title: An immuno-PCR method for detecting Bacillus thuringiensis Cry1Ac toxin.
abstract: The Cry1Ac toxin is an insecticidal protein produced by Bacillus thuringiensis var. kurstaki. Recently, the gene encoding the toxin was genetically transformed into crop plants. A specific and sensitive method for detecting the Cry1Ac toxin would facilitate monitoring for this protein in crop and non-crop plants and also in foods. The purpose of this study was to develop an immuno-PCR technique for detecting this toxin. Immuno-PCR combines the specificity of an ELISA reaction with the sensitivity of assays that use a PCR-amplification step. In our assay, anti-Cry1Ac antibodies were covalently bound to reporter DNA via a linker molecule, succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). Antigen was coated onto the surfaces of polyvinyl chloride microtiter plates or onto streptavidin-coated beads. Each of these solid-surface platforms was tested in immuno-PCR reactions. Both the microtiter plate- and bead-based assays showed a high degree of specificity and sensitivity, with minimum detection limits of 21.6 and 432 ng of toxin, respectively. This sensitive immuno-PCR method could be modified for detecting a variety of other protein toxins.
authors.affiliation: Department of Biology, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA.
authors.forename: Rebekah C
authors.initials: RC
authors.lastname: Allen
authors.forename: Snezna
authors.initials: S
authors.lastname: Rogelj
authors.forename: Susan E
authors.initials: SE
authors.lastname: Cordova
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Kieft
mesh_terms: D000818:Animals; D000906:Antibodies; D001413:Bacillus thuringiensis / Q000235:genetics / Q000276:immunology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004731:Endotoxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D017930:Genes, Reporter; D006460:Hemolysin Proteins; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011137:Polystyrenes; D011817:Rabbits; D012680:Sensitivity and Specificity; D019809:Streptavidin
chemical_list: D000906:Antibodies; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004274:DNA, Recombinant; D004731:Endotoxins; D006460:Hemolysin Proteins; D011137:Polystyrenes; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D019809:Streptavidin