Immunoassays have become highly important weapons in the detection of allergens, drugs and other problematic components in various food stuffs. They are so sophisticated now, they have become easy and efficient screening tools to rival the chromatographic methods which are perceived to have become much more expensive, time-consuming and labour-intensive. A variety of immunoassays have been developed including immunochromatographic strip tests, immunosensors and various forms of ELISA (enzyme-linked immunoassay) which are outside the scope of this short post.

Fluorescence polarization immunoassay (FPIA) came to the fore because unlike ELISA, does not involve any enzymatic reactions. Also, the bound and free label does not need to be separated, and so there is no need for the extensive separation and washing steps required. It is also much less costly because of the reduction in steps.

Perrin first developed fluorescence polarization (FP) in the 1920s (Perrin, 1926) before applications to the antigen-antibody reaction (Dandliker and Feigen, 1961) and subsequent development by Abbott Laboratories. It is a competitive assay which measures the change in fluorescence of a fluorescent-labelled antigen, the tracer when it binds to a specific antibody, and in the presence of the antigen being measured. The assay relies on the fluoroscein labelled antigen competing with the unlabelled antigen for the antibody. In the procedure, plane polarized light at 490nm excites the sample and fluorescein emits plane polarized light which is detected at 520 nm. The unbound antigen-fluoroscein complex produces a greater emission than the bound version of the complex. The antigen is thus competing for antibody with the fluoroscein bound antigen. The more antigen in the sample, the less fluoroscein labelled antigen binds to the antibody and so the emission of plane polarized light rises.

There is a useful recent review covering its application for assessing mycotoxins (Maragos, 2009). The method has been around for a number of years, mainly to detect small but complex toxins such as fumonisins (Maragos et al., 2001), deoxynivalenol (Maragos and Plattner, 2002), ochratoxin A (OTA) (Zessa et al., 2009) and just recently for zearalenone (Choi et al., 2011).

One of the key successes in recent years has been the identification of better tracers which allows for increased sensitivity. An understanding of what produces the ideal tracer is examined in the paper by Chun et al., (2009) which compares FPIA with other current techniques and looks at the chemistry behind the ideal linker. The latest paper (Choi et al., 2011) identifies levels zearalenone as low as 20 µg/kg in a sample although one article reported a level as low as 11 µg/kg (Maragos and Kim, 2004). Such detection limits are critical to meet government guidelines.

I noticed some very recent papers using the technique and these are given here, for detection of herbicides such as butachlor (Lei et al., 2011), melamine (Wang et al., 2011), the T-2 and HT-2 toxins in wheat (Lippolos et al., 2011).

If you would like to know more about this technique and how FoodWrite Ltd can help you implement it then please call on tel: 07714101039. Look forward to hearing from you !

References

Choi, E.H., Kim, D.M.m, Choi, S-W., Eremin, S.A. Chun, H.S. (2011) Optimisation and validation of a fluorescence polarisation immunoassay for rapid detection of zearalenone in corn. Inst. J. Food Sci. Technol. 46(10) pp. 2173-2181
Chun, H.S., Choi, E.H., Chang, H.J., Choi, S.W. & Eremin, S.A. (2009). A fluorescence polarization immunoassay for the detection of zearalenone in corn. Analytica Chimica Acta, 639, pp. 83–89.
Dandliker, W. B., and Feigen, G. A. (1961) Quantification ofthe antigen-antibody reaction by the polarization of fluorescence. Biochem. Biophys. Res. Commun. 5, pp. 299-304
Lei, H.,Xue, G., Yu, C., Haughey, S.A., Eremin, S.A., et al., (2011) Fluorescence polarization as a tool for the detection of a widely used herbicide, butachlor, in polluted waters. Anal. Methods DOI: 10.1039/C1AY05347G
Lippolis, V., Pascale, M., Valenzano, S., Pluchinotta, V., Baumgartner, S., Krska, R., Visconti, A. (2011) A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat. Anal. Bioanalyt. Chem 401 (8) pp. 2561-2571 DOI: 10.1007/s00216-011-5379-3
Maragos, C. (2009) Fluorescence polarization immunoassay of mycotoxins: a review. Toxins 1 pp.196-207 doi:10.3390/toxins1020196
Maragos, C.M., Jolley, M.E., Plattner, R.D., Nasir, M.S. (2001) Fluorescence polarization as a means for determination of fumonisins in maize. J Agric. Food Chem. 49 pp. 596–602.
Maragos CM, Kim E. (2004) Detection of zearalenone and related metabolites by fluorescence polarization immunoassay. J Food Protect. 67(5) pp. 1039–1043.
Maragos CM, Plattner RD. (2002) Rapid fluorescence polarization immunoassay for the mycotoxin deoxynivalenol in wheat. J Agric. Food. Chem. 50 pp. 1827–1832.
Perrin, F., (1926) Polarization de la lumière fluorescence. Vie moyenne des molécules dans l’etat excité. J. Phys. Radium 7, pp. 390-401
Wang, Q., Haughey, S.A., Sun, Y-M., Eremin, S.A., Li, Z-F., Liu, H., Xu, Z-L., Shen, Y-D., Lei, H-T. (2011) Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder. Anal. Bioanalyt. Chem.   399 (6) pp. 2275-2284 DOI: 10.1007/s00216-010-4599-2
Wang, Z., Zhang, S., Nesterenko, I.S., Eremin, S.A. & Shen, J. (2007). Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine. J. Agric.Food Chem., 55, pp. 6871–6878
Zezza, F., Longobardi, F., Pascale, M., Eremin, S.A. & Visconti, A. (2009). Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine. Anal. Bioanal. Chem., 395, pp. 1317–1323.