WO2003102208A2 - Rapid detection of bt-cry toxins - Google Patents

Rapid detection of bt-cry toxins Download PDF

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Publication number
WO2003102208A2
WO2003102208A2 PCT/IN2003/000199 IN0300199W WO03102208A2 WO 2003102208 A2 WO2003102208 A2 WO 2003102208A2 IN 0300199 W IN0300199 W IN 0300199W WO 03102208 A2 WO03102208 A2 WO 03102208A2
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Prior art keywords
gold
membrane
cry
line
fibre
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PCT/IN2003/000199
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French (fr)
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WO2003102208A3 (en
Inventor
Keshav Raj Kranthi
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Indian Council Of Agricultural Research
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Priority to AU2003249576A priority Critical patent/AU2003249576A1/en
Priority to MXPA04011769A priority patent/MXPA04011769A/en
Publication of WO2003102208A2 publication Critical patent/WO2003102208A2/en
Publication of WO2003102208A3 publication Critical patent/WO2003102208A3/en

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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
    • 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)

Definitions

  • This invention relates to a rapid immunochromatographic assay using polyclonal antibodies to detect crystal toxins Cry1 Ac/Cry1 Ab/Cry2Aa/Cry2Ab in seed or plant tissues on lateral flow strips.
  • crystal toxins isolated from a native Bacillus thuringiensis strain and a method using polyclonal antibodies raised against the said crystal toxins.
  • the invention relates to a simplified development using manual striping methods and a greatly enhanced specificity and accuracy of the assay by the use of antigen-affinity and Protein-A affinity purified polyclonal IgG raised in two different animals - goat and rabbit.
  • the invention incorporates the use of crystal toxin receptors from lepidopteran insects as capture ligands to detect the cry toxins.
  • the invention also facilitates the simultaneous detection of two crystal toxins on a single strip.
  • the soil bacterium Bacillus thuringiensis produces some protein crystals that are toxic to insect pests but harmless to the host plant and environment. Genetic transformation of host plants using genes isolated from the bacterium can enable them to synthesize the said toxin and combat the insect pests. Genes isolated from B. thuringiensis (also commonly known as Bt-genes) have been used to transform cotton (Gossypium hirsutum) and several other crop plants in breeding for pest resistance programmes and genes such as crylAc, crylAb, crylAa, cry2Aa and Cry2Ab have been among the most commonly used genes for such genetic transformation.
  • toxin in transformed plants is vital as this has a bearing on the toxicity effect on the target species of insect pest.
  • the CrylAc expression in transgenic plants is the most important attribute if pest control has to be effective and the detection of its expression is, therefore, equally important.
  • the expression of toxin is generally quantified using standard ELISA methods and, further, rapid test has become possible with the advent of immunochromatographic methods to detect the expression of the transgenes.
  • the immunochromatographic strips are, however, expensive when made by the process that involves use of monoclonal antibodies and several instruments.
  • the commercial Bt-transgenic crops have to be assessed for the purity as well as efficacy (being the quality toxin expression) at various stages and levels - research, technology development and verification, environmental impact, seed quality control, cultivation fields, commercial lots of the produce, etc. Any method leading to a rapid detection of the Bt- transgenic technology could be helpful and, further, an inexpensive one can be an asset.
  • One alternative could be to use polyclonal antibodies instead of monoclonal ones to develop immunochromatographic strips without compromising on efficacy of detection of cry expression in seed or plant tissues and, therefore, it is an object of the present investigations. Another object is to develop the said strips with minimum instrumentation, involving less cost and price.
  • Yet another object is to develop a method of detection of the said expression using the polyclonal antibodies raised against crystal toxins isolated from a native Bacillus thuringiensis strain.
  • the method aims at providing a robust and easy method suitable for development of immunochromatographic assays to detect CrylAc/ CrylAb/ Cry2Aa Cry2Ab.
  • yet another object of the development of present method was to simplify the immunochromatographic detection method for CrylAc/ CrylAb/ Cry2Aa/ Cry2Ab detection using affinity purified polyclonal antibodies specific to the analyte and also to the complete crystal toxins.
  • the capture antibody line polyclonal immunoglobin (IgG) from animals such as rabbit or goat
  • N-aminopeptidase or cadherin line striped midway across the membrane.
  • the IgG coated in Gold accumulates at the capture IgG line and generates a visible signal indicating the presence of the Cry 1 Ac/Cry 1Ab/Cry2Aa/Cry2Ab toxin.
  • the gold coated IgG travels along the membrane, binds with the goat/rabbit anti-rabbit/goat antibody, accumulates and generates a visible signal.
  • brush border membrane vesicles are prepared from guts of lepidopteran larvae using differential precipitation and centhfugation methods with mannitol, Ethylene glycol-bis ( ⁇ -aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) and Magnesium Chloride (MgCI 2 ) in accordance with Wolfersberger, et al. (1987) Preparation and partial characterization of amino acid transporting brush border membrane vescicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A, 301 -308.
  • the pellet containing N-aminopeptidase / cadherin receptors of Cry proteins are used for striping as antigen capture proteins.
  • Antigen According to a second aspect of the invention, there is isolated a native Bacillus thuringiensis strain 'x' (identity confirmed at the Institute of Microbial Technology (IMTECH), Chandigarh and characterized for uniqueness through antibiotic resistance profile, colony morphology and growth characteristics. Crystal protein producing genes cry1Ac/cry2A from plasmids of the strain were amplified on PCR using specific primers, and cloned into pUC 18 and pRK 223-3 expression vectors. Crystal toxins were purified from the clones through differential centhfugation, chromatography and SDS polyacrylamide gel electrophoresis. The toxins are purified and antisera raised against the specific toxins.
  • Raising antibodies The purified crystal toxins are mixed with Freunds complete adjuvant and injected into Rabbits/Goats. A booster dose is given using the dissolved toxin in incomplete Freunds adjuvant at monthly intervals and serum is collected. The serum is precipitated with ammonium sulphate and IgG purified using DEAE cellulose, Protein A/Protein G columns and/or, when necessary, finally with Cry1Ac/Cry2A antigen-affinity column chromatography. Preparation of colloidal gold and IgG conjugation.
  • Colloidal Gold is prepared by citrate reduction of gold chloride in the synthesis of colored particle conjugates, as per standard methods and engineering principles previously explained in Horisberger, (1979) "Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy", Biol. Cellulaire, 36, 253-258; Leuvering et al., (1980) "Sol Particle Immunoassay", J Immunoassay, 1 (1 ): 77-91 , and Frens (1973) "Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions", Nature, Physical Science, 241 : 20-22.
  • the affinity purified antibody IgG from one animal is conjugated to gold according to the methods published in protocols described in these abovementioned publications and applied on 30 cm x 1 cm conjugate release glass-fibre pads.
  • a polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman) is stuck on the plastic backing.
  • Crystal toxin specific IgGs are striped manually as a 30 cm x 0.1 cm line on the membrane midway at 1.25 cm and 1 cm from bottom of the membrane, for the two toxin detection strips and Crystal toxin specific IgG or crystal toxin receptor, only 1.25 cm from both ends of the membrane for a single toxin detection strip.
  • a goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane.
  • the membrane is blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing sugars and preservatives, and washed twice with phosphate buffered saline.
  • the dry conjugate coated glass fibre pad is placed on the lower end of the membrane so as to overlap 2 mm on it.
  • a thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing.
  • Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing.
  • the assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
  • Symptoms In positive samples (for a single toxin detection strip) two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive. Whereas in samples, which are devoid of CrylAc/ CrylAb/ Cry2Aa/ Cry2Ab, only one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin.
  • the strips designed to detect two toxins (CrylA and Cry2A) simultaneously on the same strip have three lines in samples positive for both toxins, or only two for samples positive for any one toxin or only one (control line) if sample is negative for both toxins.
  • CrylAc is isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins.
  • the toxin is solubilized in an alkaline buffer and extracted by centhfugation. Purification of the toxin is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
  • Antiserum is raised against CrylAc toxin in rabbits or goats by injecting them separately with purified toxin.
  • the purified IgG obtained from antigen affinity purification is dialyzed with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • the anti-Cry1Ac-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
  • Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • the membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1-5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • a polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
  • Colloidal gold is commercially available from Sigma Chemicals, USA.
  • the affinity purified anti-Cry1Ac-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring.
  • the conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
  • BSA Bovine Serum Albumin
  • the conjugate is centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet.
  • the pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide.
  • the colloidal gold conjugated solution is applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
  • the dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it. 16.
  • a thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
  • Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
  • the assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
  • Plant tissues such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1 .5 ml microcentrifuge plastic vial, using a Teflon pestle.
  • the bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue.
  • the solution flows up into the glass fibre pad and moves up the membrane through capillary force.
  • Example(2) Preparation of a rapid immunochromatographic assay/strip to detect Cry1Ac/Cry2Ab using Cry-toxin receptor proteins as capture ligands.
  • CrylAc and Cry2Aa/Cry2Ab are isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins.
  • the toxins are solubilized in an alkaline buffer and extracted by centhfugation. Purification of the toxins is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
  • Antiserum is raised against CrylAc /Cry2Aa/Cry2Ab toxins in rabbits or goats by injecting them separately with purified toxins.
  • the immunoglobin IgG is purified from the antiserum by precipitating with ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity columns.
  • the methods used herein are described in detail in Antibodies -A laboratory Manual (Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).
  • the purified IgG obtained from antigen affinity purification is dialysed with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • BBMVs Brush border membrane vesicles
  • EGTA Ethylene glycol-bis ( ⁇ -aminoethyl ether)-N,N,N',N'-tetra acetic acid
  • MgCI 2 Magnesium Chloride
  • cry-toxin-receptor proteins are striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
  • Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • the membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1 -5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • a polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
  • Colloidal gold is commercially available from Sigma Chemicals, USA.
  • the affinity purified anti-Cry1Ac-lgG or anti-Cry2Ab-lgG are diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring.
  • the conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
  • BSA Bovine Serum Albumin
  • the conjugate is centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet.
  • the pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide.
  • the colloidal gold conjugated solution is applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
  • the dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it.
  • a thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
  • Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
  • the assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
  • Plant tissues (ca. 50 mg) such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1.5 ml microcentrifuge plastic vial, using a Teflon pestle.
  • the bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue.
  • the solution flows up into the glass fibre pad and moves up the membrane through capillary force.
  • Example(3) Preparation of a rapid immunochromatographic strip to simultaneously detect Cry1 Ac and Cry2Ab.
  • CrylAc and Cry2Aa/Cry2Ab are isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins.
  • the toxins are solubilized in an alkaline buffer and extracted by centrifugation. Purification of the toxins is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
  • Antiserum is raised against CrylAc /Cry2Aa/Cry2Ab toxins in rabbits or goats by injecting them separately with purified toxins.
  • the immunoglobin IgG is purified from the antiserum by precipitating with ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity columns.
  • the methods used herein are described in detail in Antibodies -A laboratory Manual (Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).
  • the purified IgG obtained from antigen affinity purification is dialysed with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • the anti-Cry1Ac-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
  • the anti-Cry2Aa/Cry2Ab-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.0 cm distance from the bottom end on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
  • Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane. Anti-goat IgG is used as control line if the conjugate pad IgG is from goat. Similarly anti-rabbit IgG is used as control line if the conjugate pad IgG is from rabbit.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • the membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1-5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
  • the membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
  • a polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
  • Colloidal gold is commercially available from Sigma Chemicals, USA.
  • the affinity purified anti-Cry1Ac-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring.
  • the conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
  • BSA Bovine Serum Albumin
  • Colloidal gold is commercially available from Sigma Chemicals, USA.
  • the affinity purified anti-Cry2Aa/Cry2Ab-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring.
  • the conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
  • BSA Bovine Serum Albumin
  • the conjugates are centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet.
  • the pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide.
  • colloidal gold conjugated solutions of CrylAc and Cry2Ab/Cry2Aa are mixed in equal quantities and applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
  • the dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it.
  • a thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
  • Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
  • the assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
  • Plant tissues (ca. 50 mg) such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1.5 ml microcentrifuge plastic vial, using a Teflon pestle.
  • the bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue.
  • the solution flows up into the glass fibre pad and moves up the membrane through capillary force.
  • samples positive for either Cry1Ac/Cry1Ab or Cry2Aa/Cry2Ab only two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive either for Cry1 Ac/Cry1 Ab or Cry2Aa/Cry2Ab.
  • samples, which are devoid of Cry1Ac/Cry1Ab or Cry2Aa/Cry2Ab only one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin. If three bands (includes the control band) appear it indicates that the sample is positive for both Cry1 Ac/Cry1 Ab and Cry2Aa/Cry2Ab.
  • the processes claimed herein employ affinity-purified immunoglobins (IgG) raised in two different animals, thus enhancing sensitivity of detection.
  • IgG affinity-purified immunoglobins
  • the methods enable the simultaneous detection of two or more toxins on a single strip, thus reducing on the cost of two or more strips for the same purpose.

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. The methods and devices have high sensitivity to detect either single or two Cry toxins simultaneously on a single strip. Use of the present methods provide an assay system which involve a minimal number of steps, and yield reliable results even when used by untrained persons.

Description

DESCRIPTION
TITLE OF INVENTION
Rapid detection of Bt-Cry toxins Technical Field
This invention relates to a rapid immunochromatographic assay using polyclonal antibodies to detect crystal toxins Cry1 Ac/Cry1 Ab/Cry2Aa/Cry2Ab in seed or plant tissues on lateral flow strips. Particularly it concerns crystal toxins isolated from a native Bacillus thuringiensis strain and a method using polyclonal antibodies raised against the said crystal toxins. More particularly the invention relates to a simplified development using manual striping methods and a greatly enhanced specificity and accuracy of the assay by the use of antigen-affinity and Protein-A affinity purified polyclonal IgG raised in two different animals - goat and rabbit. The invention incorporates the use of crystal toxin receptors from lepidopteran insects as capture ligands to detect the cry toxins. The invention also facilitates the simultaneous detection of two crystal toxins on a single strip.
Background Art
The soil bacterium Bacillus thuringiensis produces some protein crystals that are toxic to insect pests but harmless to the host plant and environment. Genetic transformation of host plants using genes isolated from the bacterium can enable them to synthesize the said toxin and combat the insect pests. Genes isolated from B. thuringiensis (also commonly known as Bt-genes) have been used to transform cotton (Gossypium hirsutum) and several other crop plants in breeding for pest resistance programmes and genes such as crylAc, crylAb, crylAa, cry2Aa and Cry2Ab have been among the most commonly used genes for such genetic transformation.
Confirmation of genetic transformation of the plants is essential for the success of breeding programme and there are several molecular methods to do this. Further, the expression of the toxin in transformed plants is vital as this has a bearing on the toxicity effect on the target species of insect pest. The CrylAc expression in transgenic plants is the most important attribute if pest control has to be effective and the detection of its expression is, therefore, equally important. The expression of toxin is generally quantified using standard ELISA methods and, further, rapid test has become possible with the advent of immunochromatographic methods to detect the expression of the transgenes. The immunochromatographic strips are, however, expensive when made by the process that involves use of monoclonal antibodies and several instruments.
Disclosure of the Invention
The commercial Bt-transgenic crops have to be assessed for the purity as well as efficacy (being the quality toxin expression) at various stages and levels - research, technology development and verification, environmental impact, seed quality control, cultivation fields, commercial lots of the produce, etc. Any method leading to a rapid detection of the Bt- transgenic technology could be helpful and, further, an inexpensive one can be an asset. One alternative could be to use polyclonal antibodies instead of monoclonal ones to develop immunochromatographic strips without compromising on efficacy of detection of cry expression in seed or plant tissues and, therefore, it is an object of the present investigations. Another object is to develop the said strips with minimum instrumentation, involving less cost and price. Yet another object is to develop a method of detection of the said expression using the polyclonal antibodies raised against crystal toxins isolated from a native Bacillus thuringiensis strain. The method aims at providing a robust and easy method suitable for development of immunochromatographic assays to detect CrylAc/ CrylAb/ Cry2Aa Cry2Ab.
Qualitative rapid immunodiagnostic tests (Huang et. al (1998) One step immunochromatographic device US Patent No. 5,712,172) have been commonly used to detect antigens on lateral flow assays as positive or negative signal. Examples of such assays include pregnancy detection kits, AIDS detection, and many types of urine analyses. CrylAc detection kits based on immunochromatoghraphic detection methods have been developed and commercialized by two US companies 'Agdia, USA' and 'Strategic Diagnostic Inc, USA' in July 2001 and October 2001 respectively. Both companies claim in their promotional literature, that the kits are based on monoclonal antibody combinations. Therefore, yet another object of the development of present method was to simplify the immunochromatographic detection method for CrylAc/ CrylAb/ Cry2Aa/ Cry2Ab detection using affinity purified polyclonal antibodies specific to the analyte and also to the complete crystal toxins.
Principle: A polyclonal immunoglobin (IgG) from animals such as rabbit or goat, coated with gold, captures Cry1Ac/Cry1Ab/Cry2Aa/Cry2Ab and carries it along the membrane by capillary action until the free ends of the CrylAc/ CrylAb/ Cry2Aa/ Cry2Ab bind to the capture antibody line (polyclonal immunoglobin (IgG) from animals such as rabbit or goat) or N-aminopeptidase or cadherin line striped midway across the membrane. The IgG coated in Gold accumulates at the capture IgG line and generates a visible signal indicating the presence of the Cry 1 Ac/Cry 1Ab/Cry2Aa/Cry2Ab toxin. In the absence of Cry1Ac/Cry1Ab/ Cry2Aa/Cry2Ab in the sample, the gold coated IgG travels along the membrane, binds with the goat/rabbit anti-rabbit/goat antibody, accumulates and generates a visible signal.
Preparing N-aminopeptidase/cadherin: According to one aspect of the invention, brush border membrane vesicles are prepared from guts of lepidopteran larvae using differential precipitation and centhfugation methods with mannitol, Ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) and Magnesium Chloride (MgCI2) in accordance with Wolfersberger, et al. (1987) Preparation and partial characterization of amino acid transporting brush border membrane vescicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A, 301 -308. The pellet containing N-aminopeptidase / cadherin receptors of Cry proteins are used for striping as antigen capture proteins.
Antigen: According to a second aspect of the invention, there is isolated a native Bacillus thuringiensis strain 'x' (identity confirmed at the Institute of Microbial Technology (IMTECH), Chandigarh and characterized for uniqueness through antibiotic resistance profile, colony morphology and growth characteristics. Crystal protein producing genes cry1Ac/cry2A from plasmids of the strain were amplified on PCR using specific primers, and cloned into pUC 18 and pRK 223-3 expression vectors. Crystal toxins were purified from the clones through differential centhfugation, chromatography and SDS polyacrylamide gel electrophoresis. The toxins are purified and antisera raised against the specific toxins.
Raising antibodies: The purified crystal toxins are mixed with Freunds complete adjuvant and injected into Rabbits/Goats. A booster dose is given using the dissolved toxin in incomplete Freunds adjuvant at monthly intervals and serum is collected. The serum is precipitated with ammonium sulphate and IgG purified using DEAE cellulose, Protein A/Protein G columns and/or, when necessary, finally with Cry1Ac/Cry2A antigen-affinity column chromatography. Preparation of colloidal gold and IgG conjugation. Colloidal Gold is prepared by citrate reduction of gold chloride in the synthesis of colored particle conjugates, as per standard methods and engineering principles previously explained in Horisberger, (1979) "Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy", Biol. Cellulaire, 36, 253-258; Leuvering et al., (1980) "Sol Particle Immunoassay", J Immunoassay, 1 (1 ): 77-91 , and Frens (1973) "Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions", Nature, Physical Science, 241 : 20-22. The affinity purified antibody IgG from one animal is conjugated to gold according to the methods published in protocols described in these abovementioned publications and applied on 30 cm x 1 cm conjugate release glass-fibre pads.
Preparation of the Lateral flow assembly. A polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman) is stuck on the plastic backing. Crystal toxin specific IgGs are striped manually as a 30 cm x 0.1 cm line on the membrane midway at 1.25 cm and 1 cm from bottom of the membrane, for the two toxin detection strips and Crystal toxin specific IgG or crystal toxin receptor, only 1.25 cm from both ends of the membrane for a single toxin detection strip. A goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane. The membrane is blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing sugars and preservatives, and washed twice with phosphate buffered saline. After drying the membrane completely, the dry conjugate coated glass fibre pad is placed on the lower end of the membrane so as to overlap 2 mm on it. A thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. The assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
Symptoms: In positive samples (for a single toxin detection strip) two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive. Whereas in samples, which are devoid of CrylAc/ CrylAb/ Cry2Aa/ Cry2Ab, only one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin. The strips designed to detect two toxins (CrylA and Cry2A) simultaneously on the same strip have three lines in samples positive for both toxins, or only two for samples positive for any one toxin or only one (control line) if sample is negative for both toxins.
Example(1): Preparation of a rapid immunochromatographic assay/strip to detect CrylAc
1. CrylAc is isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins. The toxin is solubilized in an alkaline buffer and extracted by centhfugation. Purification of the toxin is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
2. Antiserum is raised against CrylAc toxin in rabbits or goats by injecting them separately with purified toxin.
3. The method of raising antisera by injecting the antigen (CrylAc) mixed with Freunds complete adjuvant initially and repeated booster doses as mixed with incomplete Freund's adjuvant before collecting serum, is common art available in text books of immunology (Antibodies -A laboratory Manual -Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988) and is not described in detail here. 4. The immunoglobin IgG is purified from the antiserum by precipitating with ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (CrylAc toxin) affinity columns. The methods used herein are described in detail in Antibodies -A laboratory Manual (Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).
5. The purified IgG obtained from antigen affinity purification is dialyzed with 0.01 M, sodium phosphate buffer, Ph 7.2.
6. The anti-Cry1Ac-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
7. Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane.
8. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
9. The membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1-5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
10. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
11. A polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
12. Colloidal gold is commercially available from Sigma Chemicals, USA. The affinity purified anti-Cry1Ac-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring. The conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
13. The conjugate is centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet. The pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide.
14. The colloidal gold conjugated solution is applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
15. The dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it. 16. A thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
17. Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
18. The assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
19. Plant tissues (ca. 50 mg) such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1 .5 ml microcentrifuge plastic vial, using a Teflon pestle.
20. The bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue. The solution flows up into the glass fibre pad and moves up the membrane through capillary force.
21. In about ten to fifteen minutes the solution reaches the top end of the strip. One clear purple line appears at the upper portion one cm below the top end of the membrane region, indicating that the membrane is functional. This is called as a control line. Another purple coloured line appears at the central portion of the membrane if the test sample is positive for CrylAc.
22. Thus in CrylAc positive samples two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive. Whereas in samples, which are devoid of CrylAc only one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin. Example(2): Preparation of a rapid immunochromatographic assay/strip to detect Cry1Ac/Cry2Ab using Cry-toxin receptor proteins as capture ligands.
1. CrylAc and Cry2Aa/Cry2Ab are isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins. The toxins are solubilized in an alkaline buffer and extracted by centhfugation. Purification of the toxins is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
2. Antiserum is raised against CrylAc /Cry2Aa/Cry2Ab toxins in rabbits or goats by injecting them separately with purified toxins.
3. The method of raising antisera by injecting the antigen (Cry1 Ac/Cry2Aa/Cry2Ab) mixed with Freunds complete adjuvant initially and repeated booster doses as mixed with incomplete Freund's adjuvant before collecting serum, is common art available in text books of immunology (Antibodies -A laboratory Manual -Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988) and is not described in detail here.
4. The immunoglobin IgG is purified from the antiserum by precipitating with ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity columns. The methods used herein are described in detail in Antibodies -A laboratory Manual (Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).
5. The purified IgG obtained from antigen affinity purification is dialysed with 0.01 M, sodium phosphate buffer, Ph 7.2.
• 6. Brush border membrane vesicles (BBMVs) are prepared from guts of lepidopteran larvae using differential precipitation and centrifugation methods with mannitol, Ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) and Magnesium Chloride (MgCI2) accordance to the protocols described by Wolfersberger et al (1987). Cry-receptor proteins are purified from the BBMVs using ammonium sulphate and standard spin cloumn chromatographic methods.
7. The cry-toxin-receptor proteins are striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
8. Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane.
9. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
10. The membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1 -5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
11. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
12. A polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
13. Colloidal gold is commercially available from Sigma Chemicals, USA. The affinity purified anti-Cry1Ac-lgG or anti-Cry2Ab-lgG are diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring. The conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
14. The conjugate is centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet. The pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide. 15. The colloidal gold conjugated solution is applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
16. The dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it.
17. A thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
18. Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
19. The assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
20. Plant tissues (ca. 50 mg) such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1.5 ml microcentrifuge plastic vial, using a Teflon pestle.
21. The bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue. The solution flows up into the glass fibre pad and moves up the membrane through capillary force.
22. In about ten to fifteen minutes the solution reaches the top end of the strip. One clear purple line appears at the upper portion one cm below the top end of the membrane region, indicating that the membrane is functional. This is called as a control line. Another purple coloured line appears at the central portion of the membrane if the test sample is positive for CrylAc or Cry2Aa/Cry2Ab depending on the conjugate antibody used.
23. Thus in CrylAc or Cry2Aa/Cry2Ab positive samples, two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive. Whereas in samples, which are devoid of any of the two toxin groups one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin.
Example(3): Preparation of a rapid immunochromatographic strip to simultaneously detect Cry1 Ac and Cry2Ab.
1. CrylAc and Cry2Aa/Cry2Ab are isolated from the clones by sonicating the bacterial clone cultures, pelleting out the cellular debris and the insoluble toxins. The toxins are solubilized in an alkaline buffer and extracted by centrifugation. Purification of the toxins is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis.
2. Antiserum is raised against CrylAc /Cry2Aa/Cry2Ab toxins in rabbits or goats by injecting them separately with purified toxins.
3. The method of raising antisera by injecting the antigen (Cry1Ac/Cry2Aa/Cry2Ab) mixed with Freunds complete adjuvant initially and repeated booster doses as mixed with incomplete Freund's adjuvant before collecting serum, is common art available in text books of immunology (Antibodies -A laboratory Manual -Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988) and is not described in detail here.
4. The immunoglobin IgG is purified from the antiserum by precipitating with ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (Cry1Ac/Cry2Aa/Cry2Ab toxin) affinity columns. The methods used herein are described in detail in Antibodies -A laboratory Manual (Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).
5. The purified IgG obtained from antigen affinity purification is dialysed with 0.01 M, sodium phosphate buffer, Ph 7.2.
6. The anti-Cry1Ac-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.25 cm distance from upper and bottom ends on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
7. The anti-Cry2Aa/Cry2Ab-lgG raised in rabbit /goat is striped as a 1 mm thick 30 cm long line centrally at 1.0 cm distance from the bottom end on one side of a 2.5 x 30 cm nylon/ nitrocellulose/ cellulose nitrate membrane strip (S&S/ Whatman/ millipore/ Pall-Gelman), using a hand held sable hair brush (Numbers 0, 00, 000, 1 , 2, 3, 4 or 5), along a 30-cm-ruler support.
8. Anti-rabbit IgG / anti-goat IgG (available commercially with a number of companies including Sigma Chemical Company, USA) is solubilised in 0.01 M, sodium phosphate buffer, Ph 7.2. and striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane. Anti-goat IgG is used as control line if the conjugate pad IgG is from goat. Similarly anti-rabbit IgG is used as control line if the conjugate pad IgG is from rabbit.
9. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
10. The membrane is then blocked with a buffer containing 2% Casein/BSA and 3% fat free milk powder containing 1-5% sucrose and sodium azide preservatives, and washed twice with 0.01 M, sodium phosphate buffer, Ph 7.2.
11. The membrane is dried at 50°C under a dry wind blower for 10-15 minutes.
12. A polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is stuck centrally equidistant from the top and bottom ends of the sheet.
13. Colloidal gold is commercially available from Sigma Chemicals, USA. The affinity purified anti-Cry1Ac-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring. The conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
14. Colloidal gold is commercially available from Sigma Chemicals, USA. The affinity purified anti-Cry2Aa/Cry2Ab-lgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal gold (pH adjusted to 9.0) while stirring. The conjugate is stabilized by adding 10% BSA (Bovine Serum Albumin) to the mixture.
15. The conjugates are centrifuged at 12,000 g for 30 minutes at 4°C to obtain a loose pellet. The pellet is dissolved in 100 ml of a solution containing 0.01 M Tris, 5% BSA, 2% sucrose, 0.87% NaCI and 0.1 M sodium azide.
16. The colloidal gold conjugated solutions of CrylAc and Cry2Ab/Cry2Aa are mixed in equal quantities and applied on 30 cm x 1 cm conjugate release glass-fibre pads and dried under dry air blast for 10-15 minutes.
17. The dry conjugate coated glass fibre pad is placed on the lower end of the membrane mentioned in step 10, so as to overlap 2 mm on it.
18. A thick filter pad 30 x 1 x 0.1 cm is placed on the lower end of the conjugate release pad to overlap 2 mm and stuck using acrylic adhesive on the free area of the plastic backing. This end is being referred here as bottom end.
19. Another filter pad 30 x 1 x 0.1 cm is placed on the upper end of the membrane to overlap 2 mm and stuck using acrylic adhesive on the membrane free area of the plastic backing. This end is being referred here as top end.
20. The assembly is laminated using a cold-lamination sheet and cut into 6 cm x 0.5 cm strips.
21. Plant tissues (ca. 50 mg) such as leaf, stem, roots, flowers, seeds etc are crushed in 0.5 ml 0.01 M, sodium phosphate buffer, Ph 7.2 in a 1.5 ml microcentrifuge plastic vial, using a Teflon pestle.
22. The bottom end of the strip is dipped into the vial containing the crushed leaf/seed tissue. The solution flows up into the glass fibre pad and moves up the membrane through capillary force.
23. In about ten to fifteen minutes the solution reaches the top end of the strip. One clear purple line appears at the upper portion one cm below the top end of the membrane region, indicating that the membrane is functional. This is called as a control line. Two purple coloured lines appear at the central portion of the membrane if the test sample is positive for both Cry1Ac/Cry1Ab or Cry2Aa/Cry2Ab. Only one line appears at the central portion of the membrane corresponding to 1.25 cm from the bottom of the membrane for Cry1 Ac/Cry1 Ab or at 1.0 cm from the bottom of the membrane for Cry2Aa/Cry2Ab. 24. Thus in samples positive for either Cry1Ac/Cry1Ab or Cry2Aa/Cry2Ab only two lines appear - a control line to indicate that the strip is functional and a sample line to indicate that the sample is positive either for Cry1 Ac/Cry1 Ab or Cry2Aa/Cry2Ab. Whereas in samples, which are devoid of Cry1Ac/Cry1Ab or Cry2Aa/Cry2Ab only one line appears indicating that the strip is functional; and the sample is detected as negative for the toxin. If three bands (includes the control band) appear it indicates that the sample is positive for both Cry1 Ac/Cry1 Ab and Cry2Aa/Cry2Ab.
Advantages
1. The methods described herein which employ polyclonal antibodies and manual striping methods are simple to execute and do not require expensive 'monoclonal antibodies or monoclonal techniques and striping equipment costing > US $ 20,000' as used in the commercial manufacture of strips similar kind.
2. The processes claimed herein employ affinity-purified immunoglobins (IgG) raised in two different animals, thus enhancing sensitivity of detection.
3. The methods enable the simultaneous detection of two or more toxins on a single strip, thus reducing on the cost of two or more strips for the same purpose.
4. The use of 'Cry toxin receptors' in strips enable high sensitivity detection of any of the Cry toxins that are toxic to the concerned lepidopteran insect from which the receptors were isolated.
References
1. US Patent No. 5,712,172.
2. Harlow Ed and David Lane. (1988) Antibodies -A laboratory manual. Cold Spring harbor Laboratory. New York 11724, USA.
3. Wolfersberger, M. G., Luthy, P., Maurer, A., Parenti, P., Sacchi, V. F., Giordana, B and Hanozet, G. M. (1987) Preparation and partial characterization of amino acid transporting brush border membrane vescicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A, 301-308.
4. Hohsberger, (1979). Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy, Biol. Cellulaire, 36, 253-258
5. Leuvering et al., (1980) Sol Particle Immunoassay, J. Immunoassay, 1 (1): 77-91 ,
6. Frens, (1973) Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions, Nature Physical Science, 241 : 20-22.
Dated this day 2 % ' WΛ/ -
Figure imgf000009_0001
Assistant Director General (I PR)
Indian council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road
New Delhi 110001

Claims

1. A method to prepare a rapid immunochromatographic assay/strip using polyclonal antibodies that identifies/detects Cry toxins from Bacillus thuringiensis as single toxin from seed or plant tissues, said method comprising the steps of-
(i) Identification of a unique native Bacillus thuringiensis strain, said strain characterized through antibiotic resistance profile, colony morphology and growth characteristics, amplifying crystal protein producing genes from plasmids of said strain using specific primers and cloning into specific expression vectors, (n) Purification of CrylAc and Cry2Aa to apparent homogeneity using a variety of methods including chromatography and electrophoresis (in) Raising polyclonal antibodies to the toxins as claimed in step (u) in rabbits and goats by recurrently injecting them with booster dose of the crystal toxins mixed with adjuvant and collected serum thereof, precipitating said serum and purifying the antibody, (iv) Purification of IgG from antiserum as claimed in step (in) using affinity chromatographic methods comprising of protein-A and Cry1 Ac/Cry2Aa antigen, (v) Using anti-Cry polyclonal IgG, as claimed in step (iv) to immobilise on a membrane (vi) immobilizing the capture line containing said polyclonal IgG raised in goat as claimed in step (iv) on a cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns) using a manual striping method using a hand held sable hair brush (vn) immobilizing the capture line containing said polyclonal IgG raised in rabbit as claimed in step (iv) on a cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns) using a manual striping method using a hand held sable hair brush (vin) Labelling the polyclonal IgG antibodies raised in goat as claimed in step (iv) with colloidal gold 20-40 nm (ix) Labelling the polyclonal IgG antibodies raised in rabbit as claimed in step (iv) with colloidal gold 20-40 nm. (x) Immobilisation of the gold-antibody conjugate of step (VIM) on glass-fibre pads using a micropipette (xi) Immobilisation of the gold-antibody conjugate of step (ix) on glass-fibre pads using a micropipette. (xn) A goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x
0 1 cm line at 0 5 cm from the top end of the membrane in each of the strips (XIII) The said goat anti-rabbit IgG or rabbit anti-goat IgG line of step (xn) would capture gold labeled anti-rabbit antibody or anti-goat antibody respectively as may be the case in any of the assembled strips (xiv) The said line of step (xn) of claim 1 , would serve as control line to indicate a proper functioning of the strip (xv) The said line of step (xn) of claim 1 , would turn pink/purple when used irrespective of the test sample being positive or negative (xvi) The membrane is blocked with blocking buffer containing proteins, sugars and preservatives, and washed twice with phosphate buffered saline and dried for
30 minutes before assembly, (xvn) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (x) of claιm-1 , on the membrane as claimed in step (vi) of claιm-1 , to overlap 2mm as shown in Fig 1. (xviii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (x) of claim-1 , on the membrane as claimed in step (vii) of claim-1 , to overlap 2mm as shown in Fig 1.
(xix) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xi) of claim-2, on the membrane as claimed in step (vi) of claim-1 , to overlap 2mm as shown in Fig 1.
(xx) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xi) of claim-2, on the membrane as claimed in step (vii) of claim-1 , to overlap 2mm as shown in Fig 1.
(xxi) Completing the assembly by placing a sample-release filter paper pad (1 to 2mm thick) at the bottom end of the assembly as claimed in steps (xvii, xviii, xix and xx) so as to overlap 2 mm. A second sample-absorbing pad is placed at the top end of the membrane as claimed in step (vi) and (vii) of claim-1 , so as to overlap 2 mm.
(xxii) Cold lamination of the final assembly
(xxiii) Using a combination of capture line of one anti-Cry antibody raised in one animal (goat) with sandwich of antigen between gold labelled anti-Cry antibody raised in another animal (rabbit) as claimed in step (xix) to detect the toxin as a visible line.
(xxiv) A combination of capture line of one anti-Cry antibody raised in one animal (rabbit) with sandwich of antigen between gold labelled anti-Cry antibody raised in another animal (goat) as claimed in step (xviii) to detect the toxin as a visible line.
(xxv) A combination of capture line of one anti-Cry antibody raised in one animal (rabbit) with sandwich of antigen between gold labelled anti-Cry antibody of the same animal (rabbit) as claimed in step (xx) to detect the toxin as a visible line.
(xxvi) A combination of capture line of one anti-Cry antibody raised in one animal (goat) with sandwich of antigen between gold labelled anti-Cry antibody of the same animal (goat) as claimed in step (xx) to detect the toxin as a visible line.
(xxvii) The steps (xxiii) to (xxvi) used to detect cry toxins CrylAa, CrylAb, CrylAc, Cry2Aa and Cry2Ab
2. A method to prepare rapid immunochromatographic assay/strips as claimed in claim 1 wherein there is used a combination of Cry-toxin receptors isolated from lepidopteran insect guts and anti-Cry polyclonal antibodies that identifies/detects Cry toxins from Bacillus thuringiensis as single toxin from seed or plant tissues, said method comprising the steps of:
(i) Isolation of brush border membrane vesicles from guts of lepidopteran larvae according to the standard methods, say the one published by Wolfersberger et al (1987) (ii) Purification of N-aminopeptidases and cadherin proteins using protein precipitation methods with 30-80% ammonium sulfate. (iii) Immobilizing the capture line containing said proteins from step (ii) of claim 2 on cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns) using a manual striping method using a hand held sable hair brush (iv) Labelling the polyclonal IgG antibodies as claimed in step (iv) of claim 1 , with colloidal gold 20-40 nm. (v) Labelling the N-aminopeptidases and cadherin proteins as claimed in step (ii) of claim-2, with colloidal gold 20-40 nm. (vi) Immobilisation of the gold-antibody conjugate as claimed in step (iv) of claim-2, on glass-fibre pads (vii) Immobilisation of the gold-Cry-receptor protein conjugate as claimed in step (v) of claim-2 on glass-fibre pads (viii) A goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x 0.1 cm line at 0.5 cm from the top end of the membrane in each of the strips. (ix) The said goat anti-rabbit IgG or rabbit anti-goat IgG line of step (viii) would capture gold labeled anti-rabbit antibody or anti-goat antibody respectively as may be the case in any of the assembled strips, (x) The said line of step (ix) of claim 2, would serve as control line to indicate a proper functioning of the strip, (xi) The said line of step (ix) of claim 2, would turn pink/purple when used irrespective of the test sample being positive or negative, (xii) The membrane is blocked with blocking buffer containing proteins, sugars and preservatives, and washed twice with phosphate buffered saline and dried for
30 minutes before assembly, (xiii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (v) of claim-2, on the membrane as claimed in step (vi) of claim-1 , to overlap 2mm as shown in Fig 1. (xiv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (v) of claim-2, on the membrane as claimed in step (vii) of claim-1 , to overlap 2mm as shown in Fig 1. (xv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (v) of claim-2, on the membrane as claimed in step (iii) of claim-2, to overlap 2mm as shown in Fig 1. (xvi) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (x) of claim-1 , on the membrane as claimed in step (iii) of claim-2, to overlap 2mm as shown in Fig 1. (xvii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xi) of claim-1 , on the membrane as claimed in step (iii) of claim-2, to overlap 2mm as shown in Fig 1. (xviii) Completing the assembly by placing a sample-release filter paper pad (1 to
2mm thick) at the bottom end of the glass-fibre as claimed in steps (xiii, xiv, xv, xvi and xvii) of claim-2, so as to overlap 2 mm. A second sample-absorbing pad is placed at the top end of the membranes as claimed in steps (xiii, xiv, xv, xvi and xvii) of claim-2, so as to overlap 2 mm. (xix) Cold lamination of the final assembly (xx) Using a combination of capture line of one anti-Cry antibody raised in one animal (goat) as claimed in step (vi) of claim-1 with sandwich of antigen between gold-Cry-receptor protein conjugate as claimed in step (v) of claim-2, to detect the toxin as a visible line, (xxi) A combination of capture line of one anti-Cry antibody raised in one animal
(rabbit) as claimed in step (vii) of claim-1 , with sandwich of antigen between gold-Cry-receptor protein conjugate as claimed in step (v) of claim-2 to detect the toxin as a visible line, (xxii) A combination of capture line of Cry-receptor of step (iii) of claim-2, with sandwich of antigen between gold-Cry-receptor protein conjugate as claimed in step (v) of claim-2 to detect the toxin as a visible line, (xxiii) A combination of capture line of Cry-receptor of step (iii) of claim-2, with sandwich of antigen between gold labelled anti-Cry antibody raised in goat of step (x) of claim-1 , to detect the toxin as a visible line, (xxiv) A combination of capture line of Cry-receptor of step (iii) of claim-2, with sandwich of antigen between gold labelled anti-Cry antibody raised in rabbit of step (xi) of claim-1 , to detect the toxin as a visible line, (xxv) The steps (xx) to (xxiv) used to detect cry toxins CrylAa, CrylAb, CrylAc,
Cry2Aa and Cry2Ab
A method to prepare a rapid immunochromatographic assay/strip using polyclonal antibodies as claimed in claim-1 that simultaneously identifies/detects two or more Cry toxins from Bacillus thuringiensis from seed or plant tissues, said method comprising the steps of: (I) Production of CrylAc and Cry2Aa toxins from the clones as claimed in step (i) of claim-1. (i) Purification of CrylAc and Cry2Aa to apparent homogeneity using a variety of methods including chromatography and electrophoresis. (ii) Raising polyclonal antibodies to the toxins as claimed in step (iii) of claim-1 , in rabbits and goats obtained as antiserum. (iii) Purification of IgG from antiserum as claimed in step (iv) of claim-1 , using affinity chromatographic methods comprising of protein-A and Cry1Ac/Cry2Aa antigen, (iv) using anti-Cry polyclonal IgG, as claimed in step (iv) to immobilise on a cellulose nitrate, nitrocellulose or nylon membrane (5 to 12 microns) using a manual striping method using a hand held sable hair brush, (v) immobilizing two capture lines on central part, one cm from both ends of a 2.5 cm wide membrane as claimed in step (v) of claim-3, at 0.5 cm apart containing said anti-Cryl Ac polyclonal IgG raised in goat as claimed in step (iv) of claim-1 , and anti-Cry2Aa IgG raised in goat, (vi) immobilizing two capture lines on central part, one cm from both ends of a 2.5 cm wide membrane as claimed in step (v) of claim-3, at 0.5 cm apart containing said anti-Cryl Ac polyclonal IgG raised in goat as claimed in step (iv) of claim-1 , and anti-Cry2Aa IgG raised in rabbit, (vii) immobilizing two capture lines on central part, one cm from both ends of a 2.5 cm wide membrane as claimed in step (v) of claim-3, at 0.5 cm apart containing said anti-Cry1Ac polyclonal IgG raised in rabbit as claimed in step
(iv) of claim-1 , and anti-Cry2Aa IgG raised in goat, (viii) immobilizing two capture lines on central part, one cm from both ends of a 2.5 cm wide membrane as claimed in step (v) of claim-3, at 0.5 cm apart containing said anti-Cry1Ac polyclonal IgG raised in rabbit as claimed in step
(iv) of claim-1 , and anti-Cry2Aa IgG raised in rabbit, (ix) Labelling the anti-Cry1Ac IgG raised in rabbit as claimed in step (iv) of claim 1 , with colloidal gold 20-40 nm. (x) Labelling the anti-Cry2Aa IgG raised in rabbit as claimed in step (iv) of claim 1 , with colloidal gold 20-40 nm. (xi) Labelling the anti-Cry1Ac IgG raised in goat as claimed in step (iv) of claim 1 , with colloidal gold 20-40 nm. (xii) Labelling the anti-Cry2Aa IgG raised in goat as claimed in step (iv) of claim 1 , with colloidal gold 20-40 nm. (xiii) Immobilisation of the gold-antibody conjugate as claimed in step (x) and (xi) of claim-3, in equal quantities on glass-fibre pads, (xiv) Immobilisation of the gold-antibody conjugate as claimed in step (x) and (xiii) of claim-3, in equal quantities on glass-fibre pads, (xv) Immobilisation of the gold-antibody conjugate as claimed in step (xi) and (xii) of claim-3, in equal quantities on glass-fibre pads, (xvi) Immobilisation of the gold-antibody conjugate as claimed in step (xii) and (xiii) of claim-3, in equal quantities on glass-fibre pads, (xvii) A goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x
0.1 cm line at 0.5 cm from the top end of the membrane in each of the strips, (xviii) The said goat anti-rabbit IgG or rabbit anti-goat IgG line of step (xviii) would capture gold labeled anti-rabbit antibody or anti-goat antibody respectively as may be the case in any of the assembled strips, (xix) The said line of step (xix) of claim 3, would serve as control line to indicate a proper functioning of the strip, (xx) The said line of step (xix) of claim 3, would turn pink/purple when used irrespective of the test sample being positive or negative, (xxi) The membrane is blocked with blocking buffer containing proteins, sugars and preservatives, and washed twice with phosphate buffered saline and dried for
30 minutes before assembly. (xxii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xiv) of claim-3, on the membrane as claimed in step (vi) of claim-3, to overlap 2mm as shown in Fig 2. (xxiii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xiv) of claim-2, on the membrane as claimed in step (vii) of claim-2, to overlap 2mm as shown in Fig 2. (xxiv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xiv) of claim-2, on the membrane as claimed in step (viii) of claim-1 , to overlap
2mm as shown in Fig 2. (xxv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xiv) of claim-2, on the membrane as claimed in step (ix) of claim-1 , to overlap 2mm as shown in Fig 2. (xxvi) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xv) of claim-3, on the membrane as claimed in step (vi) of claim-3, to overlap 2mm as shown in Fig 2. (xxvii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xv) of claim-3, on the membrane as claimed in step (vii) of claim-3, to overlap 2mm as shown in Fig 2. (xxviii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xv) of claim-3, on the membrane as claimed in step (viii) of claim-3, to overlap
2mm as shown in Fig 2. (xxix) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xv) of claim-3, on the membrane as claimed in step (ix) of claim-3, to overlap 2mm as shown in Fig 2. (xxx) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xvi) of claim-3, on the membrane as claimed in step (vi) of claim-3, to overlap 2mm as shown in Fig 2. (xxxi) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xvi) of claim-3, on the membrane as claimed in step (vii) of claim-3, to overlap 2mm as shown in Fig 2. (xxxii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xvi) of claim-3, on the membrane as claimed in step (viii) of claim-3, to overlap
2mm as shown in Fig 2. (xxxiii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (xvi) of claim-3, on the membrane as claimed in step (ix) of claim-3, to overlap 2mm as shown in Fig 2. (xxxiv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step
(xvii) of claim-3, on the membrane as claimed in step (vi) of claim-3, to overlap
2mm as shown in Fig 2. (xxxv) Assembling the gold-conjugate impregnated glass-fibre as claimed in step
(xvii) of claim-3, on the membrane as claimed in step (vii) of claim-3, to overlap
2mm as shown in Fig 2. (xxxvi) Assembling the gold-conjugate impregnated glass-fibre as claimed in step
(xvii) of claim-3, on the membrane as claimed in step (viii) of claim-3, to overlap 2mm as shown in Fig 2. (xxxvii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step
(xvii) of claim-3, on the membrane as claimed in step (ix) of claim-3, to overlap
2mm as shown in Fig 2. (xxxviii) Completing the assembly by placing a sample-release filter paper pad (1 to
2mm thick) at the bottom end of the glass-fibre as claimed in steps (xxiii) to
(xxxviii) of claim-3, so as to overlap 2 mm. A second sample-absorbing pad is placed at the top end of the membranes as claimed in steps (xxiii to xxxviii) of claim-3, so as to overlap 2 mm. (xxxix) Cold lamination of the final assembly, (xl) Using the any of the strips thus prepared from steps (xxiii) to (Xi) to simultaneously detect Cry1Ac/Cry1Ab and Cry2Aa/Cry2Ab as two lines if the test sample is positive for both toxins or a single sample line if the test sample is positive for either of the toxins or none at the sample line position on the strip if the test sample is negative.
(xii) The said position of sample lines would be fixed as lower line for Cry1Aa/Cry1Ac and upper line Cry2Aa/Cry2Ab or the reverse, with upper line being for Cry1Aa/Cry1Ac.
(xiii) The said sample lines appear due to the accumulation of the gold-labeled anti- Cry-toxin antibody, which binds the toxin (Cry1 Ac/Cry1 Ab or Cry2Aa or Cry2Ab), moves along the membrane due to capillary force, until the sample capture line is encountered. Once the gold-labeled anti-Cry-toxin antibody- toxin conjugate comes in contact with the sample capture line, the toxin gets bound, thus forming a sandwich and resulting in accumulation of the gold conjugate along the line. The accumulated gold conjugate appears as a visible line.
1. A method to prepare rapid immunochromatographic assay/strips as claimed in claim 1 wherein there is used a manual striping method with a hand held sable hair brush to immobilize antibody (IgG) solutions on the membrane.
(i) The said method of manual striping as claimed in steps (vi) of claim-1 , (iii) of claim-2 and (v) of claim-3 is done with the aid of a sable hair brush.
(ii) The hand held brush technique enables the absorption of antibody on the membrane without formation of craters.
(iii) A 30-cm-ruler is held gently on the membrane and striping is carried out by running the metallic part of the brush along the ruler to gently touch the tip of the antibody wetted bristles of the brush, thus forming a capture line.
^-
Dated this day ^ °~f ^ <2o ' <g^- V-
(Jai Prakash Mishra)
Assistant Director General (I PR)
Indian council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road
New Delhi 110001
PCT/IN2003/000199 2002-05-31 2003-05-29 Rapid detection of bt-cry toxins WO2003102208A2 (en)

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US11530238B2 (en) 2016-09-07 2022-12-20 Glaxosmithkline Intellectual Property Development Limited Methods for purifying antibodies
CN108828230A (en) * 2018-06-21 2018-11-16 北京市农林科学院 The method that nucleic acid chromatography quickly detects transgenic product
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AU2003249576A1 (en) 2003-12-19
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