Monday, August 16, 2010

ELISA: not just a pretty name

We’ve talked about methyl farnesoate, the reproductive hormone, but what about the hormone that makes crustaceans molt? Ecdysteroids!

from Kumar et al., 2004

These bad boys stimulate the production of a new cuticle, or soft shell, under the old hard one, as well as stimulate the production of enzymes to separate the new and old shells.

production of a new cuticle before and after the molting process
(Thanks to Miranda Westphal for the artistic idea!)

You can tell when a crab is getting ready to molt because the ecdysteroids increase significantly:

ecdysteroid profile for Dungeness crabs

How do we measure ecdysteroids? With a competitive enzyme-linked immunosorbant assay (ELISA), of course! An ELISA works by taking advantage of the specific nature of antibodies. Rabbits are injected with crab hemolymph (blood) to make a primary rabbit antibody (1° Ab), and then goats are injected with rabbit blood to make a secondary goat anti-rabbit antibody (2° Ab). This way the 1° Ab will only bind to the 2° Ab, and ecdysteroids from crab hemolymph will only bind to the 1° Ab.

The competition comes in with the crustacean ecdysteroids: the ecdysteroids will bind to the rabbit antibodies, but in order to measure how many ecdysteroids are in the hemolymph we add marked ecdysteroids to the mix.

ELISAs are run in tiny wells. Within the wells, the 2° Ab first binds to the well wall. Next we put both the crab hemolymph and the marked ecdysteroids (“horseradish peroxidase-conjugated ecdysone”) into the wells. They can’t bind to anything yet, but we finally add the 1° Ab. This is when things go nuts! The 1° Ab is busy binding to the 2° Ab, while the two types of ecdysteroids are competing for spots on the 1° Ab. After we let the wells sit for a bit, anything that hasn’t bound to an antibody gets dumped.


The horseradish peroxidase-conjugated ecdysone turns blue during the development process. When you see a well with bright blue, it means that sample of hemolymph did not have a lot of ecdysteroids in it, which allowed for more of our marked ecdysteroids to bind to the rabbit antibodies.

an ELISA plate after development

Wells that appear mostly clear represent crabs that had a lot of ecdysteroids in their hemolymph. These crabs are the ones that are ready to molt!

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