KR20050026396A - Rapid detection of bt-cry toxins - Google Patents

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

RAPID DETECTION OF BT-CRY TOXINS

The present invention relates to high speed immunochromatographic assays using polyclonal antibodies to detect crystalline toxins Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab in seeds or plant tissues on lateral flow strips. In particular, the present invention relates to the use of crystalline toxins isolated from native Bacillus thuringiensis strains and polyclonal antibodies raised against crystalline toxins. More specifically, the present invention provides a broad range of simplified development using a manual striping method and analysis by the use of antigen-affinity and protein-A affinity purified polyclonal IgG derived from two different animals-goat and rabbit. To enhanced specificity and accuracy. The present invention includes the use of crystalline toxin receptors from insects of the forest as capture ligands for detecting cry toxins. The present invention also facilitates the simultaneous detection of two crystalline toxins on a single strip.

Soil bacterium Bacillus thuringiensis produces some protein crystals that are toxic to insect pests but harmless to host plants and the environment. Genetic transformation of host plants with genes isolated from bacteria allows them to synthesize the toxin and fight insect pests. Genes isolated from Bacillus thuringiensis (commonly also known as Bt-genes) have been used to transform cotton ( Gossypium hirsutum ) and several other crops in breed improvement for pest resistance programs. The most commonly used for conversion are genes such as cry1Ac, cry1Ab, cry1Aa, cry2Aa and Cry2Ab.

Identification of the plant's genetic transformation is essential for the success of the breed improvement program and there are several molecular ways to accomplish this. In addition, the expression of toxins in transgenic plants is extremely important as the toxins have a toxic effect on the target species of insect pests. Cry1Ac expression is the most important attribute in transgenic plants where pest control is effective and thus detection of toxin expression is equally important. Toxin expression is generally quantified using standard ELISA methods, and furthermore, high-speed testing has been made possible by the emergence of immunochromatographic methods for detecting expression of transgenes. However, immunochromatographic strips are expensive if they are produced by a process involving the use of monoclonal antibodies and several types of equipment.

1.Schematic diagram of the lateral flow assembly for single Cry toxin detection

1. Sample absorbent pad

2. Control line: Control line: Control line is goat anti-rabbit IgG or rabbit anti-goat IgG by the assay format.

3. Sample line for Cry1Ab / Cry1Ac or Cry2Aa / Cry2Ab: Sample line: The sample capture line is a rabbit IgG or directed against a crystalline toxin (Cry1Ac / Ab / Cry2Aa / Cry2Ab) or Cry toxin receptor protein isolated from larvae Striped with goat IgG.

4. cellulose nitrate / nitrocellulose or nylon membrane

5. Conjugate Release Glass-Fiber Pads: Conjugate release pads contain colloidal gold coated with antibodies from a goat or rabbit (affinity purified anti-Bt IgG).

6. Plastic backing.

7. Sample release pad

2. Schematic diagram of the lateral flow assembly for detecting two or more Cry toxins.

1. Sample absorbent pad

2. Control Line: The control line is goat anti-rabbit IgG or rabbit anti-goat IgG by the assay format.

3. Sample Line for Cry1Ab / Cry1Ac: The sample capture line is striped with rabbit IgG or goat IgG derived against crystalline toxin Cry1Ac.

4. Sample Line for Cry2Aa / Cry2Ab: Sample capture lines are striped with rabbit IgG or goat IgG derived against crystalline toxin Cry2Aa / Cry2Ab.

5. Cellulose nitrate / nitrocellulose or nylon membrane.

6. Conjugate Release Glass-Fiber Pads: The conjugate release pad contains colloidal gold coated with antibodies from goats or rabbits (affinity purified anti-Bt IgGs of Cry1Ac and Cry2Aa / Cry2Ab).

7. Plastic backing

8. Sample release pad

Commercially available Bt-transgenic crops should be evaluated for purity and potency (good toxin expression) at various stages and levels-research, technology development and certification, environmental impacts, seed quality control, plantation, commercial sites of agricultural products. Any method that leads to high-speed detection of Bt-transgenic technology can be helpful and expensive can also be an advantage. Another alternative could be to use polyclonal antibodies instead of monoclonal antibodies against immunochromatographic strips without compromising the detection efficacy of cry expression in seed or plant tissues, and therefore for the purposes of this study. Another object is to develop the strip with minimal instrumentation, which involves low cost and price. Yet another object is to develop a method for detecting such expression using polyclonal antibodies directed against crystalline toxins isolated from native Bacillus thuringiensis strains. The purpose of the method is to provide a robust and easy method suitable for the development of immunochromatographic assays for detecting Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab.

Qualitative high-speed immunodiagnostic testing (Huang et al., (1998) One step immunochromatographic device US Pat. No. 5,712,172) has been commonly used for antigen detection in lateral flow analysis as a positive or negative signal. Examples of such assays include pregnancy test kits, AIDS tests, and various types of urine analytes. Cry1Ac detection kits based on immunochromatographic detection methods were developed and commercialized in July 2001 and October 2001 by two US companies, "Agdia, USA" and "Strategic Diagnostic Inc., USA," respectively. Both companies claim in their promotional literature that kits are based on monoclonal antibody combinations. Therefore, another object of the present method development is to simplify the immunochromatographic detection method for Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab detection using affinity purified polyclonal antibodies specific for analytes and fully crystalline toxins. .

Principle : Polyclonal immunoglobin (IgG) from animals such as rabbits or goats coated with gold captures Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab, and the free end of Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab captures the antibody line ( Membranes by capillary action until they bind with polyclonal immunoglobin (IgG) from animals such as rabbits or goats or with N-aminopeptidase or Cadherin lines that are interspersed across the membrane Carry along. Gold coated IgG accumulates in the capture IgG line and generates a visible signal indicating the presence of Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab toxin. In the absence of Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab in the sample, the gold coated IgG migrates along the membrane and binds to goat / rabbit anti-rabbit / goat antibodies to accumulate and produce a visible signal.

Preparation of N-aminopeptidase / cadherin : According to one aspect of the invention, 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, differential precipitation and centrifugation with mannitol, ethylene glycol-bis (β-aminoethylether) -N, N, N ', N'-tetraacetic acid (EGTA) and magnesium chloride (MgCl ₂ ) Brush border membrane vesicles were prepared from the viscera of the larvae using the separation method. Pellets containing the Cadherin receptor of N-aminopeptidase / Cry protein are used to stripe as antigen capture protein.

Antigen : According to the second aspect of the invention, the native Bacillus thuringiensis strain "x" (identified by Institute of Microbial Technology (IMTECH), Chandigarh) is isolated and through antibiotic resistance profile, colony morphology and growth characteristics Appearance is identified for uniqueness. The crystalline protein producing gene cry1Ac / cry2A from the plasmid of the strain was amplified on PCR using specific primers and cloned into pUC 18 and pRK 223-3 expression vectors. Crystalline toxins were purified from clones by fractional centrifugation, chromatography and SDS polyacrylamide gel electrophoresis. Toxins were purified and antiserum induced against specific toxins.

Induction of Antibodies : Purified crystalline toxin was injected into rabbits / goats mixed with Freund's complete adjuvant. Booster doses and serum were collected using lysed toxins in incomplete Freund's adjuvant at 1 month intervals. Serum was precipitated with ammonium sulfate and IgG purified using DEAE cellulose, Protein A / Protein G column and / or, if necessary, finally Cry1Ac / Cry2A antigen-affinity column chromatography.

Preparation of Colloidal Gold and IgG Conjugates. 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, colloidal gold is prepared by citrate reduction of gold chloride in the synthesis of colored particle conjugates, according to the standard methods and engineering principles previously described. Affinity purified antibody IgG from one animal was conjugated with gold and applied on a 30 cm × 1 cm conjugate release glass-fiber pad according to the method disclosed in the protocol described in the above publication.

Preparation of Lateral Flow Assemblies. Coated polyester plastic sheet (30 x 6 cm) with acrylic adhesive and adhering 2.5 x 30 cm nylon / nitrocellulose / cellulose nitrate membrane strips (S & S / Whatman / millipore / Pall-Gelman) onto plastic backing Let's do it. Crystalline toxin specific IgG was hand-striped for two toxin detection strips as a 30 cm x 0.1 cm line in the middle of the membrane at 1.25 cm and 1 cm from the bottom of the membrane and crystallographic toxin specific IgG or crystalline toxin receptor was a single toxin detection strip. Striping only 1.25 cm from both ends of the membrane for Goat anti-rabbit IgG or rabbit anti-goat IgG is striped by hand as a 30 cm x 0.1 cm line 0.5 cm from the top of the membrane. The membrane is blocked with 2% casein / BSA and a buffer containing 3% nonfat milk powder containing sugar and preservatives and washed twice with phosphate buffered saline. After the membrane is completely dried, a dry conjugate coated glass fiber pad is placed at the bottom of the membrane to allow 2 mm overlaid thereon. Thick filter pads 30 × 1 × 0.1 cm are placed at the bottom of the conjugate release pads in a 2 mm stack and secured with acrylic adhesive in the empty place of the plastic backing. Another filter pad 30 x 1 x 0.1 cm is placed on top of the membrane to allow 2 mm overlap and secured with acrylic adhesive to the membrane free area of the plastic backing. The assemblies are laminated using cold-lamination sheets and cut into 6 cm x 0.5 cm strips.

Symptoms : Two lines appear in a positive sample (for a single toxin detection strip)-a control line indicating that the strip is functioning and a sample line indicating that the sample is positive. On the other hand, in the sample without Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab, only one line appears indicating that the strip is functioning; Samples are detected as negative for toxins. Strips designed to detect both toxins (Cry1A and Cry2A) simultaneously on the same strip have three lines in the sample that are positive for both toxins, or only two lines or a sample that is positive for any one toxin Only one line (control line) appears when negative for both toxins.

Example 1 Preparation of a Fast Immunochromatography Assay / Strip for Detecting Cry1Ac

1. Isolate Cry1Ac from clones by sonicating bacterial clone cultures and pelleting cell debris and insoluble toxins. Toxins are solubilized in alkaline buffer and extracted by centrifugation. Purification of the toxin is carried out by precipitation of ammonium sulfate and polyacrylamide electrophoresis at 25% saturation.

2. Antiserum is induced against Cry1Ac toxin by injecting purified toxin separately into rabbit or goat.

3. Antiserum induction methods by initially injecting an antigen mixed with Freund's complete adjuvant (Cry1Ac) and injecting repeated booster doses mixed with incomplete Freund's adjuvant prior to serum collection are described in the Immunology textbook (Antibodies-A laboratory Manual-Harlow). Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988) and are therefore conventional techniques and are not described in detail herein.

4. Immunoglobin IgG is purified from antiserum by precipitation with ammonium sulfate, solubilizing the precipitate in buffer and then sequentially passing through DEAE cellulose, protein-A and antigen (Cry1Ac toxin) affinity columns. Methods used herein are described in Antibodies-A laboratory Manual, Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).

5. Purified IgG obtained from antigen affinity purification is dialyzed with 0.01 M, sodium phosphate buffer, pH 7.2.

6. 2.5 x 30 cm nylon / nitrocellulose / cellulose nitrate membrane along a 30 cm ruler using a portable sable hair brush (0, 00, 000, 1, 2, 3, 4, or 5) On one side of the strip (S & G / Whatman / millipore / Pall-Gelman) stripe the anti-Cry1Ac-IgG produced in rabbits / goats as a 1 mm thick, 30 cm long line at the center 1.25 cm from the top and bottom.

7. Solubilize anti-rabbit IgG / anti-goat IgG (available from many companies including Sigma Chemical Company, USA) in 0.01 M, sodium phosphate buffer (pH 7.2), 30 at 0.5 cm from the top of the membrane. Strip by hand as a cm x 0.1 cm line.

8. The membrane is dried at 50 ° C. under a dry blower for 10 to 15 minutes.

9. The membrane was then blocked with a buffer containing 2% casein / BSA and 3% nonfat milk powder containing 1-5% sucrose and sodium azide preservative, 0.01M, sodium phosphate buffer, pH 7.2 Wash twice with).

10. The membrane is dried at 50 ° C. under a dry blower for 10 to 15 minutes.

11. Coat the polyester plastic sheet (30 x 6 cm) with acrylic adhesive and fix the membrane in the center equidistantly from the top and bottom of the sheet.

12. Colloidal gold is commercially available from Sigma Chemicals, USA. Affinity purified anti-Cry1Ac-IgG was diluted to 2 mg / ml in borate buffer (pH 8.5-9.0) and added to colloidal gold (adjusted to pH 9.0) with stirring. The conjugate is stabilized by adding 10% BSA (bovine serum albumin) to the mixture.

13. Centrifuge the conjugate at 12,000 g for 30 minutes at 4 ° C. to obtain loose pellets. The pellet is dissolved in 100 ml of a solution containing 0.01M Tris, 5% BSA, 2% sucrose, 0.87% NaCl and 0.1M sodium azide.

14. The colloidal gold bonded solution is applied on a 30 cm × 1 cm conjugate release glass-fiber pad and dried under dry blast for 10-15 minutes.

15. Place the dry conjugate coated glass fiber pads on the bottom of the membrane mentioned in step 10 and overlaid thereon 2 mm.

16. Place a thick filter pad 30 x 1 x 0.1 cm at the bottom of the conjugate release pad to allow 2 mm nesting and secure with an acrylic adhesive in the blank of the plastic backing. This end is referred to as the lower end.

17. Place another filter pad 30 x 1 x 0.1 cm on top of the membrane to allow 2 mm overlap and secure with acrylic adhesive in the absence of the membrane in the plastic backing. This end is referred to as the top end.

18. Laminate the assembly using the cold-laminate sheet and cut it into 6 cm × 0.5 cm strips.

19. Plant tissues (about 50 mg) such as leaves, stems, roots, flowers, seeds, etc. are ground in a 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. Dip the lower end of the strip into a vial containing crushed leaf / seed tissue. The solution flows into the glass fiber pads and moves upward in the membrane by capillary forces.

21. After about 10-15 minutes, the solution reaches the top of the strip. One clear purple line appears at the top 1 cm below the top of the membrane area, indicating that the membrane is functioning. This is called the control line. If the test sample is positive for Cry1Ac, another purple line appears at the center of the membrane.

22. Thus, two lines appear in the Cry1Ac positive sample-a control line indicating that the strip is functioning and a sample line indicating that the sample is positive.

On the other hand, in the sample without Cry1Ac, only one line appears indicating that the strip is functioning; Samples are detected as negative for toxins.

Example 2: Preparation of a Fast Immunochromatography Assay / Strip for Detecting Cry1Ac / Cry2Ab Using Cry-Toxin Receptor Protein as Capture Ligand

1. Isolate Cry1Ac and Cry2Aa / Cry2Ab from clones by sonicating bacterial clone cultures and pelleting cell debris and insoluble toxins. Toxins are solubilized in alkaline buffer and extracted by centrifugation. Purification of the toxin is carried out by precipitation of ammonium sulfate and polyacrylamide electrophoresis at 25% saturation.

2. Antiserum is induced against Cry1Ac / Cry2Aa / Cry2Ab toxin by injection of purified toxin separately into rabbit or goat.

3. Antiserum induction methods by initially injecting an antigen (Cry1Ac / Cry2Aa / Cry2Ab) mixed with Freund's complete adjuvant and repeated doses of booster mixed with incomplete Freund's adjuvant prior to serum collection are described in the Immunology textbook (Antibodies-A). laboratory Manual-Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988), which is a common technique and therefore is not described in detail here.

4. Immunoglobin IgG is purified from antiserum by precipitation with ammonium sulfate, solubilizing the precipitate in buffer and then sequentially passing DEAE cellulose, protein-A and antigen (Cry1Ac / Cry2Aa / Cry2Ab toxin) affinity columns. Methods used herein are described in Antibodies-A laboratory Manual, Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).

5. The purified IgG obtained from the antigen affinity purification is dialyzed with 0.01 M, sodium phosphate buffer, pH 7.2.

6. Mannitol, ethylene glycol-bis (β-aminoethylether) -N, N, N ', N'-tetraacetic acid (EGTA) and magnesium chloride (MgCl ₂ ) according to the protocol described by Wolfersberger et al. (1987). Brush border membrane vesicles (BBMV) are prepared from the viscera of the larvae using differential precipitation and centrifugation. Cry-receptor protein is purified from BBMV using ammonium sulfate and standard spin column chromatography.

7. Using a portable sable hair brush (0, 00, 000, 1, 2, 3, 4, or 5), 2.5 x 30 cm nylon / nitrocellulose / cellulose nitrate membrane along a 30 cm ruler On one side of the strip (S & G / Whatman / millipore / Pall-Gelman), the cry-toxin-receptor protein is striped as a 1 mm thick, 30 cm long line in the middle at a distance of 1.25 cm from the top and bottom.

8. Soluble anti-rabbit IgG / anti-goat IgG (available from many companies including Sigma Chemical Company, USA) in 0.01 M, sodium phosphate buffer (pH 7.2), 30 at 0.5 cm from the top of the membrane Strip by hand as a cm x 0.1 cm line.

9. The membrane is dried at 50 ° C. under a dry blower for 10 to 15 minutes.

10. The membrane was then blocked with a buffer containing 2% casein / BSA and 3% nonfat milk powder containing 1-5% sucrose and sodium azide preservative, 0.01 M, sodium phosphate buffer (pH 7.2). Wash twice with).

11. Dry the membrane at 50 ° C. under a dry blower for 10 to 15 minutes.

12. The polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is centered equidistantly from the top and bottom of the sheet.

13. Colloidal gold is commercially available from Sigma Chemicals, USA. Affinity purified anti-Cry1Ac-IgG or anti-Cry2Ab-IgG is diluted to 2 mg / ml in borate buffer (pH 8.5-9.0) and added to colloidal gold (adjusted to pH 9.0) with stirring. The conjugate is stabilized by adding 10% BSA (bovine serum albumin) to the mixture.

14. Centrifuge the conjugate at 12,000 g for 30 minutes at 4 ° C. to obtain loose pellets. The pellet is dissolved in 100 ml of a solution containing 0.01M Tris, 5% BSA, 2% sucrose, 0.87% NaCl and 0.1M sodium azide.

15. The colloidal gold-bonded solution is applied onto a 30 cm x 1 cm conjugate release glass-fiber pad and dried under dry blast for 10-15 minutes.

16. Place the dry conjugate coated glass fiber pads on the bottom of the membrane mentioned in step 10 to allow 2 mm overlap.

17. Place a thick filter pad 30 x 1 x 0.1 cm at the bottom of the conjugate release pad to allow 2 mm nesting and secure with an acrylic adhesive in the blank of the plastic backing. This end is referred to as the lower end.

18. Place another filter pad 30 x 1 x 0.1 cm on top of the membrane to allow 2 mm overlap and secure with acrylic adhesive in the absence of the membrane in the plastic backing. This end is referred to as the top end.

19. Laminate the assemblies using a cold-laminate sheet and cut into 6 cm × 0.5 cm strips.

20. Plant tissues (about 50 mg) such as leaves, stems, roots, flowers, seeds, etc. are ground in a 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. Dip the lower end of the strip into a vial containing crushed leaf / seed tissue. The solution flows into the glass fiber pads and moves upward in the membrane by capillary forces.

22. After about 10-15 minutes, the solution reaches the top of the strip. One clear purple line appears at the top 1 cm below the top of the membrane area, indicating that the membrane is functioning. This is called the control line. Depending on the conjugate antibody used, another purple line appears in the central portion of the membrane if the test sample is positive for Cry1Ac or Cry2Aa / Cry2Ab.

23. Thus, two lines appear in a Cry1Ac or Cry2Aa / Cry2Ab positive sample-a control line indicating that the strip is functioning and a sample line indicating that the sample is positive. On the other hand, in the sample without two toxin groups, one line appears indicating that the strip is functioning; Samples are detected as negative for toxins.

Example 3: Preparation of Fast Immunochromatography Strips for Simultaneous Detection of Cry1Ac / Cry2Ab

1. Isolate Cry1Ac and Cry2Aa / Cry2Ab from clones by sonicating bacterial clone cultures and pelleting cell debris and insoluble toxins. Toxins are solubilized in alkaline buffer and extracted by centrifugation. Purification of the toxin is carried out by precipitation of ammonium sulfate and polyacrylamide electrophoresis at 25% saturation.

2. Antiserum is induced against Cry1Ac / Cry2Aa / Cry2Ab toxin by injection of purified toxin separately into rabbit or goat.

3. Antiserum induction methods by initially injecting an antigen (Cry1Ac / Cry2Aa / Cry2Ab) mixed with Freund's complete adjuvant and repeated doses of booster mixed with incomplete Freund's adjuvant prior to serum collection are described in the Immunology textbook (Antibodies-A). laboratory Manual-Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988), which is a common technique and therefore is not described in detail here.

4. Immunoglobin IgG is purified from antiserum by precipitation with ammonium sulfate, solubilizing the precipitate in buffer and then sequentially passing DEAE cellulose, protein-A and antigen (Cry1Ac / Cry2Aa / Cry2Ab toxin) affinity columns. Methods used herein are described in Antibodies-A laboratory Manual, Harlow Ed and David Lane; Cold Spring Harbor laboratory, USA, 1988).

5. The purified IgG obtained from the antigen affinity purification is dialyzed with 0.01 M, sodium phosphate buffer, pH 7.2.

6. Rabbit / Goat-Generated Anti-Cry1Ac-IgG Using a Portable Sable Hair Brush (0, 00, 000, 1, 2, 3, 4, or 5), 2.5 Along a 30 cm Chair Support, On one side of the x 30 cm nylon / nitrocellulose / cellulose nitrate membrane strip (S & G / Whatman / millipore / Pall-Gelman), stripe as a 1 mm thick, 30 cm long line at the center at 1.25 cm from the top and bottom.

7. Rabbit / goat-induced anti-Cry2Aa / Cry2Ab-IgG with a portable sable hair brush (0, 00, 000, 1, 2, 3, 4, or 5), along the 30 cm ruler Stripe as a 1 cm thick, 30 cm long line at the center at 1.0 cm distance from the bottom on one side of a 2.5 x 30 cm nylon / nitrocellulose / cellulose nitrate membrane strip (S & G / Whatman / millipore / Pall-Gelman).

8. Soluble anti-rabbit IgG / anti-goat IgG (available from many companies including Sigma Chemical Company, USA) in 0.01 M, sodium phosphate buffer (pH 7.2), 30 at 0.5 cm from the top of the membrane Strip by hand as a cm x 0.1 cm line. If the conjugate pad IgG is from goat, anti-goat IgG is used as a control line. Likewise, if the conjugate pad IgG is from a rabbit, anti-rabbit IgG is used as the control line.

9. The membrane is dried at 50 ° C. under a dry blower for 10 to 15 minutes.

10. The membrane was then blocked with a buffer containing 2% casein / BSA and 3% nonfat milk powder containing 1-5% sucrose and sodium azide preservative, 0.01 M, sodium phosphate buffer (pH 7.2). Wash twice with).

11. Dry the membrane at 50 ° C. under a dry blower for 10 to 15 minutes.

12. The polyester plastic sheet (30 x 6 cm) is coated with acrylic adhesive and the membrane is centered equidistantly from the top and bottom of the sheet.

13. Colloidal gold is commercially available from Sigma Chemicals, USA. Affinity purified anti-Cry1Ac-IgG was diluted to 2 mg / ml in borate buffer (pH 8.5-9.0) and added to colloidal gold (adjusted to pH 9.0) with 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. Affinity purified anti-Cry2Aa / Cry2Ab-IgG was diluted to 2 mg / ml in borate buffer (pH 8.5 to 9.0) and added to colloidal gold (adjusted to pH 9.0) with stirring. The conjugate is stabilized by adding 10% BSA (bovine serum albumin) to the mixture.

15. Centrifuge the conjugate at 12,000 g for 30 minutes at 4 ° C. to obtain loose pellets. The pellet is dissolved in 100 ml of a solution containing 0.01M Tris, 5% BSA, 2% sucrose, 0.87% NaCl and 0.1M sodium azide.

16. Mix colloidal gold-bonded solutions of Cry1Ac and Cry2Ab / Cry2Aa in equal amounts and apply onto 30 cm x 1 cm conjugate release glass-fiber pads and dry under dry blast for 10-15 minutes.

17. Place the dry conjugate coated glass fiber pads on the bottom of the membrane mentioned in step 10 and overlaid thereon 2 mm.

18. Place a thick filter pad 30 x 1 x 0.1 cm at the bottom of the conjugate release pad to allow 2 mm nesting and secure with an acrylic adhesive in the blank of the plastic backing. This end is referred to as the lower end.

19. Place another filter pad 30 x 1 x 0.1 cm on top of the membrane to allow 2 mm overlap and secure with acrylic adhesive in the absence of the membrane in the plastic backing. This end is referred to as the top end.

20. Laminate the assemblies using a cold-laminate sheet and cut into 6 cm × 0.5 cm strips.

21. Plant tissue such as leaves, stems, roots, flowers, seeds, etc. (about 50 mg) are ground 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. Dip the lower end of the strip into a vial containing crushed leaf / seed tissue. The solution flows into the glass fiber pads and moves upward in the membrane by capillary forces.

23. After about 10-15 minutes, the solution reaches the top of the strip. One clear purple line appears at the top 1 cm below the top of the membrane area, indicating that the membrane is functioning. This is called the control line. If the test sample is positive for both Cry1Ac / Cry1Ab or Cry2Aa / Cry2Ab, two purple lines appear in the central part of the membrane. Only one line appears in the central portion of the membrane corresponding to 1.25 cm from the bottom of the membrane for Cry1Ac / Cry1Ab or 1.0 cm from the bottom of the membrane for Cry2Aa / Cry2Ab.

24. Therefore, only two lines appear in the sample that is positive for either Cry1Ac / Cry1Ab or Cry2Aa / Cry2Ab-control line indicating that the strip is functioning and the sample is either Cry1Ac / Cry1Ab or Cry2Aa / Cry2Ab. Sample line indicating positive for. On the other hand, in the sample without Cry1Ac / Cry1Ab or Cry2Aa / Cry2Ab, only one line appears indicating that the strip is functioning; Samples are detected as negative for toxins. Three bands (including control bands) appear if the sample is positive for both Cry1Ac / Cry1Ab and Cry2Aa / Cry2Ab.

Favorable effect

1. The methods described herein using polyclonal antibodies and passive striping methods are simple to implement and cost more than $ 20,000 for use in the commercial production of expensive monoclonal antibodies or monoclonal techniques and strip like species. No equipment required

2. The method claimed herein uses affinity-purified immunoglobin (IgG) derived from two different animals, whereby detection sensitivity is enhanced.

3. The method allows simultaneous detection of two or more toxins on a single strip, thus reducing the cost of two or more strips for the same purpose.

4. The use of the "Cry toxin receptor" in the strip allows for the high sensitivity detection of Cry toxin toxic to the relevant human insects from which the receptor is isolated.

Reference

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.

Wolfersberger, MG, Luthy, P., Maurer, A., Parenti, P., Sacchi, VF, Giordana, B and Hanozet, GM (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physio. 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. Immunassay, 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.

Claims (4)

A method for preparing a high speed immunochromatographic assay / strip using polyclonal antibodies that identify / detect Cry toxin from Bacillus thuringiensis as a single toxin from seed or plant tissue, comprising the following steps: (i) Unique native Bacillus thuringin, characterized by antibiotic resistance profile, colony morphology and growth characteristics, amplification of crystalline protein generating genes from the plasmid of the strain and cloning into specific expression vectors using specific primers Identification of Ensis strains; (ii) purifying Cry1Ac and Cry2Aa with apparent homogeneity using a variety of methods including chromatography and electrophoresis; (iii) repeatedly injecting a booster dose of crystalline toxin mixed with adjuvant and collection serum to induce polyclonal antibodies against the toxin claimed in step (ii) in rabbits and goats, precipitate the serum and then Purifying; (iv) purification of IgG from the antiserum claimed in step (iii) using affinity chromatography comprising protein-A and Cry1Ac / Cry2Aa antigens; (v) immobilization on the membrane using the anti-Cry polyclonal IgG claimed in step (iv); (vi) a polyclonal IgG derived from chlorine as claimed in step (iv) on a cellulose nitrate, nitrocellulose or nylon membrane (5-12 microns) using a manual striping method using a portable sable hair brush Passivating the capture line containing; (vii) Rabbit-derived polyclonal IgG derived in step (iv) on cellulose nitrate, nitrocellulose or nylon membranes (5-12 microns) using a manual striping method using a portable sable hair brush Passivating the capture line containing; (viii) labeling the polyclonal IgG antibodies derived from goats claimed in step (iv) with colloidal gold 20-40 nm; (ix) labeling the polyclonal IgG antibody derived from the rabbit claimed in step (iv) with colloidal gold 20-40 nm; (x) passivating the gold-antibody / conjugate of step (viii) on a glass-fiber pad using a micropipette; (xi) passivation of the gold-antibody / conjugate of step (ix) on a glass-fiber pad using a micropipette; (xii) manually stripe goat anti-rabbit IgG or rabbit anti-goat IgG as a 30 cm x 0.1 cm line 0.5 cm from the top of the membrane in each strip; (xiii) capture gold-labeled anti-rabbit antibody or anti-goat antibody, respectively, as in the strip assembled with goat anti-rabbit IgG or rabbit anti-goat IgG line of step (xii); (xiv) the line of step xii of claim 1 serves as a control line to indicate that the strip is functioning properly; (xv) the line of step (xii) of claim 1 will turn pink / purple regardless of the positive or negative of the test sample in use; (xvi) the membrane was blocked with blocking buffer containing protein, sugar and preservatives, washed twice with phosphate buffered saline and dried for 30 minutes prior to assembly; (xvii) assembling the gold-conjugate impregnated glass-fibers claimed in step (x) of claim 1 onto the membrane claimed in step (vi) of claim 1, as shown in FIG. (xviii) assembling the gold-conjugate impregnated glass-fibers claimed in step (x) of claim 1 onto the membrane claimed in step (vii) of claim 1, as shown in FIG. (xix) assembling the gold-conjugate impregnated glass-fibers claimed in step (xi) of claim 2 onto the membrane claimed in step (vi) of claim 1, as shown in FIG. (xx) assembling the gold-conjugate impregnated glass-fibers claimed in step (x) of claim 2 onto the membrane claimed in step (vii) of claim 1, as shown in FIG. (xxi) completing the assembly by placing a sample-release filter paper pad (1-2 mm thick) 2 mm superimposed on the bottom of the assembly claimed in steps (xvii, xviii, xix and xx). Placing a second sample-absorption pad 2 mm superimposed on top of the membrane claimed in steps (vi) and (vii) of claim 1; (xxii) cold lamination of the final assembly; (xxiii) combining a capture line of one anti-Cry antibody derived from one animal (goat) with a sandwich of antigen between gold labeled anti-Cry antibodies derived from another animal (rabbit) claimed in step (xix) Detecting toxins as visible lines; (xxiv) combining a capture line of one anti-Cry antibody derived from one animal (rabbit) with a sandwich of antigen between gold labeled anti-Cry antibodies derived from another animal (goat) claimed in step (xviii) Detecting toxins as visible lines; (xxv) The capture line of one anti-Cry antibody derived from one animal (rabbit) is combined with a sandwich of antigens between gold-labeled anti-Cry antibodies of the same animal (rabbit) claimed in step (xx). Detecting toxins as lines; (xxvi) The capture line of one anti-Cry antibody derived from one animal (goat) is combined with a sandwich of antigens between gold-labeled anti-Cry antibodies of the same animal (goat) claimed in step (xx). Detecting toxins as lines; And (xxvii) detecting the cry toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab using steps (xxiii) to (xxvi). The Cry-toxin receptor and anti-Cry poly isolated from an insect insect viscera of claim 1, which identify / detect Cry toxin from Bacillus thuringiensis as a single toxin from seed or plant tissue, comprising the following steps: Methods of making high speed immunochromatographic assays / strips in which a combination of clonal antibodies are used (i) separating the brush border membrane vesicles from the viscera of the larvae according to a standard method, ie the standard method published by Wolfersberger et al. (1987); (ii) purification of N-aminopeptidase and Cadherin protein using protein precipitation with 30-80% ammonium sulfate; (iii) a capture line containing the protein from step (ii) of claim 2 on a cellulose nitrate, nitrocellulose or nylon membrane (5-12 microns) using a manual striping method using a portable sable hair brush Passivation step; (iv) labeling the polyclonal IgG antibody claimed in step (iv) with colloidal gold 20-40 nm; (v) labeling the N-aminopeptidase and the Cadherin protein claimed in step (ii) with colloidal gold 20-40 nm; (vi) passivating the gold-antibody conjugate claimed in step (iv) of claim 2 on a glass-fiber pad; (vii) passivating the gold-Cry-receptor protein conjugate as claimed in step (v) of claim 2 on a glass-fiber pad; (viii) manually stripe goat anti-rabbit IgG or rabbit anti-goat IgG as a 30 cm × 0.1 cm line 0.5 cm from the top of the membrane in each strip; (ix) the goat anti-rabbit IgG or rabbit anti-goat IgG lines of step (viii) will capture gold labeled anti-rabbit antibodies or anti-goat antibodies, respectively, as in the assembled strip; (x) the line of step (ix) of paragraph 2 serves as a control line to indicate that the strip is functioning properly; (xi) the line of step (ix) of paragraph 2 will turn pink / purple regardless of the positive or negative of the test sample when in use; (xii) the membranes are blocked with blocking buffer containing protein, sugar and preservatives prior to assembly, washed twice with phosphate buffered saline and dried for 30 minutes; (xiii) assembling the gold-conjugate impregnated glass-fibers claimed in step (v) of claim 2 onto the membrane claimed in step (vi) of claim 1, as shown in FIG. (xiv) assembling the gold-conjugate impregnated glass-fibers claimed in step (v) of claim 2 onto the membrane claimed in step (vii) of claim 1, as shown in FIG. (xv) assembling the gold-conjugate impregnated glass-fibers claimed in step (v) of claim 2 onto the membrane claimed in step (iii) of claim 2, as shown in FIG. (xvi) assembling the gold-conjugate impregnated glass-fibers claimed in step (x) of claim 1 onto the membrane claimed in step (iii) of claim 2, as shown in FIG. (xvii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xi) of claim 1 onto the membrane claimed in step (iii) of claim 2, as shown in FIG. (xviii) placing the sample-release filter paper pad (1 to 2 mm thick) 2 mm overlaid on the bottom of the glass-fiber as claimed in the steps (xiii, xiv, xv, xvi and xvii) of claim 2 to complete the assembly. step. Placing the second sample-absorption pad 2 mm overlaid on top of the membrane claimed in the steps (xiii, xiv, xv, xvi and xvii) of claim 2; (xix) cold lamination of the final assembly; (xx) capture lines of one anti-Cry antibody derived from one animal (goat) claimed in step (vi) of claim 1 between the gold-Cry-receptor protein conjugates claimed in step (v) of claim 2 Detecting the toxin as a visible line in combination with a sandwich of antigen; (xxi) capture lines of one anti-Cry antibody derived from one animal (rabbit) claimed in step (vii) of claim 1 between the gold-Cry-receptor protein conjugates claimed in step (v) of claim 2 Detecting the toxin as a visible line in combination with a sandwich of antigen; (xxii) combining the capture line of the Cry-receptor of step (iii) of claim 2 with a sandwich of antigen between the gold-Cry-receptor protein conjugates claimed in step (v) of claim 2 to detect toxins as a visible line. step; (xxiii) the toxin as a visible line in combination with the capture line of the Cry-receptor of step (iii) of claim 2 in combination with the sandwich of antigen between the gold-labeled anti-Cry-antibody derived from the chlorine of step (x) of claim 1 Detecting; (xxiv) Toxin as a visible line, combining the capture line of the Cry-receptor of step (iii) of claim 2 with a sandwich of antigens between gold labeled anti-Cry-antibodies derived from the rabbit of step (xi) of claim 1 Detecting; And (xxv) detecting cry toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab using steps (xx) to (xxiv). The preparation of a high speed immunochromatographic assay / strip using a polyclonal antibody according to claim 1, which simultaneously identifies / detects two or more Cry toxins from Bacillus thuringiensis from seed or plant tissue, comprising the following steps: Way: (I) producing Cry1Ac and Cry2Aa toxins from the clones claimed in step (i) of claim 1; (i) purifying Cry1Ac and Cry2Aa with apparent homogeneity using a variety of methods including chromatography and electrophoresis; (ii) inducing polyclonal antibodies against the toxins claimed in step (iii) of claim 1 in rabbits and goats obtained as antiserum; (iii) purifying IgG from the antiserum claimed in step (iv) of claim 1 using affinity chromatography comprising protein-A and Cry1Ac / Cry2Aa antigens; (iv) a cellulose nitrate, nitrocellulose or nylon membrane (5-12 microns) using the anti-Cry polyclonal IgG claimed in step (iv), using a manual striping method using a portable sable hair brush Passivating the phase; (v) Claim 3, wherein step (v) contains the anti-Cry1Ac polyclonal IgG derived from goat, and the anti-Cry2Aa IgG derived from goat, 0.5 cm apart. Immobilizing the two capture lines in a central portion 1 cm from both ends of the 2.5 cm wide membrane; (vi) Claim 3, wherein step (v) contains anti-Cry1Ac polyclonal IgG derived from goat, and anti-Cry2Aa IgG derived from rabbit, 0.5 cm apart. Immobilizing the two capture lines in a central portion 1 cm from both ends of the 2.5 cm wide membrane; (vii) Claim 3, wherein in step (iv) the anti-Cry1Ac polyclonal IgG derived from rabbit, and the anti-Cry2Aa IgG derived from goat are 0.5 cm apart. Immobilizing the two capture lines in a central portion 1 cm from both ends of the 2.5 cm wide membrane; (viii) Claim 3, wherein in step (iv) the anti-Cry1Ac polyclonal IgG derived from rabbits and the anti-Cry2Aa IgG derived from rabbits are 0.5 cm apart. Immobilizing the two capture lines in a central portion 1 cm from both ends of the 2.5 cm wide membrane; (ix) labeling the anti-Cry1Ac IgG derived from the rabbit claimed in step (iv) with colloidal gold 20-40 nm; (x) labeling the anti-Cry2Aa IgG derived from the rabbit claimed in step (iv) of claim 1 with colloidal gold 20-40 nm; (xi) labeling the anti-Cry1Ac IgG derived from goats claimed in step (iv) with colloidal gold 20-40 nm; (xii) labeling the anti-Cry2Aa IgG derived from goats claimed in step (iv) of claim 1 with colloidal gold 20-40 nm; (xiii) immobilizing the gold-antibody conjugate claimed in steps (x) and (xi) on the glass-fiber pad in an equivalent amount; (xiv) immobilizing the gold-antibody conjugates claimed in steps (x) and (xiii) on the glass-fiber pad in an equivalent amount; (xv) immobilizing the gold-antibody conjugates claimed in steps (xi) and (xii) on the glass-fiber pad in an equivalent amount; (xvi) immobilizing the gold-antibody conjugates claimed in steps (xii) and (xiii) on the glass-fiber pad in an equivalent amount; (xvii) goat anti-rabbit IgG or rabbit anti-goat IgG is manually striped as a 30 cm x 0.1 cm line 0.5 cm from the top of the membrane in each strip; (xviii) the goat anti-rabbit IgG or rabbit anti-goat IgG lines of step (xviii) will capture gold labeled anti-rabbit antibodies or anti-goat antibodies, respectively, as in the assembled strip; (xix) the line of step xix of claim 3 serves as a control line to indicate that the strip is functioning properly; (xx) the line of step (ix) of paragraph 3 will turn pink / purple regardless of the positive or negative of the test sample when in use; (xxi) blocking the membrane with blocking buffer containing proteins, sugars and preservatives prior to assembly, washing twice with phosphate buffered saline and drying for 30 minutes; (xxii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xiv) of claim 3 onto the membrane claimed in step (vi) of claim 3, as shown in FIG. (xxiii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xiv) of claim 2 onto the membrane claimed in step (vii) of claim 2, as shown in FIG. 2; (xxiv) assembling the gold-conjugate impregnated glass-fibers claimed in step (xiv) of claim 2 onto the membrane claimed in step (viii) of claim 1, as shown in FIG. 2; (xxv) assembling the gold-conjugate impregnated glass-fibers claimed in step (xiv) of claim 2 onto the membrane claimed in step (ix) of claim 1, as shown in FIG. 2; (xxvi) assembling the gold-conjugate impregnated glass-fibers claimed in step (xv) of claim 3 onto the membrane claimed in step (vi) of claim 3, as shown in FIG. (xxvii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xv) of claim 3 onto the membrane claimed in step (vii) of claim 3, as shown in FIG. (xxviii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xv) of claim 3 onto the membrane claimed in step (viii) of claim 3, as shown in FIG. 2; (xxix) assembling the gold-conjugate impregnated glass-fibers claimed in step (xv) of claim 3 onto the membrane claimed in step (ix) of claim 3, as shown in FIG. 2; (xxx) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvi) of claim 3 onto the membrane claimed in step (vi) of claim 3, as shown in FIG. 2. (xxxi) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvi) of claim 3 onto the membrane claimed in step (vii) of claim 3, as shown in FIG. (xxxii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvi) of claim 3 onto the membrane claimed in step (viii) of claim 3, as shown in FIG. 2; (xxxiii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvi) of claim 3 onto the membrane claimed in step (ix) of claim 3, as shown in FIG. 2; (xxxiv) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvii) of claim 3 onto the membrane claimed in step (vi) of claim 3, as shown in FIG. 2; (xxxv) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvii) of claim 3 onto the membrane claimed in step (vii) of claim 3, as shown in FIG. 2; (xxxvi) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvii) of claim 3 onto the membrane claimed in step (viii) of claim 3, as shown in FIG. 2; (xxxvii) assembling the gold-conjugate impregnated glass-fibers claimed in step (xvii) of claim 3 onto the membrane claimed in step (ix) of claim 3, as shown in FIG. 2; (xxxviii) completing the assembly by placing a sample-release filter paper pad (1 to 2 mm thick) 2 mm superimposed on the bottom of the glass-fibers claimed in steps (xxiii) to (xxxviii) of claim 3. Placing a second sample-absorption pad 2 mm superimposed on top of the membrane claimed in the steps (xxiii to xxxviii) of claim 3; (xxxix) cold lamination of the final assembly; (xl) using the strips prepared from steps (xxiii) to (Xi), as two lines if the test sample is positive for both toxins or as a single sample line if the test sample is positive for one of the toxins or the test sample If negative, nothing appears at the sample line location on the strip, simultaneously detecting Cry1Ac / Cry1Ab and Cry2Aa / Cry2Ab; (xli) the position of the sample line is fixed as the bottom line for Cry1Aa / Cry1Ac and as the top line for Cry2Aa / Cry2Ab or vice versa as the top line for Cry1Aa / Cry1Ac; And (xlii) sample lines appear due to the accumulation of gold-labeled anti-Cry-toxin antibodies that bind toxins (Cry1Ac / Cry1Ab or Cry2Aa or Cry2Ab) and along the membrane due to capillary forces until meeting the sample capture line Move. Once the gold-labeled anti-Cry-toxin antibody-toxin conjugate is in contact with the sample capture line, the toxin binds to form a sandwich and accumulation of the gold conjugate along the line occurs. Accumulated gold conjugates appear as visible lines. The method of claim 1, wherein the portable sable hair brush is immobilized with an antibody (IgG) solution on the membrane using a manual striping method. (i) The manual striping method claimed in step (vi) of claim 1, step (iii) of claim 2 and step (v) of claim 3 is carried out with the aid of a sable hair brush. (ii) The portable brush technique enables the uptake of antibodies on the membrane without the formation of craters. (iii) A 30 cm ruler is gently applied onto the membrane and striping is performed by moving the metal part of the brush along the ruler to gently touch the tip of the hair moistened with the antibody of the brush to form a capture line.