosol (pH transfers to 9.0).Stablize conjugate by adding 10%BSA (bovine serum albumin(BSA)) to potpourri.
14. conjugate is with 12, centrifugal 30 minutes of 4 ℃ of 000g are to obtain the pine precipitation.Precipitation is dissolved in 100ml solution, and solution contains 0.01M Tris, 5%BSA, 2% sucrose, 0.87%NaCl and 0.1M sodium azide.
15. aurosol puts together that solution is applied to that 30cm * 1cm conjugate discharges fiberglass packing and under dry-air blower dry 10-15 minute.
16. the fiberglass packing of dried conjugate bag quilt places the described film of step 10 lower end with overlapping 2mm in the above.
17.30 the thick fiber pad of * 1 * 0.1cm places conjugate to discharge the pad lower end and is bonded on the plastics backing effective coverage with overlapping 2mm and with acryloid cement.Here the indication end is the bottom.
18. the fiber mat of another 30 * 1 * 0.1cm places film upper end with overlapping 2mm and be bonded at acryloid cement on the film effective coverage of plastics backing.Here the indication end is the top.
19. make set stacked and be cut into 6cm * 0.5cm band with cold stacked.
20. plant tissue (ca.50mg) pulverizes among the pH7.2 as 0.5ml 0.01M sodium phosphate buffers in 1.5ml microcentrifugation plastic bottle such as leaf, stem, root, flower, seeds, uses the Teflon pestle.
21. the band bottom is immersed and is contained the bottle that pulverizes leaf/seed.Solution upwards flow in the fiberglass packing and by capillarity and rises along film.
22. in about 10 to 15 minutes, solution arrives the band top.A clearly purple line appears in the upper part of 1cm under the diaphragm area top, and the indication film has function.This is called control line.If specimen is to the Cry1Ac or the Cry2Aa/Cry2Ab positive, this depends on the used antibody of puting together, and then another purple line occurs at the film middle body.
23. 2 line-index strips therefore in Cry1Ac or Cry2Aa/Cry2Ab positive, occur the control line of function and the sample wire of the indication sample positive are arranged.And in the sample that lacks any these 2 toxin groups, the line that 1 index strip has function only appears; Sample detection is the toxin feminine gender.
Embodiment (3): preparation fast immune chromatographic mensuration/band is to detect Cry1Ac and Cry2Ab simultaneously.
1. separation of C ry1Ac and Cry2Aa/Cry2Ab from the clone, this is by ultrasonic degradation bacterial clone culture, precipitation is removed cell fragment and lysotoxin not.Toxin is dissolved in alkaline buffer and centrifugal extraction.Finish the toxin purifying by 25% saturated ammonium sulfate precipitation and polyacrylamide gel electrophoresis.
2. by injecting purified toxins is cultivated anti-Cry1Ac/Cry2Aa/Cry2Ab toxin in rabbit or goat antiserum respectively.
3. cultivate sero-fast method and be initial injection and Freund's complete adjuvant antigens mixed (Cry1Ac/Cry2Aa/Cry2Ab) and mix with incomplete Freund repeat to strengthen dosage, collect serum then, this ordinary skill can be available from immunology teaching material (" antibody-laboratory manual "-Harlow Ed and David Lane; Cold Spring Harbor Laboratory, USA, 1988) and here do not describe in detail.
4. purifying Immunoglobulin IgG from antiserum, this is by ammonium sulfate precipitation, sediment is dissolved in damping fluid and makes it continuously through DEAE cellulose, albumin A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity column.Method used herein is described in detail in " antibody-laboratory manual " (Harlow Ed and David Lane; ColdSpring Harbor Laboratory, USA, 1988).
5. available from the IgG purification of antigen affinity purification 0.01M sodium phosphate buffer, pH7.2 dialyses.
6. peel off the anti-Cry1Ac-IgG that cultivates in rabbit/goat, as 2.5 * 30cm nylon/nitrocellulose/nitrocellulose filter band (S﹠amp; S/Whatman/Millipore/Pall-Gelman) on the side from the center line of top and bottom end 1.25cm distance, the long 30cm of thick 1mm uses hand-held sable hairbrush (number 0,00,000,1,2,3,4 or 5), supports along the 30cm chi.
7. peel off the anti-Cry2Aa/Cry2Ab-IgG that cultivates in rabbit/goat, as 2.5 * 30cm nylon/nitrocellulose/nitrocellulose filter band (S﹠amp; S/Whatman/Millipore/Pall-Gelman) on the side from the center line of top and bottom end 1.0cm distance, the long 30cm of thick 1mm uses hand-held sable hairbrush (number 0,00,000,1,2,3,4 or 5), supports along the 30cm chi.
8. anti-rabbit igg/anti-goat IgG (can be commercial available from some companies, comprise Sigma Chemical Company, USA) be dissolved in the 0.01M sodium phosphate buffer, pH7.2 and peel manually from, it is the line from 30cm * 0.1cm of film top 0.5cm.If conjugate pad IgG is from goat, anti-goat IgG is as control line.Similarly, if conjugate pad IgG from rabbit, anti-rabbit igg is as control line.
Film under dry-air blower 50 ℃ dry 10-15 minute.
10. use the damping fluid closing membrane subsequently, damping fluid contains 2% casein/BSA and 3% skimmed milk power, and skimmed milk power contains 1-5% sucrose and sodium azide antiseptic, uses the 0.01M sodium phosphate buffer, the pH7.2 washed twice.
11. film under dry-air blower 50 ℃ dry 10-15 minute.
12. (30 * 6cm) bags are sticked at by acryloid cement and film in the middle of the thin slice polyester plastics sheet, and are equidistant from the top and bottom end.
13. aurosol can be commercial available from Sigma Chemical Company, USA.The anti-Cry1Ac-IgG of affinity purification is diluted to 2mg/ml in the borate buffer of pH8.5 to 9.0, stir to add aurosol (pH transfers to 9.0).Stablize conjugate by adding 10%BSA (bovine serum albumin(BSA)) to potpourri.
14. aurosol can be commercial available from Sigma Chemical Company, USA.The anti-Cry2Aa/Cry2Ab-IgG of affinity purification is diluted to 2mg/ml in the borate buffer of pH8.5 to 9.0, stir to add aurosol (pH transfers to 9.0).Stablize conjugate by adding 10%BSA (bovine serum albumin(BSA)) to potpourri.
15. conjugate is with 12, centrifugal 30 minutes of 4 ℃ of 000g are to obtain the pine precipitation.Precipitation is dissolved in 100ml solution, and solution contains 0.01M Tris, 5%BSA, 2% sucrose, 0.87%NaCl and 0.1M sodium azide.
16.Cry1Ac and the aurosol of Cry2Aa/Cry2Ab is puted together the solution mixed in equal amounts and is applied to that 30cm * 1cm conjugate discharges fiberglass packing and under dry-air blower dry 10-15 minute.
17. the fiberglass packing of dried conjugate bag quilt places the described film of step 10 lower end with overlapping 2mm in the above.
18.30 the thick fiber pad of * 1 * 0.1cm places conjugate to discharge the pad lower end and is bonded on the plastics backing effective coverage with overlapping 2mm and with acryloid cement.Here the indication end is the bottom.
19. the fiber mat of another 30 * 1 * 0.1cm places film upper end with overlapping 2mm and be bonded at acryloid cement on the film effective coverage of plastics backing.Here the indication end is the top.
20. make set stacked and be cut into 6cm * 0.5cm band with cold stacked.
21. plant tissue (ca.50mg) pulverizes among the pH7.2 as 0.5ml 0.01M sodium phosphate buffers in 1.5ml microcentrifugation plastic bottle such as leaf, stem, root, flower, seeds, uses the Teflon pestle.
22. the band bottom is immersed and is contained the bottle that pulverizes leaf/seed.Solution upwards flow in the fiberglass packing and by capillarity and rises along film.
23. in about 10 to 15 minutes, solution arrives the band top.A clearly purple line appears in the upper part of 1cm under the diaphragm area top, and the indication film has function.This is called control line.If specimen is all positive to Cry1Ac/1Ab or Cry2Aa/Cry2Ab, 2 purple line appear in the film middle body.For Cry1Ac/1Ab, 1 line only occurring corresponding to middle body, or, 1 line only occurring corresponding to middle body from film bottom 1.0cm for Cry2Aa/Cry2Ab from film bottom 1.25cm.
24. therefore only occurring 2 line-index strips in Cry1Ac/1Ab or Cry2Aa/Cry2Ab positive has the control line of function and indicates the sample wire of sample to the Cry1Ac/1Ab or the Cry2Aa/Cry2Ab positive.And in the sample that lacks Cry1Ac/1Ab or Cry2Aa/Cry2Ab, the line that 1 index strip has function only appears; Sample detection is the toxin feminine gender.If 3 bands (comprising the contrast band) show that sample is all positive to Cry1Ac/1Ab and Cry2Aa/Cry2Ab.
Advantage
1. methods described herein are used polyclonal antibody and manual stripping means, and carrying out simply and not needs the commodity production class Seemingly ' peeling off of monoclonal antibody or monoclonal technigue and cost 〉=20,000 dollar established with costliness used in the kind Standby '.
2. the method for this paper institute claim is used the affinity purification immunoglobulin (Ig) of cultivating in the different animals in 2 Thereby improve detection sensitivity (IgG).
Invention can detect simultaneously single with on two or more toxin, therefore reduce by 2 that are used for identical purpose Or multi-band cost more.
4. use in band that ' Cry toxoreceptor ' is can be high responsive to be detected poisonous any of concern lepidopterous insects From then on Cry toxin, acceptor separate in the insect.
List of references
1. U.S. Patent number 5,712, and 172.
2.Harlow Ed and David Lane. (1988) " antibody-laboratory manual " .Cold Spring HarborLaboratory, New York 11724, USA.
3.Wolfersberger, M.G., Luthy, P., Maurer, A., Parenti, P., Sacchi, V.F., Giordana, B and Hanozet, G.M. (1987) " transhipment is determined from the amino acid preparation and the Partial Feature of the BBM bubble of cabbage butterfly larva (Pierrisbrassicae) midgut " .Comp.Biochem.Physiol.86A, 301-308.
4.Horisberger, (1979) " assessment is as aurosol of the cytochrome mark of transmission and scanning electron microscope " .Biol.Cellulaire, 36,253-258.
5.Leuvering etc., (1980) " molten particle immunoassays " .J Immunoassay, 1 (1): 77-91.
6.Frens (1973) " the single control nucleation of disperseing grain size in the golden suspending liquid of adjusting " .NaturePhysical Science, 241:20-22.
Date: 2003.5.28
(Jai?Prakash?Mishra)
Vice president (IPR)
ICAR
Krishi Bhavan, Dr.Rajendra Prasad road
New Delhi 110001
Claims (4)
1. one kind prepares the method for fast immune chromatographic mensuration/band with polyclonal antibody, and described antibody evaluation/detection as the single toxin from seed or plant tissue, is characterized in that described method comprises step from the Cry toxin of bacillus thuringiensis:
(i) identify unique natural bacillus thuringiensis bacterial strain, determine described strain characteristics by antibiotic resistance distribution, colonial morphology and growth characteristics, generate the gene of crystalline protein and be cloned into the particular expression carrier from the plasmid amplification of described bacterial strain with special primer;
(ii) use the several different methods purifying Cry1Ac and the Cry2Aa that comprise chromatography and electrophoresis extremely obviously even;
(iii) by periodically injecting the crystalline toxin that mixes with adjuvant of strengthening dosage for rabbit and goat and collecting its serum, precipitate described serum and the antibody purification (ii) polyclonal antibody of described toxin of incubation step in rabbit and goat;
(iv) use affinity chromatography IgG purification from the (iii) described antiserum of step, comprise albumin A and Cry1Ac/Cry2Aa antigen;
(v) use the (iv) described anti-Cry polyclone IgG of step to be fixed on the film;
(vi) go up the fixed trapped line at cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns), described line contains the (iv) described described polyclone IgG that cultivates of step in goat, adopt manual stripping means, with hand-held sable hairbrush;
(vii) go up the fixed trapped line at cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns), line contains step
The (iv) described described polyclone IgG that cultivates in rabbit uses manual stripping means, with hand-held sable hairbrush;
(viii) with the (iv) described polyclone IgG antibody of in goat, cultivating of aurosol 20-40nm markers step;
(ix) with the (iv) described polyclone IgG antibody of in rabbit, cultivating of aurosol 20-40nm markers step;
(x) with micropipet fixing step (gold-antibody conjugates viii) on fiberglass packing;
(xi) gold-antibody conjugates of usefulness micropipet fixing step (ix) on fiberglass packing;
(xii) peel manually is from goat anti-rabbit igg or the anti-goat IgG of rabbit, its in each band from the line of 30cm * 0.1cm of film top 0.5cm;
(xiii) the anti-goat IgG line of the goat anti-rabbit igg of described step (xii) or rabbit can be caught the anti-rabbit antibody or the anti-goat antibody of golden mark respectively, and is also like this in any congregation zone;
(xiv) the described line of claim 1 step (xii) can be used as control line to indicate the suitable function of described band;
When (xv) using, the described line of claim 1 step (xii) can become pink colour/purple, and no matter specimen is a positive or negative;
(xvi) film is with the sealing damping fluid sealing that contains albumen, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, gathers then;
(xvii) (glass fibre of the described gold of vi) described film upper set claim 1 step (x)-conjugate infiltration is with overlapping 2mm as shown in Figure 1 in claim 1 step;
(xviii) (glass fibre of the described gold of vii) described film upper set claim 1 step (x)-conjugate infiltration is with overlapping 2mm as shown in Figure 1 in claim 1 step;
(xix) (glass fibre of the described gold of vi) described film upper set claim 2 step (xi)-conjugate infiltration is with overlapping 2mm as shown in Figure 1 in claim 1 step;
(xx) (glass fibre of the described gold of vii) described film upper set claim 2 step (xi)-conjugate infiltration is with overlapping 2mm as shown in Figure 1 in claim 1 step;
(xxi) finish gathering, this is to place the described set of step (xvii, xviii, xix and xx) bottom with overlapping 2mm by sample being discharged filter paper pads (1-2mm is thick), and the 2nd absorption of sample pad places claim 1 step (vi) with (vii) described film top is with overlapping 2mm;
(xxii) cold stacked set eventually;
(xxiii) use a kind of anti-Cry antibody capture line of cultivation in a kind of animal (goat) and the combination of antigen sandwich to detect toxin as object line, described antigen is between the anti-Cry antibody of the described golden mark of cultivating in another kind of animal (rabbit) of step (xix);
(xxiv) a kind of anti-Cry antibody capture line of cultivating in a kind of animal (rabbit) and the combination of antigen sandwich detect toxin as object line, and described antigen is between the anti-Cry antibody of the described golden mark of cultivating in another kind of animal (goat) of step (xviii);
(xxv) a kind of anti-Cry antibody capture line of cultivating in a kind of animal (rabbit) and the composition of antigen sandwich detect toxin as object line, and described antigen is between the anti-Cry antibody of the described golden mark of cultivating in same animals (rabbit) of step (xx);
(xxvi) a kind of anti-Cry antibody capture line of cultivating in a kind of animal (goat) and the composition of antigen sandwich detect toxin as object line, and described antigen is between the anti-Cry antibody of the described golden mark of cultivating in same animals (goat) of step (xx);
(xxvii) step (xxii) is used to detect cry toxin C ry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab to step (xxvi).
2. the method for preparing fast immune chromatographic mensuration/band as claimed in claim 1, wherein, use the combination that separates from the Cry of lepidopterous insects internal organ toxoreceptor and anti-Cry polyclonal antibody, as the single toxin from seed or plant tissue, described method comprises step from the Cry toxin of bacillus thuringiensis in this polyclonal antibody evaluation/detection:
(i) method of delivering according to (1987) such as standard method such as Wolfersberger is separated the BBM bubble from the lepidopterous larvae internal organ;
(ii) use the albumen precipitation method purifying N-aminopeptidase and the cadherin of 30-80% ammonium sulfate;
(iii) go up the fixed trapped line at cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns), described line contains from claim 2 step albumen (ii), uses manual stripping means, with hand-held sable hairbrush;
(iv) use the (iv) described polyclone IgG of aurosol 20-40nm mark claim 1 step antibody;
(v) use (ii) described N-aminopeptidase of aurosol 20-40nm mark claim 1 step and cadherin;
(the vi) fixing (iv) described gold-antibody conjugates of claim 2 step on fiberglass packing;
(vii) fixing claim 2 step (v) described gold-Cry receptor protein conjugate on fiberglass packing;
(viii) peel manually is from goat anti-rabbit igg or the anti-goat IgG of rabbit, its in each band from the line of 30cm * 0.1cm of film top 0.5cm;
(ix) the anti-goat IgG line of the goat anti-rabbit igg of described step (xii) or rabbit can be caught the anti-rabbit antibody or the anti-goat antibody of golden mark respectively, and is also like this in any congregation zone;
(x) the described line of claim 2 step (ix) can be used as control line to indicate the suitable function of described band;
When (xi) using, the described line of claim 2 step (ix) can become pink colour/purple, and no matter specimen is a positive or negative;
(xii) film is with the sealing damping fluid sealing that contains albumen, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, gathers then;
(xiii) ((glass fibre of v) described gold-conjugate infiltration is with overlapping 2mm as shown in Figure 1 for vi) described film upper set claim 2 step in claim 1 step;
(xiv) ((glass fibre of v) described gold-conjugate infiltration is with overlapping 2mm as shown in Figure 1 for vii) described film upper set claim 2 step in claim 1 step;
(xv) (glass fibre of v) described gold-conjugate infiltration is with overlapping 2mm as shown in Figure 1 in the (iii) described film upper set of claim 2 step claim 2 step;
(xvi) at the glass fibre of the described gold of the (iii) described film upper set of claim 2 step claim 1 step (x)-conjugate infiltration, with overlapping 2mm as shown in Figure 1;
(xvii) at the glass fibre of the described gold of the (iii) described film upper set of claim 2 step claim 1 step (xi)-conjugate infiltration, with overlapping 2mm as shown in Figure 1;
(xviii) finish gathering, this is to place the described set of claim 2 step (xiii, xiv, xv, xvi and xvii) bottom with overlapping 2mm by sample being discharged filter paper pads (1-2mm is thick), and the 2nd absorption of sample pad places the described film of claim 2 step (xiii, xiv, xv, xvi and xvii) top with overlapping 2mm;
(xix) cold stacked set eventually;
(xx) use that (a kind of anti-Cry antibody capture line of cultivation and the combination of antigen sandwich detect toxin as object line in a kind of animal (goat) vi), and described antigen is in claim 2 step (between v) described gold-Cry-receptor protein conjugate in claim 1 step;
(xxi) (a kind of anti-Cry antibody capture line of cultivating in a kind of animal (rabbit) vii) and the combination of antigen sandwich detect toxin as object line to claim 1 step, and described antigen is in claim 2 step (between v) described gold-Cry-receptor protein conjugate;
(xxii) the Cry-receptor capture line of claim 2 step described in (iii) and the composition of antigen sandwich detect toxin as object line, and described antigen is in claim 2 step (between v) described gold-Cry-receptor protein conjugate;
(xxiii) the Cry-receptor capture line of claim 2 step described in (iii) and the composition of antigen sandwich detect toxin as object line, between the Cry antibody of the golden mark that described antigen is cultivated in the described goat of claim 1 step (x);
(xxiv) the Cry-receptor capture line of claim 2 step described in (iii) and the composition of antigen sandwich detect toxin as object line, between the Cry antibody of the golden mark that described antigen is cultivated in the described rabbit of claim 1 step (xi);
(xxv) step (xx) is used to detect cry toxin C ry1 Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab to step (xxiv).
3. the method for preparing fast immune chromatographic mensuration/band with polyclonal antibody as claimed in claim 1, two or more Cry toxin from the bacillus thuringiensis of seed or plant tissue are identified/detected to described polyclonal antibody simultaneously, it is characterized in that described method comprises step:
(l) accessory rights requires to generate among the described clone of 1 step (i) Cry1Ac and Cry2Aa toxin;
(i) use the several different methods purifying Cry1Ac and the Cry2Aa that comprise chromatography and electrophoresis to arrive obviously evenly;
(ii) in rabbit that obtains as antiserum and goat, cultivate the (iii) polyclonal antibody of described toxin of claim 1 step;
(iii) require IgG purification in the (iv) described antiserum of 1 step, comprise albumin A and Cry1Ac/Cry2Aa antigen with the affinity chromatography accessory rights;
(iv) use the (iv) described anti-Cry polyclone IgG of step being fixed on cellulose nitrate, nitrocellulose or the nylon membrane (5 to 12 microns), use peel manually from, with hand-held sable hairbrush;
(v) catch line and be fixed in middle body 2, from claim 3 step (the film two ends 1cm that v) described 2.5cm is wide, separate 0.5cm, it comprises the anti-Cry2Aa IgG that cultivates in (iv) described anti-Cry1Ac polyclone IgG that cultivates of claim 1 step and the goat in goat;
(vi) catch line and be fixed in middle body 2, from claim 3 step (the film two ends 1cm that v) described 2.5cm is wide, separate 0.5cm, it comprises the anti-Cry2Aa IgG that cultivates in (iv) described anti-Cry1Ac polyclone IgG that cultivates of claim 1 step and the rabbit in goat;
(vii) catch line and be fixed in middle body 2, from claim 3 step (the film two ends 1cm that v) described 2.5cm is wide, separate 0.5cm, it comprises the anti-Cry2Aa IgG that cultivates in (iv) described anti-Cry1Ac polyclone IgG that cultivates of claim 1 step and the goat in rabbit;
(viii) catch line and be fixed in middle body 2, from claim 3 step (the film two ends 1cm that v) described 2.5cm is wide, separate 0.5cm, it comprises the anti-Cry2Aa IgG that cultivates in (iv) described anti-Cry1Ac polyclone IgG that cultivates of claim 1 step and the rabbit in rabbit;
(ix) as claim 1 step (iv) as described in the anti-Cry1AcIgG that cultivates in the aurosol 20-40nm mark rabbit;
(x) as claim 1 step (iv) as described in the anti-Cry2AaIgG that cultivates in the aurosol 20-40nm mark rabbit;
(xi) as claim 1 step (iv) as described in the anti-Cry1Ac IgG that cultivates in the aurosol 20-40nm mark goat;
(xii) as claim 1 step (iv) as described in the anti-Cry2Aa IgG that cultivates in the aurosol 20-40nm mark goat;
(xiii) with claim 3 step (x) and (xi) described gold-antibody conjugates equivalent be fixed in fiberglass packing;
(xiv) with claim 3 step (x) and (xiii) described gold-antibody conjugates equivalent be fixed in fiberglass packing;
(xv) with claim 3 step (xi) and (xii) described gold-antibody conjugates equivalent be fixed in fiberglass packing;
(xvi) with claim 3 step (xii) and (xiii) described gold-antibody conjugates equivalent be fixed in fiberglass packing;
(xvii) peel manually is from goat anti-rabbit igg or the anti-goat IgG of rabbit, its in each band from the line of 30cm * 0.1cm of film top 0.5cm;
(xviii) the anti-goat IgG line of the goat anti-rabbit igg of described step (xviii) or rabbit can be caught the anti-rabbit antibody or the anti-goat antibody of golden mark respectively, and is also like this in any congregation zone;
(xix) the described line of claim 3 step (xix) can be used as control line to indicate the suitable function of described band;
When (xx) using, the described line of claim 3 step (xix) can become pink colour/purple, and no matter specimen is a positive or negative;
(xxi) film is with the sealing damping fluid sealing that contains albumen, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, gathers then;
(xxii) (glass fibre of the described gold of vi) described film upper set claim 3 step (xiv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxiii) (glass fibre of the described gold of vii) described film upper set claim 2 step (xiv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 2 step;
(xxiv) (glass fibre of the described gold of viii) described film upper set claim 2 step (xiv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 1 step;
(xxv) at the glass fibre of the described gold of claim 1 step (ix) described film upper set claim 2 step (xiv)-conjugate infiltration, with overlapping 2mm as shown in Figure 2;
(xxvi) (glass fibre of the described gold of vi) described film upper set claim 3 step (xv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxvii) (glass fibre of the described gold of vii) described film upper set claim 3 step (xv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxviii) (glass fibre of the described gold of viii) described film upper set claim 3 step (xv)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxix) at the glass fibre of the described gold of claim 3 step (ix) described film upper set claim 3 step (xv)-conjugate infiltration, with overlapping 2mm as shown in Figure 2;
(xxx) (glass fibre of the described gold of vi) described film upper set claim 3 step (xvi)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxi) (glass fibre of the described gold of vii) described film upper set claim 3 step (xvi)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxii) (glass fibre of the described gold of viii) described film upper set claim 3 step (xvi)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxiii) at the glass fibre of the described gold of claim 3 step (ix) described film upper set claim 3 step (xvi)-conjugate infiltration, with overlapping 2mm as shown in Figure 2;
(xxxiv) (glass fibre of the described gold of vi) described film upper set claim 3 step (xvii)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxv) (glass fibre of the described gold of vii) described film upper set claim 3 step (xvii)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxvi) (glass fibre of the described gold of viii) described film upper set claim 3 step (xvii)-conjugate infiltration is with overlapping 2mm as shown in Figure 2 in claim 3 step;
(xxxvii) at the glass fibre of the described gold of claim 3 step (ix) described film upper set claim 3 step (xvii)-conjugate infiltration, with overlapping 2mm as shown in Figure 2;
(xxxviii) finish gathering, this is to place claim 3 step (the xxiii)-described set of step (xxxviii) bottom with overlapping 2mm by sample being discharged filter paper pads (1-2mm is thick), and the 2nd absorption of sample pad places the described film of claim 3 step (xiii-xxxviii) top with overlapping 2mm;
(xxxix) cold stacked set eventually;
(xl) use any band of preparing from step (xxiii) to (Xi) to detect Cry1Ac/Cry1Ab and Cry2Aa/Cry2Ab simultaneously, if specimen is positive then 2 lines are arranged to two kinds of toxin, if or specimen is positive then be the simple sample line to arbitrary toxin, if or specimen negative then with on the sample wire position do not have thing;
(xli) described sample wire position will be fixed, and the line of Cry1Aa/Cry1Ac is lower and line Cry2Aa/Cry2Ab is higher or opposite, and top line is Cry1Aa/Cry1Ac;
(xlii) described sample wire occurring is that it moves along film through capillarity in conjunction with toxin (Cry1Ac/Cry1Ab or Cry2Aa or Cry2Ab), up to running into the analyte capture line because the anti-Cry toxin antibody of golden mark gathers.In case the anti-Cry toxin antibody of golden mark-toxin conjugate contact analyte capture line, therefore the toxin combination forms sandwich and causes golden conjugate to gather along the line.The golden conjugate of assembling occurs with object line.
4. the method for preparing fast immune chromatographic mensuration/band as claimed in claim 1 is characterized in that, uses manual stripping means so that antibody (IgG) solution is fixed in film, with hand-held sable hairbrush.
(i) claim 1 step (vi), (iii) (claim 2 step assists to finish by the sable hairbrush with claim 3 step by v) described method;
(ii) hand-held brush technology can make antibody absorb on film and not form depression;
(iii) the 30cm chi remains on the film lightly, finishes that to peel off be that metal part by brush is advanced to contact the antibody of getting wet in the bristle top gently along chi, catches line thereby form.
Date: 2003.5.28
(Jai?Prakash?Mishra)
Vice president (IPR)
ICAR
Krishi Bhavan, Dr.Rajendra Prasad road
New Delhi 110001
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| IN600/DEL/02 | 2002-05-31 | ||
| IN600DE2002 | 2002-05-31 |
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| CN1672049A true CN1672049A (en) | 2005-09-21 |
| CN100403028C CN100403028C (en) | 2008-07-16 |
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| CNB038176416A Expired - Fee Related CN100403028C (en) | 2002-05-31 | 2003-05-29 | Rapid detection of bt-cry toxins |
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| Country | Link |
|---|---|
| KR (1) | KR20050026396A (en) |
| CN (1) | CN100403028C (en) |
| AU (1) | AU2003249576A1 (en) |
| MX (1) | MXPA04011769A (en) |
| WO (1) | WO2003102208A2 (en) |
| ZA (1) | ZA200410268B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100348616C (en) * | 2005-12-12 | 2007-11-14 | 中国农业大学 | Bt CrylA antibody, and its preparing method and special antigen and use |
| CN103197075A (en) * | 2012-01-16 | 2013-07-10 | 华中农业大学 | Method for detecting Bt protein in transgenic rice by quantum dot |
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN106674334A (en) * | 2017-02-08 | 2017-05-17 | 金陵科技学院 | Cry2Ad-bonded cyclo-heptapeptide as well as encoding gene and application thereof |
| CN110346569A (en) * | 2019-06-28 | 2019-10-18 | 安徽恩禾生物技术有限公司 | A kind of thymidine kinase chemoluminescence method detection kit and preparation method thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007000048A1 (en) * | 2005-06-28 | 2007-01-04 | Zbx Corporation | Membrane array and analytical device |
| ES2582784T3 (en) * | 2009-04-09 | 2016-09-15 | Hitachi Chemical Company, Ltd. | Detector and detection method |
| ES2765206T3 (en) | 2013-03-15 | 2020-06-08 | Glaxosmithkline Ip No 2 Ltd | Antibody purification procedures |
| EP4389757A2 (en) | 2016-09-07 | 2024-06-26 | GlaxoSmithKline Intellectual Property Development Limited | Methods for purifying antibodies |
| CN108828230B (en) * | 2018-06-21 | 2021-04-30 | 北京市农林科学院 | Method for rapidly detecting transgenic product by nucleic acid chromatography |
| CN114280312B (en) * | 2020-09-27 | 2023-09-15 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5141850A (en) * | 1990-02-07 | 1992-08-25 | Hygeia Sciences, Inc. | Porous strip form assay device method |
| US5712171A (en) * | 1995-01-20 | 1998-01-27 | Arqule, Inc. | Method of generating a plurality of chemical compounds in a spatially arranged array |
| US5712172A (en) * | 1995-05-18 | 1998-01-27 | Wyntek Diagnostics, Inc. | One step immunochromatographic device and method of use |
| AU742971B2 (en) * | 1996-11-20 | 2002-01-17 | Monsanto Technology Llc | Broad-spectrum delta-endotoxins |
| US6017534A (en) * | 1996-11-20 | 2000-01-25 | Ecogen, Inc. | Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity |
| WO2001044779A2 (en) * | 1999-12-14 | 2001-06-21 | Strategic Diagnostics, Inc. | Method of processing and testing powdered samples using immunochromatographic strip tests |
-
2003
- 2003-05-29 KR KR1020047019456A patent/KR20050026396A/en active Search and Examination
- 2003-05-29 MX MXPA04011769A patent/MXPA04011769A/en active IP Right Grant
- 2003-05-29 CN CNB038176416A patent/CN100403028C/en not_active Expired - Fee Related
- 2003-05-29 WO PCT/IN2003/000199 patent/WO2003102208A2/en active Application Filing
- 2003-05-29 AU AU2003249576A patent/AU2003249576A1/en not_active Abandoned
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2004
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100348616C (en) * | 2005-12-12 | 2007-11-14 | 中国农业大学 | Bt CrylA antibody, and its preparing method and special antigen and use |
| CN103197075A (en) * | 2012-01-16 | 2013-07-10 | 华中农业大学 | Method for detecting Bt protein in transgenic rice by quantum dot |
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN106674334A (en) * | 2017-02-08 | 2017-05-17 | 金陵科技学院 | Cry2Ad-bonded cyclo-heptapeptide as well as encoding gene and application thereof |
| CN110346569A (en) * | 2019-06-28 | 2019-10-18 | 安徽恩禾生物技术有限公司 | A kind of thymidine kinase chemoluminescence method detection kit and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100403028C (en) | 2008-07-16 |
| WO2003102208A2 (en) | 2003-12-11 |
| AU2003249576A8 (en) | 2003-12-19 |
| AU2003249576A1 (en) | 2003-12-19 |
| KR20050026396A (en) | 2005-03-15 |
| ZA200410268B (en) | 2006-07-26 |
| MXPA04011769A (en) | 2005-07-26 |
| WO2003102208A3 (en) | 2004-03-25 |
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