ZA200410268B - Rapid detection of BT-cry Toxins - Google Patents
Rapid detection of BT-cry Toxins Download PDFInfo
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- ZA200410268B ZA200410268B ZA200410268A ZA200410268A ZA200410268B ZA 200410268 B ZA200410268 B ZA 200410268B ZA 200410268 A ZA200410268 A ZA 200410268A ZA 200410268 A ZA200410268 A ZA 200410268A ZA 200410268 B ZA200410268 B ZA 200410268B
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Classifications
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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)
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Description
Rapid detection of Bt-Cry toxins
This invention relates to a rapid immunochromatographic assay using polyclonal antibodies to detect crystal toxins Cry1Ac/Cry1Ab/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.
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, cry1Ab, 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 Cry1Ac 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 ots 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 Cry1Ac/
Cry1Ab/ Cry2Aa/ Cry2Ab. oo
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. Cry1Ac 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 Cry1Ac/
Cry1Ab/ 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 Cry1Ac/ Cry1Ab/ 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 Cry1Ac/Cry1Ab/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 centrifugation methods with mannitol, Ethylene glycol-bis (B-aminoethyl ether)-N,N,N’,N'-tetra acetic acid (EGTA) and Magnesium Chloride (MgCl) 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 centrifugation, 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.
EE WO 03/102208 } oo : .PCT/IN03/00199 LL
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 ST : methods and engineering principles previously explained in Horisberger, (1979) “Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning EK
Electron Microscopy”, Biol. Cellulaire, 36, 253-258; Leuvering et al., (1980) “Sol Particle So
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
Co 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 Cry1Ac/ Cry1Ab/ 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 (Cry1A 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
Cryt1Ac 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 centrifugation. Purification of the toxin is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis. 2. Antiserum is raised against Cry1Ac toxin in rabbits or goats by injecting them separately with purified toxin. 3. The method of raising antisera by injecting the antigen (Cry1Ac) 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 wiffl oo ammonium sulphate, solubilizing the precipitate in a buffer and passing it sequentially through DEAE cellulose, protein-A and antigen (Cry1Ac 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-IgG raised in rabbit /goat is striped as a 1mm 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 bufter, 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-IgG is diluted to 2mg/ml in a borate buffer Ph 8.5 to 9.0 and added to the colloidal goid (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 0.01M Tris, 5%
BSA, 2% sucrose, 0.87% NaCl 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 Cry1Ac. 22. Thus in Cry1Ac 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 Cry1Ac only one line appears indicating
Co that the strip is functional; and the sample is detected as negative for the toxin. oo
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 centrifugation. a
Purification of the toxins is done by ammonium sulphate precipitation at 25% saturation and by polyacrylamide electrophoresis. 2. Antiserum is raised against Cry1Ac /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. Brush border membrane vesicles (BBMVs) are prepared from guts of lepidopteran larvae using differential precipitation and centrifugation methods with mannitol, ha
Ethylene glycol-bis (B-aminoethyl ether)-N,N,N’',N’-tetra acetic acid (EGTA) and
Magnesium Chloride (MgCl;) 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 1mm 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-IgG 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 0.01M Tris, 5%
BSA, 2% sucrose, 0.87% NaCl 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 | oo 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 Cry1Ac or Cry2Aa/Cry2Ab depending on the conjugate antibody used. 23. Thus in Cry1Ac 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 Cry1Ac and Cry2Ab. 1. Cry1Ac 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 Cry1Ac /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 |gG obtained from antigen affinity purification is dialysed with 0.01 M, sodium phosphate buffer, Ph 7.2. 6. The anti-Cry1Ac-IgG raised in rabbit /goat is striped as a 1mm 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-IgG raised in rabbit /goat is striped as a 1mm 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-IgG 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-IgG 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 0.01M Tris, 5%
BSA, 2% sucrose, 0.87% NaCl and 0.1 M sodium azide. 16. The colloidal gold conjugated solutions of Cry1Ac 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 overiap 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 CrytAc/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 Cry1Ac/Cry1Ab or at 1.0 cm from the bottom of the membrane for Cry2Aa/Cry2Ab. nL . 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 Cry1Ac/Cry1Ab 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 oo as negative for the toxin. if three bands (includes the control band) appear it indicates that the sample is positive for both Cry1Ac/Cry1Ab and Cry2Aa/Cry2Ab.
Advantages 1. The methods described herein which employ polycional 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. Horisberger, (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 % nf. May, 20 5 Spb L ™ (Jai Prakash Mishra)
Assistant Director General (IPR)
Indian council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road
New Delhi 110001
Claims (6)
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: (1) 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, (ii) Purification of Cry1Ac and Cry2Aa to apparent homogeneity using a variety of: methods including chromatography and electrophoresis. (iii) Raising polyclonal antibodies to the toxins as claimed in step (ii) 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 (iii) using affinity chromatographic methods comprising of protein-A and Cry1Ac/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 (vii) 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 (viii) 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 (viii) on glass-fibre pads using a micropipette. (xi) immobilisation of the gold-antibody conjugate of step (ix) on glass-fibre pads using a micropipette. (xii) A goat anti-rabbit IgG or rabbit anti-goat IgG is striped manually, as 30 cm x
0.1 cm line at 0.5cm 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 (xii) 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 (xii) of claim 1, would serve as control line to indicate a proper functioning of the strip. ° AMENDED SHEET
(xv) The said line of step (xii) 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 minutes before assembly. (xvii) Assembling the gold-conjugate impregnated glass-fibre as claimed in step (x) of claim-1, on the membrane as claimed in step (vi) of claim-1 so as to overlap
2mm. 9a AMENDED SHEET
(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, so as to overlap 2mm. (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, so as to overlap 2mm. (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, so as to overlap 2mm. (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 Cry1Aa, Cry1Ab, Cry1Ac, 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: 2 AMENDED SHEET
BN ()) Isolation of brush border membrane vesicles from guts of lepidopteran larvae according to the standard methods. (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. 2a AMENDED SHEET
(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 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, so as to overlap 2mm.
(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 so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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 (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.
(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.
3 AMENDED SHEET
(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 Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab
3. 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: 3a AMENDED SHEET
© ‘WO 03/102208 PCT/IN03/00199 {)] Production of Cry1Ac and Cry2Aa toxins from the clones as claimed in step (i) of claim-1. (i) Purification of Cry1Ac 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.
(Vv) 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 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-Cry1Ac 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.
4 AMENDED SHEET
So (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 minutes before assembly. 4a AMENDED SHEET
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(xxv) 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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm .
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
(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, so as to overlap 2mm.
AMENDED SHEET
(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, so as to overlap 2mm. (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, so as to overlap 2mm. (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 xxxviiii) of claim-3, so as to overlap 2 mm, (xxxix) Cold lamination of the final assembly. : (x1) 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 lithe test sample is negative. 5a AMENDED SHEET
(xli) 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. (xlii) The said sample lines appear due to the accumulation of the gold-labeled anti- Cry-toxin antibody, which binds the toxin (Cry1Ac/Cry2Ab 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.
4. 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. 0) 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.
5. An immunochromatographic assay/strip substantially as described herein and with reference to any one or more of examples 1 to 3.
6. An immunochromatographic assay/strip substantially as described herein and as illustrated in Figures 1 and 2. 6 AMENDED SHEET
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN600DE2002 | 2002-05-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200410268B true ZA200410268B (en) | 2006-07-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200410268A ZA200410268B (en) | 2002-05-31 | 2004-12-21 | Rapid detection of BT-cry Toxins |
Country Status (6)
| Country | Link |
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| KR (1) | KR20050026396A (en) |
| CN (1) | CN100403028C (en) |
| AU (1) | AU2003249576A1 (en) |
| MX (1) | MXPA04011769A (en) |
| WO (1) | WO2003102208A2 (en) |
| ZA (1) | ZA200410268B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2612450C (en) * | 2005-06-28 | 2014-04-29 | Zbx Corporation | Membrane array and analytical device |
| 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 |
| AU2009343906B2 (en) * | 2009-04-09 | 2013-07-04 | Hitachi Chemical Diagnostics Systems Co., Ltd. | Detector 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 |
| CN105188872B (en) | 2013-03-15 | 2019-01-15 | 葛兰素史克知识产权第二有限公司 | Method for antibody purification |
| 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 |
| WO2018047080A1 (en) | 2016-09-07 | 2018-03-15 | Glaxosmithkline Intellectual Property Development Limited | Methods for purifying antibodies |
| CN106674334B (en) * | 2017-02-08 | 2020-09-01 | 金陵科技学院 | Cry2 Ad-combined cyclic heptapeptide and encoding gene and application thereof |
| 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 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5141850A (en) * | 1990-02-07 | 1992-08-25 | Hygeia Sciences, Inc. | Porous strip form assay device method |
| US5712171A (en) * | 1995-01-20 | 1998-01-27 | Arqule, Inc. | Method of generating a plurality of chemical compounds in a spatially arranged array |
| US5712172A (en) * | 1995-05-18 | 1998-01-27 | Wyntek Diagnostics, Inc. | One step immunochromatographic device and method of use |
| AP9901541A0 (en) * | 1996-11-20 | 1999-06-30 | Ecogen Inc | Broad-spectrum delta-endotoxins. |
| US6017534A (en) * | 1996-11-20 | 2000-01-25 | Ecogen, Inc. | Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity |
| EP1240515A4 (en) * | 1999-12-14 | 2004-07-14 | Strategic Diagnostics Inc | Method of processing and testing powdered samples using immunochromatographic strip tests |
-
2003
- 2003-05-29 AU AU2003249576A patent/AU2003249576A1/en not_active Abandoned
- 2003-05-29 CN CNB038176416A patent/CN100403028C/en not_active Expired - Fee Related
- 2003-05-29 KR KR1020047019456A patent/KR20050026396A/en active Search and Examination
- 2003-05-29 MX MXPA04011769A patent/MXPA04011769A/en active IP Right Grant
- 2003-05-29 WO PCT/IN2003/000199 patent/WO2003102208A2/en active Application Filing
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2004
- 2004-12-21 ZA ZA200410268A patent/ZA200410268B/en unknown
Also Published As
| Publication number | Publication date |
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| WO2003102208A3 (en) | 2004-03-25 |
| AU2003249576A1 (en) | 2003-12-19 |
| MXPA04011769A (en) | 2005-07-26 |
| WO2003102208A2 (en) | 2003-12-11 |
| AU2003249576A8 (en) | 2003-12-19 |
| CN100403028C (en) | 2008-07-16 |
| KR20050026396A (en) | 2005-03-15 |
| CN1672049A (en) | 2005-09-21 |
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