Author
|
Submitted to: Annual Meeting of the UNJR Panel on Toxic Microorganisms
Publication Type: Abstract Only Publication Acceptance Date: 11/7/2004 Publication Date: 11/7/2004 Citation: Maragos, C.M. 2004. Fluorescence polarization immunoassay of mycotoxins [abstract]. 39th Joint Panel Meeting, UNJR Panel on Toxic Microorganisms. Book of Abstracts. p. 34. Interpretive Summary: Technical Abstract: Enzyme-linked immunosorbent assays (ELISAs) for mycotoxins have existed for many years and are commercially available. Most such assays rely upon the competition between a toxin-labeled enzyme and toxin, from a sample, for a limited amount of antibody attached to a solid surface. After a washing step to remove unattached enzyme, and addition of substrate, a colored product is produced that is inversely proportional to the amount of toxin present. There are at least two possibilities for increasing the speed of immunoassays relative to ELISAs; (1) elimination of the washing step, and (2) elimination of the amplification (enzymatic reaction) step. Fluorescence polarization (FP) is a technology that has been used in medical and veterinary diagnostics to detect small molecules in solution without requiring either enzymatic reaction or washing steps. FP immunoassays have recently been described for a number of mycotoxins, including the aflatoxins, deoxynivalenol (DON), fumonisins, and zearalenone (ZEN). Fluorescence polarization detectors indirectly measure the rate of rotation of a fluorophore in solution. The rate of rotation is directly related to the size of the fluorescent molecule, with larger molecules rotating more slowly at a given temperature. FP immunoassay uses this effect by combining mycotoxin-fluorophore conjugates and mycotoxin-specific antibodies. The fluorescent conjugate (tracer) has a low molecular weight and rotates rapidly in solution. Addition of anti-toxin antibody results in the formation of an immune complex of the tracer with the antibody, effectively slowing the rate of rotation of the tracer and increasing the polarization. The presence of free toxin in a sample decreases the fraction of the tracer bound to the antibody, and decreases the FP signal. Because of this, low molecular weight materials such as mycotoxins can be detected in solution without the need for separating "bound" and "unbound" label, and the amplification step of ELISAs is unnecessary. These are potentially significant advantages over traditional ELISA techniques. Application of FP immunoassay to fumonisins in maize, DON in wheat, aflatoxins in maize, and ZEN in maize will be presented. As with other immunoassays, the selection of appropriate antibody and tracer pairs is essential. While FP immunoassays can be conducted using either the rate of association (kinetic assays) or the endpoint of equilibrated mixture (batch or equilibration assays), in general the latter method may be preferable if untrained personnel will be performing the assays. The time it takes for the antibody/tracer/toxin combination to achieve equilibration is a critical aspect of FP immunoassays. This equilibration time can vary from 1 min to over 15 min, depending upon the antibody/tracer combination selected. When appropriate antibody/tracer combinations are used, rapid FP immunoassays can be developed. The potential speed of FP assays combined with the portability of commercially available devices, suggests this technology may be useful for mycotoxin screening, particularly in situations where samples need to be analyzed in a serial, rather than a batch, fashion. |
