CN100403028C - Rapid detection of bt-cry toxins - Google Patents
Rapid detection of bt-cry toxins Download PDFInfo
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- CN100403028C CN100403028C CNB038176416A CN03817641A CN100403028C CN 100403028 C CN100403028 C CN 100403028C CN B038176416 A CNB038176416 A CN B038176416A CN 03817641 A CN03817641 A CN 03817641A CN 100403028 C CN100403028 C CN 100403028C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
- G01N2333/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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, ' x＞(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.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-Alaboratory 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 with costliness used in the kind ' peeling off of monoclonal antibody or monoclonal technigue and cost 〉=20,000 dollar Equipment '.
2. method of the present invention is used the immunoglobulin (Ig) (IgG) of cultivating of affinity purification in two kinds of different animals, Thereby raising detection sensitivity.
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.
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Claims (9)
1. one kind with the method for extracting from the Bt-Cry-of insect toxin-acceptor-protein Preparation fast immune chromatographic lateral flow band, and described lateral flow band is used for detecting the bacillus thuringiensis Cry toxin of seed or plant tissue, and described method comprises step:
(i) with standard method to extracting the Bt-Cry receptor protein the hypersusceptible lepidopterous insects of Cry toxin, separate the BBM bubble from the lepidopterous larvae internal organ, and pass through albumen precipitation method purifying N-aminopeptidase and cadherin with 30-80% ammonium sulfate;
(ii) use hand-held sable hairbrush, make setting-out method by hand, on 5 to 12 microns cellulose nitrate, nitrocellulose or nylon membrane fixedly 30cm * 0.1cm catch line, the described line of catching contains in steps receptor protein described in (i), the described top 1.25cm that catches film in each lateral flow band of linear distance;
(iii) use hand-held sable hairbrush, make setting-out method by hand, the fixing control line of 30cm * 0.1cm on 5 to 12 microns cellulose nitrate, nitrocellulose or nylon membrane, described control line contains goat anti-rabbit igg, and described control line is apart from the top 0.5cm of film in each lateral flow band;
(iv) film is with the sealing damping fluid sealing that contains protein, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, assembles then;
(v) use receptor protein described in the aurosol 20-40nm markers step (i);
(vi) use aurosol 20-40nm mark rabbit igg;
(vii) with the step of equivalent (v) and step (protein of golden mark vi) mixes;
(viii) use micropipet fixing step (gold-antibody conjugates vi) on fiberglass packing;
(ix) (glass fibre that has soaked into gold-conjugate viii) overlays on the film of step described in (iv), overlapping 2mm with step;
(x) sample that 1-2mm is thick discharge bottom that filter paper pads places step (ix) gained assembly with the overlapping 2mm of glass fibre, place step (iv) the top of described film to finish assembling thus on second absorption of sample pad with overlapping 2mm;
(xi) cold layer of subassembly of finishing;
(xii) step goat anti-rabbit igg line is (iii) caught the rabbit antibody of golden mark, and line is to indicate described band function normal in contrast;
(xiii) the described line of step (xii) can become pink colour/purple, and no matter specimen is a positive or negative;
(xiv) adopt step (ii) described in receptor protein catch the combination of the receptor protein of line and golden mark, catch golden labeled receptor albumen line of accumulation of line position formation in the middle of the cry toxin is sandwiched in and at the (ii) described part of step, show with the object line form thus and caught toxin;
(xv) comprise that step I to the said method of xiv does not need to cultivate the antibody of anti-Cry toxin, can detect multiple toxin.
2. the method for claim 1, described lepidopterous insects is selected from rice green caterpillar (semilooper Anomisflava), pink bollworm (bollworm) and bollworm (helicoverpa armigera).
3. the method for claim 1, described toxin is selected from Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry2Aa and Cry2Ab.
4. will extract the anti-Cry polyclonal antibody of catching albumen and golden mark as part and unite the method that is used to prepare fast immune chromatographic lateral flow band from the Cry-of lepidopterous insects internal organ toxoreceptor, described lateral flow band is used for detecting each specific bacillus thuringiensis Cry toxin of seed or plant tissue, and described method comprises step:
(i) with standard method to extracting the Bt-Cry receptor protein in the hypersusceptible lepidopterous insects of Cry toxin, separate the BBM bubble from the lepidopterous larvae internal organ, and pass through intermediate processing purifying N-aminopeptidase and cadherin with 30-80% ammonium sulfate albumen;
(ii) use hand-held sable hairbrush, make setting-out method by hand, on 5 to 12 microns cellulose nitrate, nitrocellulose or nylon membrane fixedly 30cm * 0.1cm catch line, the described line of catching contains in steps receptor protein described in (i), the described top 1.25cm that catches film in each lateral flow band of linear distance;
(iii) use hand-held sable hairbrush, make setting-out method by hand, the fixing control line of 30cm * 0.1cm on 5 to 12 microns cellulose nitrate, nitrocellulose or nylon membrane, described control line contains goat anti-rabbit igg, and described control line is apart from the top 0.5cm of film in each lateral flow band;
(iv) film is with the sealing damping fluid sealing that contains protein, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, assembles then;
(v) 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 specific expression vector from the plasmid amplification of described bacterial strain with special primer;
(vi) use the several different methods purifying Cry1Ac and the Cry2Aa that comprise chromatography and electrophoresis extremely obviously even;
(vii) by periodically inject the crystalline toxin that mixes with adjuvant incubation step (polyclonal antibody of toxin vi), collection serum, precipitation serum, antibody purification in rabbit of strengthening dosage to rabbit;
(viii) with the affinity chromatography that comprises albumin A and Cry1Ac/Cry2Aa antigen from step (IgG purification the antiserum vii);
(ix) with the aurosol 20-40nm markers step (IgG of polyclone viii) antibody;
(x) gold-antibody conjugates described in the fixing step (ix) on fiberglass packing;
(xi) glass fibre that has soaked into gold-conjugate described in the step (x) is overlayed on the (iv) described film of step overlapping 2mm;
(xii) sample that 1-2mm is thick discharge that filter paper pads places the bottom of step (xi) gained assembly and on glass fibre overlapping 2mm, place step (iv) the top of described film to finish assembling thus on second absorption of sample pad with overlapping 2mm;
(xiii) cold layer of subassembly of finishing;
(xiv) described step goat anti-rabbit igg line is (iii) caught the rabbit antibody of golden mark, and line is to indicate described band function normal in contrast;
(xvi) the described line of step (xiv) can become pink colour/purple, and no matter specimen is a positive or negative;
(xvii) be used in step (ii) described in receptor protein catch line and step (ix) and (x) described in the combination that constitutes of the anti-rabbit igg of anti-Cry of golden mark, in the middle of the cry toxin is sandwiched in, and catch one of line position formation at the (ii) described part of step and accumulate golden labeled receptor albumen line, show with the object line form thus and caught toxin;
(xviii) IgG that cultivates at each particular toxin is carried out golden mark, but the multiple particular toxin of said method specific detection.
5. method as claimed in claim 4, described lepidopterous insects are selected from rice green caterpillar (semilooper Anomisflava), pink bollworm (bollworm) and bollworm (helicoverpa armigera).
6. method as claimed in claim 4, described toxin is selected from Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry2Aa and Cry2Ab.
7. the receptor protein of catching line and golden mark as part with polyclonal antibody makes up the method that is used to prepare fast immune chromatographic order-checking fluxion strap, described lateral flow band is used for detecting simultaneously seed or two or more bacillus thuringiensis Cry toxin of plant tissue, it is characterized in that described method comprises step:
(i) (generate Cry1Ac and Cry2Aa toxin v) as claim 4 step;
(ii) (use comprises that the several different methods purifying Cry1Ac of chromatography and electrophoresis and Cry2Aa arrive obviously evenly vi) as claim 4 step;
(iii) (in rabbit, cultivate the polyclonal antibody of toxin vii), obtain antiserum as claim 4 step;
(iv) (viii), use the affinity chromatography IgG purification from antiserum that comprises albumin A and Cry1Ac/Cry2Aa antigen as claim 4 step;
(v) use the (iv) described anti-Cry polyclone IgG of step, be fixed on 5 to 12 microns cellulose nitrate, nitrocellulose or the nylon membrane, make setting-out by hand, with hand-held sable hairbrush;
(vi) catch line and be fixed in middle body two, separately apart from step (the two ends 1cm of the film that v) described 2.5cm is wide, the 0.5cm that is separated from each other, described catch line comprise step (iii) and the anti-Cry1Ac polyclone IgG of rabbit (iv) and step (iv) described in the anti-Cry2Aa IgG of rabbit;
(the vii) manual goat anti-rabbit igg line of drawing 30cm * 0.1cm is apart from film top 0.5cm in each band;
(viii) film is with the sealing damping fluid sealing that contains protein, sugar and antiseptic, and with phosphate-buffered saline washed twice and dry 30 minutes, assembles then;
(ix) with receptor protein described in aurosol 20-40nm mark claim 2 step (i);
(x) with aurosol 20-40nm mark rabbit igg;
(xi) step (ix) of equivalent and the protein of the golden mark in the step (x) are mixed;
(xii) gold-antibody conjugates of usefulness micropipet fixing step (xi) on fiberglass packing;
(xiii) glass fibre that has soaked into the gold-compound that stops described in the step (xii) is overlayed step (on the viii) described film, overlapping 2mm;
(xiv) 1-2mm is thick sample release filter paper pads places on the described assembly of step (xiii), and with the overlapping 2mm in its glass fibre bottom, second absorption of sample pad places the described film of step (xiii) top, overlapping 2mm;
(xv) cold layer of subassembly of finishing;
(xvi) (vii) described goat anti-rabbit igg will be caught the rabbit antibody of golden mark to step, and line shows that described lateral flow band function is normal in contrast;
(xvii) the described line of step (xvi) can become pink colour or purple, no matter sample is the positive or feminine gender;
(xviii) be fixed as part by step described in (iv) and catch the Cry toxin specific IgG of line-1 and be fixed as the 2nd Crg toxin specific IgG that part is caught line-2 by adopting, the combination that constitutes with the receptor protein of golden mark, corresponding C ry toxin is sandwiched in wherein, and step (ii) described in the part position of catching line form the golden labeled receptor albumen line of accumulation, detect toxin with the indication of object line form thus;
(xix) above-mentioned step 1 to the method for xviii that comprises can once detect more than one particular toxin on a film, and the specific IgG according to obtaining can accurately detect several toxin on a lateral flow band;
(xx) use described in the step (xix) the lateral flow band can detect Cry1Ac/Cry1Ab and Cry2Aa/Cry2Ab simultaneously, if specimen is positive then two lines are arranged to two kinds of toxin, if specimen is positive then be the simple sample line to arbitrary toxin, if specimen is negative with on the sample wire position do not show;
(xxi) described sample wire position will be fixed to the Cry1Aa/Cry1Ac line down, and the Cry2Aa/Cry2Ab line is last, or be that the Cry1Aa/Cry1Ac line is last on the contrary;
(xxii) described sample wire occurring is because the receptor protein of golden mark gathers, described receptor protein is in conjunction with toxin, move along film through capillarity, up to running into the analyte capture line, in case the anti-Cry toxin antibody of golden mark-toxin conjugate contact analyte capture line, then toxin is combined, therefore forms the sandwich sample, and causing golden conjugate to gather along the line, the golden conjugate of gathering is rendered as object line.
8. method as claimed in claim 7, toxin is selected from Cry1Ac, Cry1Ab, Cry1Ac, Cry1C, Cry2Aa and Cry2Ab described in the step (xix).
9. method as claimed in claim 7, in the step (xxii), described receptor protein is in conjunction with toxin C ry1Ac/Cry1Ab or Cry2Aa or Cry2Ab.
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| CN (1) | CN100403028C (en) |
| AU (1) | AU2003249576A1 (en) |
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| 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 |
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| JP4891317B2 (en) * | 2005-06-28 | 2012-03-07 | ズィービーエックス・コーポレーション | Membrane array and analytical equipment |
| 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 |
| CN102388309B (en) * | 2009-04-09 | 2016-05-25 | 日立化成株式会社 | Checkout gear and detection method |
| CN103197075B (en) * | 2012-01-16 | 2014-10-08 | 华中农业大学 | Method for detecting Bt protein in transgenic rice by quantum dot |
| RU2651483C2 (en) | 2013-03-15 | 2018-04-19 | ГлаксоСмитКлайн Интеллекчуал Проперти (N2) Лимитед | Methods for purifying antibodies |
| 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 |
| EP3510042A1 (en) | 2016-09-07 | 2019-07-17 | 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 |
| 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 |
| 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 |
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- 2003-05-29 CN CNB038176416A patent/CN100403028C/en not_active Expired - Fee Related
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| US5712171A (en) * | 1995-01-20 | 1998-01-27 | Arqule, Inc. | Method of generating a plurality of chemical compounds in a spatially arranged array |
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| AU2003249576A1 (en) | 2003-12-19 |
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| WO2003102208A2 (en) | 2003-12-11 |
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| WO2003102208A3 (en) | 2004-03-25 |
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