Northwest Fisheries Science Center

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Receptor Binding Assay

First, a little background

Toxins exert their devastating affects in a variety of ways, but most commonly they interfere with the transmission of nerve impulses. The activation of a nerve impulse is an electrical phenomena in which a series of connected nerve fibers are sequentially polarized and depolarized. Actually these cells are not "connected" in the usual meaning of the word, but are "connected" through a space or junction called the "synaptic cleft" (see below). A nerve impulse is passed from one nerve cell to the other across the cleft by a "neurotransmitter". Usually, this is a small, low molecular weight chemical, such as acetylcholine or epinephrine (adrenaline). A number of chemicals have been identified as neurotransmitters in the past 20 years.

Nerve cells have an electrical potential between the inside and outside of the cell. This voltage comes about due to the differences between the ionic composition of inside the cell (where potassium ions, K+, are in higher concentration) and outside the cell (where sodium ions, Na+, are in higher concentration). When stimulus is applied to these cells, Na+ ions flow into the cell, voltage increases, thereby causing K+ to flow out of the cell. This change in voltage is referred to as depolarization.

Shematic drawing of a receptor
As K+ flows out of the cell it releases a neuro-transmitter which crosses the synaptic cleft and goes to a receptor on the next nerve cell. When the neuro-transmitter binds to the receptor, a change occurs in the cell causing it to depolarize. When that cell depolarizes it also releases neuro-transmitters and the process goes on to the next cell.

What are receptors?

Propagation of nerve impulses can be a rather complicated process and there are plenty of opportunities for things to go wrong. In order for cells not to depolarize all the time, the receptor site must accept only very specific chemicals. By and large, the receptor site has a structure that is fairly specific for its designated neurotransmitter. Nerve Toxins are harmful in a number of ways, among them:

Assays

Very early on, researchers took advantage of toxin binding to nerves as the basis of assays for the toxins and/or neurotransmitters. However, these usually required very complex setups and the isolation of nerve receptors from a variety of animals (e.g., the giant squid axon). Needless to say these procedures were time consuming, very expensive, and required highly trained and skilled people. While they were used widely, and still are to some extent today, by medical and pharmacological researchers, recent advances in molecular biology have permitted much simpler methods. These new methods can be faster, cheaper, far more sensitive, and most importantly use very small amounts of toxic extracts. It is now possible to clone receptors from one species and have them reproduced in cells of another species. It is this method that we use to produce receptors used in our receptor binding assay for domoic acid determination.

Receptor binding assays measure the binding of a toxin to its nerve cell receptor by either marking the toxin or a suitable derivative of the toxin. This is most commonly done now using radioactive markers. In the procedure, the radioactive toxin will be displaced or "bumped off" its receptor by toxin present in an unknown sample, thereby reducing the total radioactivity. The amount of radioactively labelled toxin that is displaced is proportional to the amount of toxin in the unknown sample. The toxin present in an unknown sample can then be quantified by comparison to a standard curve obtained using pure toxin. The advantage of this technique is that it can be made highly specific and sensitive for a particular toxic activity. Currently, receptor binding assays have been developed for domoic acid and PSP toxins. Below is a general description of the receptor binding assay.

Schematic diagram of receptor binding assay.

Domoic Acid Receptor Binding Assay

Domoic acid is a potent neurotoxin that binds very tightly to a glutamate receptor in the brain. Under normal circumstances, these cells use glutamic acid (a common amino acid) as a neurotransmitter. Unfortunately, this receptor also binds two other compounds even more tightly than glutamic acid. These two compounds, kainic acid and domoic acid, are at first glance not that similar to glutamic acid; nevertheless, they both bind to the glutamate receptor. Of the three compounds, domoic acid binds to the glutamate receptor most tightly and will displace both kainic acid and glutamate from the binding sites. We take advantage of this fact in designing this assay.

Image of receptor binding assay standard curve.
We can take advantage of the fact that kainic acid has a structure very similar to domoic acid. Furthermore, kainic acid is rela tively easy to synthesize and is available from commercial sources. Kainic acid can be easily marked using tritium (a radioactive isotope of hydrogen). In addition, by using techniques developed in molecular biology, it is possible to grow cells in the laboratory that contain glutamate receptor sites. Using radiolabelled kainic acid we can then use the domoic acid to displace the radioactive kainic acid binding from the receptor. By measuring the radioactivity in counts per minute (CPM) of samples with known amounts of domoic acid we can create a "standard curve" such as the one shown here which relates added amounts of domoic acid and counts of radioactivity.

Once we have established this "standard curve" we can then add our unknown samples to the radiolabelled kainic acid and receptor preparation. We can then compare the amount of radioactivity in our samples to our standard curve. This allows us to estimate very low concentrations of domoic acid in a variety of extracts, be they sea water, shellfish, or phytoplankton cells.

Detection & Analyses