Contents - Faperta

472 Biotechnological Approaches for Pest Management and Ecological Sustainability
An antibody-sandwich ELISA has been developed to detect and quantify Bt insecticidal
protein expressed in transgenic insect-resistant cotton, using a purifi ed Bt toxin (67 kDa)
as the standard protein and antigen (Chen et al., 1999). The minimal detectable concentration
of purifi ed Bt toxin was 1 to 15 g L 1 . The mean recovery rate was 94.94%. Quality control
experiments indicate that the ELISA method is stable and reliable, and is characterized
by its strong specifi city and high sensitivity. A sandwich-type ELISA method has also
been developed for Cry1Ab in maize (Walschus, Witt, and Wittmann, 2002). Monoclonal
antibodies were developed by immunization of mice. The secondary antibody was labeled
with horseradish peroxidase and 3,3,5,5-tetramethylbenzidine (TMB) used as a chromogene.
The detection limit was 0.4 ng mL 1 of Cry1Ab. There was no Cry1Ac, Cry1C,
Cry2A, and Cry3A, or little Cry9C cross-reactivity with other Cry proteins. This assay has
been applied to protein extracts from maize powder containing different amounts of
Bt maize from three different lines (Bt 176, Bt 11, and MON 810). Using 100 mg maize
powder and Tris-borate buffer for rapid protein extraction, the minimum detection limit
was 0.10% for Bt 11 and 0.23% for MON 810. In contrast, Bt176 maize could not be detected
due to the fact that the Cry1Ab gene is not expressed in the seed tissue of Bt 176 maize.
Lateral Flow Sticks
The lateral fl ow test (dipstick format) uses a membrane-based detection system. The membrane
contains two capture zones, one captures the bound transgene protein, while the
other captures color reagent. Paper strips or plastic paddles can be used as support for capturing
the antibody immobilized onto a test strip in the specifi c zone. The lateral fl ow test
strip is dipped in the sample in extraction solution, and the sample migrates up the strip by
capillary action. As the sample fl ows through the detection antibody strip and the capture
antibody strip, the protein of interest will accumulate and give a high intensity band. These
tests generally provide qualitative or semiquantitative results using antibodies and color
reagents incorporated into a lateral fl ow strip. By following appropriate sampling procedures,
it is possible to obtain a 99% confi dence level of less than 0.15% transgenic material.
Phenotypic Characterization
Phenotypic characterization allows detection of the presence or absence of a specifi c trait.
Such methods can be used to test for presence or absence of herbicide-resistant transgenic
varieties. They consist of conducting germination tests on solid germination media in the
presence of a specifi c herbicide, where the nontransgenic and transgenic seeds show distinct
characteristics. The detection level depends on the effi ciency of seed germination.
Seeds testing positive should be exposed to subsequent tests for confi rmation. The herbicide
bioassay tests are accurate, inexpensive, and useful as preliminary tests. Negative and
positive trait seeds should be included as controls with every test. In the future, bioassays
for insect- resistant or other transgenic crops may also be developed.
Mass Spectrometry
Spectrometrical methods (matrix-assisted laser desorption/ionization) can also be used
for analysis of oligonucleotides. In this system, the analyte is embedded in a UV absorbing
matrix in vacuum on a carrier between electrodes. When the UV light is applied, the UV
energy is absorbed by the matrix, and also carried over to the sample such that it is ionized.
The ionized ions move towards oppositely charged electrode, and enter the fl ight tube