Protein Based Detection

Detection of the novel proteins expressed by GM crops is based almost exclusively on the application of immunoassay technology. Several immunoassays are available for different traits present in diverse GM plant crops and are used in a variety of applications, including testing for unauthorised events and determining the relative GM content (Grothaus et al. 2007). Immunoassays are based on the reaction of an antigen (e.g. the GM-derived protein) with a specific antibody to give a antigen-antibody complex that can be indirectly measured. The immunoassay formats commonly used for GM-protein detection are the enzyme-linked immunosorbent assay (ELISA) and the lateral flow device (LFD). Lateral Flow Strip

Lateral flow strip devices (LFD) are used for qualitative or semi-quantitative detection of antigens and, in the case of novel GM proteins, antibodies are used in the same sandwich immunoassay format as in ELISA, except that the secondary antibody is labelled with a coloured particle such as colloidal gold rather than an enzyme as a means of generating a visible signal. A typical LFD has linked simultaneously a second antibody on the strip to provide visual control that the test has worked correctly. LFDs are available for several traits, require low instrumentation and allow rapid testing also in the field. They are show to be sufficiently specific, but concerning sensitivity only up to the 0.1% range is achievable. LFD represent a useful tool to detect GM proteins in raw materials such as seeds and leaves, however in food and feed products their applicability is restricted to samples containing sufficient GM plant material where the GM protein is expressed. The more drastic limitation for the application of LFDs for food and feed testing is obviously the physico-chemical instability of proteins when products are processed and heat-treated. The CP4-EPSPS protein is considered as a useful GM protein marker in food/feed products and the CrylAb protein to a lesser extent (van den Bulcke et al. 2007). Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assays (ELISAs) are commonly 96-well microplates with removable strips of 8-12 wells coated with a primary antibody to capture a target antigen in the sample. A secondary antibody, conjugated to an enzyme such as horseradish peroxidase, is used to detect the presence of the bound antigen, which results in a sandwich of the analyte between the primary and secondary antibodies.

In general ELISAs are quantitative and provide high-throughput capability to the laboratory analysis, considering that the protein is not denatured. Detection limits for Cry1Ab protein is reported to be below 0.1% for dried maize flour (Ermolli et al. 2006). To determine the concentration of the targeted protein in a sample, standards correlating to known concentrations of the antigen are used to produce a calibration curve to determine the unknown concentration of the antigen in the sample. Either recombinant proteins, which contain a similar or identical amino acid sequence and immunoreactivity as the plant-expressed protein, or uniform preparations of actual samples with known concentrations of GM proteins (such as maize or soybean flours available as certified reference materials) may also be used as calibration standards. Since processing affects the detectability of proteins, ELISA is not applicable to most processed food or feed matrices. Furthermore, ELISA does not allow event-specific identification and may fail to detect novel GM proteins.

7.2.3 Method Validation and Standardisation

Validation of detection methods is an essential component to assess the reliability of test methods. By using validated and standardised methods, control laboratories assure that the analytical procedures applied are harmonised at the national or even international level. The process of validation establishes numerical values for the different performance criteria (specificity, sensitivity, applicability, robustness, etc.) and consists at the beginning of an in-house validation in the developers' laboratory followed by a collaborative trial to determine the method's repeatability and repro-ducibility in order to estimate the transferability of a method between laboratories (Codex 2009). If a collaborative trial-validated method is to be implemented in a laboratory, it is of course also necessary to confirm that the method performs as well under the local conditions as it did in the inter-laboratory method validation study.

To harmonise the procedures applied for the detection of GM plants in foodstuffs and derived products, the International Standardization Organisation (ISO) has published a series of internationally agreed standards for nucleic acid extraction (ISO 2005c), for qualitative nucleic acid analysis (ISO 2005a), for quantitative nucleic acid analysis (ISO 2005b) and for protein-based methods (ISO 2004). Furthermore, general requirements and definitions involving these different working steps are described in a generic standard document (ISO 2006). These ISO standards prescribe what method performance and validation studies have to be conducted to establish data and the performance characteristics for the specific method application. At the European level a guidance document of the European Network of GMO Laboratories (ENGL) provides practical recommendations how event-specific PCR methods shall be evaluated in the context of the approval of a GM food or feed according to EU Regulation 1829/2003 and defines minimum performance requirements for acceptance of these methods (ENGL 2008).

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