I recently received a grant from the Animal Behavior Society to buy enzyme immunoassay kits. These kits will be used to measure how cortisol (a “stress hormone”) concentrations influence the ability of trematode parasites to infect California killifish (i.e., are killifish with higher cortisol levels more likely to become infected by their trematode parasites?), and to measure how cortisol concentrations change after infection. Why I’m asking these questions is a good story for another day. Today I want to tell you about how the kits I’ll be purchasing work, because they’re AWESOME:

Below is a picture of a finished plate. In this case, the second and third columns were used as standards. If you look carefully, you can see that they start very faint, and get more bright as you move towards the bottom. This is because more of the kit hormone (bound to the tracer) has been added to each well as you progress down the plate.

Critiques of my explanation for how this kits works are welcome!

11 Responses to “Enzyme Immunoassay Kits: How do they work?”

  1. danny

    Great post, one small comment.

    You said the antibodies will bind and unbind until they reach an equilibrium, for any good antibody this is wrong. Theoretically an antibody can unbind after binding, but this will not happen in RT after any reasonable amount of time. To all intents and purposes, once an antibody is bound, it will never unbind in normal conditions.

    This is important, because you must not let your sample or the kit hormone bind first and thus get a competitive edge (unless the kit is used just to bind the free left antibodies, this will reduce the dynamic range).

    • weinersmith

      Thanks, Danny! I made an edit in the video to reflect this information.

  2. Morris Keesan

    Cool explanation. One small nitpick: You say that the hormone and the kit hormone will bind to the antibody receptors in the proportion that they’re present in the solution. Isn’t it slightly more accurate to say that the hormones will bind in a proportion which ON AVERAGE is the same as their proportions in the solution?
    With some kind of statistical mumbo-jumbo describing the expected deviations from that mean?

    • weinersmith

      Hi, Morris.

      You’re right, it’s probably not EXACTLY the proportion and saying on average would be more accurate. To account for this problem most people (myself included) run all of their samples in duplicate or triplicate. If the 2 values for the same sample are very close, and if all duplicate samples on the plate yield similar values, then we can feel pretty good about getting pretty darn close to the average concentration of the hormone in the sample. In my experience, the values are consistent.

      Thanks for helping me clarify!


  3. Pip


    Awesome! Thanks for the explanation! Your excitement is contagious.


  4. David Quick


    The term your looking for i believe instead of “glowing” is fluorescing.

    • weinersmith

      Thanks, David!

  5. Pedro Almada

    Hello again,

    I’ve never done ELISA (which I prefer to use over EIA, just rolls off the tongue better =P), but a quick look at the internet tells me you aren’t using a fluorescent reagent; Ellman’s reagent is a chromogenic reagent instead (yay wikipedia). This means it’s not emitting light, only absorbing specific wavelengths resulting in a particular color. The plate reader is usually a spectrophotometer, which makes sense given that their purpose is to measure absorption at a particular wavelength.

  6. Martin

    Hi Kelly,
    What brand of dry-erase markers were you using? They look awesome!

  7. Electronics-designer

    It’s not really glowing, at least for the assay above. There are a number of different labels that can be used, and you can use the luminescence (glowing) assays, which use luciferase (a firefly enzyme) that will glow; but the above uses antibodies labeled with an enzyme that cleave a substrate into a yellow byproduct (spectrophotometric), you’ll notice that if you leave the assay out for a long time at room temperature it turns completely yellow, the enzyme never stop working and eventually the samples will reach saturation.

    Other labels include fluorescent labels, radioactive labels, etc, the advantage to using an enzyme that continually reacts with a substrate is that even minute quantities of sample will produce finely divided gradients of color change given enough time, the disadvantage is that you can consume all the substrate eventually and oversaturate, i.e. platueau, which depending on starting hormone concentrations may lead to the loss of data.

    The technical name for this assay is a competitive binding assay, and unlike most assays (ELISAs being the other commonly used one) the signal produced is inversely proportional to the target hormone/chemical.

    Great video, wish I’d had it explained by you instead of learning the hard way. good luck in grad school.

  8. Electronics-designer

    On a side note, make sure that the kit you buy has good antibodies, there’s a lot of crap out there, antibodies that cross-react with other hormone/proteins (non-specific binding) in addition to your target, give bands of the wrong size depending on conditions, etc. Antibodies are non- standard, vary by lot, and about half the papers out there have BIG problems with antibodies. Check publications which your specific lot number, it may save you a lot of grief.

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.


September 1st, 2018

Graduate student position available studying alternative reproductive tactics at BGSU

We seek a graduate student for a newly NSF-funded project examining the life history decisions made by male smallmouth bass. […]

June 14th, 2018

Part of that World

The other day I was singing “Part of your World” from The Little Mermaid, but was changing some of the […]

June 13th, 2018

Parasite manipulation of host behavior in pop culture

I’m going to be giving a talk at the sure-to-be-amazing Zombie Apocalypse Medicine Meeting. The meeting celebrates all things zombie-related, and […]

June 4th, 2017

Soonish giveaway on Goodreads!

Five copies of the advance reader version of Zach and my new book Soonish are up for grabs on Goodreads! Click […]

March 7th, 2017

Soonish: Ten Emerging Technologies That’ll Improve and/or Ruin Everything

Zach and I wrote a book! Soonish: Ten Emerging Technologies That’ll Improve and/or Ruin Everything explores 10 emerging technologies, and discusses the roadblocks […]

January 26th, 2017

Tales from the Crypt: a parasitoid manipulates the behaviour of its parasite host

I have a new paper out with Dr. Scott Egan, Dr. Andrew Forbes, and Sean Liu! The paper is Open Access […]

May 30th, 2016

Postdoc with Dr. Ryan Hechinger (and me!)

We’re looking for a postdoc! See below! —————— Postdoctoral Opportunity with the Marine Biology Research Division at SIO Postdoctoral Scholar […]

May 7th, 2016

Science…sort of Episode 240: Moon Rocks Don’t Glow

I co-hosted an episode of Science…sort of recently. I pasted the show notes below, but you’ll have to head over […]

February 24th, 2016

Books on parasites

I’m often asked by students to suggest books they can read about parasites. Below is a list of books that […]

August 22nd, 2015

Great Adaptations – A kid’s book about evolution

Zach Weinersmith and I contributed to Tiffany Taylor’s children’s book about evolution. Tiffany worked with scientists to create Seuss-style stories […]