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Rapid detection of bt-cry toxins

A toxin and rapid immunization technology, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., can solve the problem of expensive immune chromatographic strips

Inactive Publication Date: 2005-09-21
INDIAN COUNCIL OF AGRI RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunochromatographic strips are expensive when the production process includes the use of monoclonal antibodies and some instrumentation

Method used

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  • Rapid detection of bt-cry toxins
  • Rapid detection of bt-cry toxins
  • Rapid detection of bt-cry toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment (1

[0013] Example (1): Preparation of Rapid Immunochromatographic Assay / Band to Detect Cry1Ac

[0014] 1. Isolation of Cry1Ac from clones, which is done by ultrasonically degrading bacterial clone cultures, and sedimentation to remove cell debris and insoluble toxins. The toxin is dissolved in alkaline buffer and extracted by centrifugation. Toxin purification was accomplished by 25% saturated ammonium sulfate precipitation and polyacrylamide electrophoresis.

[0015] 2. Antiserum against Cry1Ac toxin was raised in rabbits or goats by injecting purified toxins respectively.

[0016] 3. The method of culturing antiserum is to initially inject the antigen (Cry1Ac) mixed with Freund's complete adjuvant and repeated booster doses mixed with Freund's incomplete adjuvant, and then collect the serum. This common technique can be obtained from the immunology textbook ( Antibodies-A laboratory Manual - Harlow Ed and David Lane; Cold Spring Harbor Laboratory, USA, 1988) and will not be d...

Embodiment (2

[0036] Example (2): Preparation of a rapid immunochromatographic assay / band to detect Cry1Ac / Cry2Ab using Cry-toxin receptor protein as capture ligand.

[0037] 1. Isolation of Cry1Ac and Cry2Aa / Cry2Ab from clones, which is by ultrasonically degrading bacterial clone cultures, and sedimentation to remove cell debris and insoluble toxins. The toxin is dissolved in alkaline buffer and extracted by centrifugation. Toxin purification was accomplished by 25% saturated ammonium sulfate precipitation and polyacrylamide electrophoresis.

[0038] 2. Cultivate antisera against Cry1Ac / Cry2Aa / Cry2Ab toxins in rabbits or goats by injecting purified toxins, respectively.

[0039] 3. The method of raising antiserum is initial injection of antigen (Cry1Ac / Cry2Aa / Cry2Ab) mixed with Freund's complete adjuvant and repeated booster doses mixed with Freund's incomplete adjuvant, and then the serum is collected. This general technique can be obtained from Immunology textbook ("Antibodies - A Labo...

Embodiment (3

[0060] Example (3): Preparation of a rapid immunochromatographic assay / strip to simultaneously detect Cry1Ac and Cry2Ab.

[0061] 1. Isolation of Cry1Ac and Cry2Aa / Cry2Ab from clones, which is by ultrasonically degrading bacterial clone cultures, and sedimentation to remove cell debris and insoluble toxins. The toxin is dissolved in alkaline buffer and extracted by centrifugation. Toxin purification was accomplished by 25% saturated ammonium sulfate precipitation and polyacrylamide electrophoresis.

[0062] 2. Cultivate antisera against Cry1Ac / Cry2Aa / Cry2Ab toxins in rabbits or goats by injecting purified toxins, respectively.

[0063] 3. The method of raising antiserum is initial injection of antigen (Cry1Ac / Cry2Aa / Cry2Ab) mixed with Freund's complete adjuvant and repeated booster doses mixed with Freund's incomplete adjuvant, and then the serum is collected. This general technique can be obtained from Immunology textbook ("Antibodies - A Laboratory Manual" - Harlow Ed and Da...

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Abstract

The present invention is based on the use of 1) two polyclonal IgGs against two Cry toxins, 2) Cry toxin receptors isolated from lepidopteran larvae and 3) manual striping methods to manufacture immunochromatographic strips that are useful in detecting a variety of analytes. Immunochromatographic strips, which facilitate rapid detection of analytes are expensive if made using monoclonal antibodies and cannot be affordable for Indian farmers and extension workers. The main objective of the current method was to simplify the development of the immunochromatographic detection method for Cry (Bt) toxin detection using affinity purified polyclonal antibodies specific to the analyte and also to provide a robust and easy method suitable for manufacture of 'Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab toxin' detection strips at affordable cost, under Indian conditions. Anti-Bt (anti-Cry1 Ac or anti-Cry2Aa / Ab or both) antibody or Cry receptor proteins are immobilized on a cellulose nitrate membrane by manual striping. The membrane is blocked using casein or BSA and preservatives. Anti-Bt (anti-Cry1 Ac or anti-Cry2Aa / Ab or both) antibody is labeled with gold and adsorbed on glass-fibre membrane which is placed at the bottom the membrane so as to overlap 2mm. Specificity and accuracy of the assay are greatly enhanced due to the use of antigen-affinity and Protein-A affinity purified polyclonal IgG raised in two different animals (goat and rabbit). The schematic diagram of the Cry toxin detection lateral flow strip assembly is shown in a diagram appended herewith. The strips thus made represent rapid, sensitive devices and methods for detecting the presence of Cry1Ac / Cry1Ab / Cry2Aa / Cry2Ab and crystal toxins either from transgenic plants or in Bt-fermented products.

Description

technical field [0001] The present invention relates to a rapid immunochromatographic assay using polyclonal antibodies for the detection of crystalline toxins Cry1 Ac / Cry1Ab / Cry2 Aa / Cry2 Ab in seeds or plant tissues on lateral flow zones. It focuses specifically on crystalline toxins isolated from native Bacillus thuringiensis and methods using polyclonal antibodies against which antibodies are raised. The invention more particularly relates to the simplified development of a manual stripping method and an assay with greatly improved specificity and accuracy by using antigen affinity and protein A affinity purified polyclonal IgG grown in two different animals - goat and rabbit . The invention involves the use of crystalline toxin receptors from Lepidopteran insects as capture ligands to detect crystalline toxins. The invention also facilitates simultaneous detection of two crystalline toxins on a single band. Background technique [0002] The soil bacterium Bacillus thu...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N2333/325G01N33/68
Inventor K·R·克兰西
Owner INDIAN COUNCIL OF AGRI RES
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