Assay Identification
The first step in getting your project off the ground with EBPI laboratory services is to choose an assay and test method from our wide variety of offered services. When choosing a test design, try to answer the following questions:
1. What endpoint should I be testing for?
EBPI offers various options to test for
- a) Acute endpoints - Usually lethality, higher doses, short exposure times
- b) Chronic endpoints - May not be lethal, sublethal effects like altered function, longer exposure times
- c) Sub-chronic endpoints - Genetic damage, low doses, relatively long exposure times eg. the lifetime of the organism
Your sample or compound of interest will have different potencies depending on what organism you are using and may be either a very short acting toxicant and cause adverse effects almost immediately, or make take a longer exposure time to demonstrate the toxic endpoint you are looking for. Explore the literature for previous study designs on similar sample types or studies conducted on other members of your compound’s family. You may also want to explore an endpoint for a compound that has yet to be tested. If this is the case, get in touch with a representative, we can certainly help you with the research on picking the best endpoint to evaluate for your sample.
2. What is my sample matrix? Is it compatible with the test?
Depending on your compound or sample, it may have properties that make it immiscible in aqueous environments. All of our biological testing is done on organisms that prefer an aqueous solution. However, most of these organisms can tolerate certain concentrations of organic solvents like ethanol, DMF or acetone. Some organic solvents like DMSO are very well tolerated and larger volumes of this solvent can be used in the test. Figure out which environment can solubilize your compound completely and is also compatible with the aqueous test environment. In addition we have specific assays for marine or estuarine samples that prefer salt water. Having some knowledge of your sample matrix goes a long way to ensuring our analysis will produce data that you can use and will save you testing costs.
3. What organism should I use?
When picking an organism, it is important to consider the compound sensitivity to certain classes of organisms. We have several options in this regard which include higher order plants, invertebrates, small crustaceans, algae and bacteria. Many of these organisms demonstrate varying levels of sensitivity to different kinds of toxicants. For example, the Toxi-Chromotest™ bacteria is very sensitive to heavy metals. Consider also the lifetime of the organism which determines the test duration and will influence the total cost of analysis and anticipated completion date. You may also want to use an organism which is native to the sample collection area or the most sensitive species in an ecosystem which adds a great deal of biological significance to your analysis.
4. What strains of bacteria should I use?
For our bacterial-based assays that detect mutagenicity and genotoxicity you have several options for incorporating bacterial strains that are sensitive to specific mutations. These strains have been formulated to detect many different mutagens and regardless if the mechanism of mutagenicity involves a base-pair or frameshift error, we can provide a bacterial strain that fits your compound. If you are unsure of the mechanism for mutagenicity, we generally recommend testing using both the TA100 and TA98 bacterial strains that are sensitive to most base-pair and frameshift mutagens.
5. How many replicates and dilutions do I need?
Our laboratory division can accommodate any size of project. We always recommend including multiple dilution levels and replicates of each dilution to produce reliable dose response data and statistically robust results. However, both of these important parameters increase the number of sample endpoints and the cost. In some cases, a screening assay might be a better option but clients are reminded that both replicates and dilutions are usually required for publication purposes. For more information, contact a representative at EBPI.
6. Do I need metabolic activation?
Many toxins are inert when present in the environment or in their original form. They only exert detrimental effects in the body after they have been metabolized into a more reactive species. For this reason, many assays have the option of including S9 liver homogenate and cofactor system which confers metabolic ability to the sample and increases both the sensitivity of the assay, as well as the amounts of different compounds that cause a positive response. Simply add S9 metabolism to your test design and our technicians will run the samples both with and without metabolic activation.
After you have decided the best way to test your samples move on to step 2, Sample Submission