PHATS project progress, ELISA validations and my return to Liege

Moulting grey seals hauled out on the Isle of May
Remember please send all ringed shag sightings to shags@ceh.ac.uk

Welcome back to my corner of the internet and the PHATS blog! The first four months of 2017 have flown by as the team has headed back into their labs to analyse all the samples we collected over the winter on the Isle of May grey seal breeding colony (to read about our fieldwork adventures, see these blogs here). I was lucky enough to escape to perform a survey of the Isle of May in January, to see if there were any grey seals that were moulting early in the year. There were plenty of them as it turned out, which bodes well for the fieldwork we are planning next year to try and look at moulting seal physiology. The island already looks so different to how it was when the seals breed there in the winter, much greener and all the seabirds are starting to come back. The cliffs were lined with guillemots, razorbills and shags; some were even getting started on gathering nesting material. The puffins had not returned yet, they arrive later in the year closer to summer, but we did see a few on the water during the boat crossing to the island.

Large grey seal haul out on the North East coast of the Isle of May
Is there anything better than well organised samples?

Inside the lab, I’ve been working on biochemical analysis of the cell culture media from all the blubber sample experiments last year (see here for more info) and am now two thirds through the samples we generated. By measuring the metabolic profiles of the various blubber culture experiments, we can see if the pollutant or hormone treatments had any impact on the blubber cells we collected from the seals. I’m also working on validating ELISAs (Enzyme-linked immunosorbent assays) to detect a variety of hormones in the blood samples we collected from the seals last year, so we can see if an individual’s hormone profiles are linked to their pollutant burden.

Using ELISAs on wild animal species like grey seals can be tricky, as they use antibodies as part of a binding process to detect the hormone you are interested in. These antibodies will have come from a specific species of mammal, usually rodent or domestic animal species that lots of scientists study, and the company making the ELISA kit will provide a list of species they know the kit works with. As a hormone’s protein structure is not always the same in different species of animals, the antibodies used in a widely available ELISA kit may not react properly with samples from unusual species that has never been tested with that kit before. Unfortunately seals often fall into the ‘unusual’ category, so we need to test the kits (validate them) before we use them to run lots of our samples, to make sure the results we are getting from the kits are accurate. There are several things to check when validating an ELISA, some of the most important are:

  1. Test for linearity. By diluting some of your samples (e.g. to half the concentration, then a quarter, then an eighth etc) you can make a serial dilution series to run on the kit. You can then see whether the curve the dilution series produces is parallel to the standard curve, which is what the kit uses to determine hormone concentrations. If the curve is not parallel to the standard curve, then the hormone in your samples is not binding correctly to the kit components.
  2. Test for recovery. By spiking a sample with a known quantity of the hormone you are interested in studying, you can tell how much the kit is detecting and how much is ‘lost’ during the analysis process.
  3. Test for consistency across kits and within kits. Many studies have lots of samples and need to use more than one kit to analyse them all. You must make sure the results of one kit are comparable to the others (inter-assay coefficient of variance), and the easiest way to do this is to run the same sample on each kit you use. You can then calculate the coefficient of variance across all the kits you have run. It’s also important to check the kit’s internal consistency (intra-assay coefficient of variance) by running one sample multiple times on a plate and seeing how similar the results are, and by running all samples in duplicate on the kit.

Our seal blood samples are proving to be rather tricky currently, and we’re still working on which are the best kits to use to measure the hormones we are interested in. The biochemistry analysis is going very well though, and we’re all looking forward to having some data to play with in the coming months.

Two ELISA plates at different stages of incubation. The different intensities of colour indicate different concentrations of hormone.

The biochemistry and ELISA work I’m doing is currently on hold however, as I have returned to the University of Liege in Belgium to work with the Centre de Recherche Analytique et Technologique (CART) to detect the amounts of persistent organic pollutants (POPs) in the blubber of our study seals from the Isle of May last year. We will be using Gas Chromatography – Mass Spectrometry (GC-MS) again, which requires a lengthy extraction and clean up process before the blubber tissue can be analysed (see these blogs from last year for more details) so I will be here for a month to work on our samples (and I will update the blog every week while I am visiting CART). It is always interesting, and more than a little sad, to find out how many pollutants all the seals have inside them after we got to know so well during the field season…

Unfortunately Alpha and Kilo, like all young marine mammals, will have large concentrations of POPs in their tissue from the high fat milk they drink from their mothers.

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