Immunoassays in the Analysis of Drugs and Poisons

I'm back, and this time we're going to be looking at one of the most powerful techniques in the toxicologists arsenal, the immunoassay. No matter the sample size or the specificity or broadness of molecules your trying to ferret out, the immunoassay is both practical, accurate and relatively cheap.

In forensics the immunoassays are used mainly for identifying the presence of drugs in or poisons in both ante and post mortem samples. Immunoassays can be highly specific, picking out only a single compound (e.g. mephamphetamine), or a group of compounds. Immunoassays are useful because not only are they highly sensitive, they are also able to analyse large samples relatively quickly and require no preliminary extraction phase.

Immunoassay relies on the antibody-antigen reaction, which occurs in the mammalian immune system. When an antigen enters the body, an antibody specific to it i produced. The antibody binds with the antigen forming an antigen-antibody complex. In immunoassays specific antibodies are used to detect and measure the level of the analyte of interest.

In immunoassays, fixed quantities of the specific antibody to the analyte and a labelled form of that sample are added to the sample being tested. The labelled molecules and the analyte (if it's present) will competitively attempt to bind with the antibodies, in inversely proportional numbers. So you can measure the reduction in concentration of the labelled poison to work out the concentration of a drug in the sample. 

However in some types of immunoassay, it isn't possible to distinguish between the labelled poison bound to the antibody and that remaining free in solution, so physical separation of the two phases is required before measurement. These are known as heterogenous immunoassays (for example radioimmunoassay). However immunoassays where this is not necessary are called homogenous immunoassays (for example fluorescence polarisation immunoassay). These two examples are actually both very good examples of immunoassays. 

Radioimmunoassays: In radioimmunoassays, the poisonous molecules are labelled with an appropriate radioisotope (for example iodine-125) and the radioactivity is measured. The concentration of unlabelled poison molecules in the test mixture is determined by reference to a standard curve. This graph is produced by adding increasing concentrations of poison to a fixed quantity of labelled poison and antibodies. 

Fluorescence Polarised Immunoassays: A suitable fluorescent substance is used to label the analyte. It's possible to distinguish between labelled analytes bound to the antibody and analytes free in solution. This is because the former emits polarised fluorescence, but the latter generates unpolarised fluorescence.

Thanks for reading, over and out!