Friday, February 5, 2010


ELISA.  Enzyme-Linked Immunosorbent Assay.  A helpful, interesting biological assay.

To perform an ELISA, you have to have a target molecule.  Let's say the target molecule is histamine, a molecule that causes an inflammatory response, which is usually the culprit in allergic reactions.  And let's say you want to know, "How much histamine is in this sample?  Which of these four samples has the highest or lowest histamine concentration?"  ELISA can answer those questions.

You take a 96-well plate and in however many wells you need (2 wells per sample, 2 wells per standard curve sample--usually 16-18 wells), you add in antibody.  This antibody sticks to the bottom of each well, and it's specific for histamine.  So if you put histamine in the well, it'll stick to the antibody.  Nothing else will.

So you coat the wells with histamine antibody, and then you add your samples.  You let them incubate, and the histamine in each sample sticks to the antibody.  Of course, if one sample has more histamine than another sample, you'll get more histamine binding.  Then you wash out the wells, leaving nothing except whatever histamine stuck to the antibody.

Then you add more antibody for histamine, which sticks to the histamine in the wells, creating a sandwich (these are actually called sandwich ELISAs).  These antibodies are special; you can then add various reagents to the wells and those reagents will react to these detection antibodies.  Eventually, you'll be left with a bright color in every well; the brighter the color is, the more histamine is in the sample.  You then stick it in a special reader which tells you the concentration in each sample.  Tadah.

Well, I'm not doing an ELISA on histamine; it's just my fav molecule and something people tend to be familiar with, thanks to things like allergies and Benadryl.  Next week, I have to run an ELISA for VEGF, a growth factor.  Here's what I've got:

Samples from 1 day, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks.
For every time point, I have three different conditions.
For every condition, I have three different samples (that is, the experiment was done in triplicate).
For every sample, I have one sample from the top of the well and one from the bottom of the well.
For every one of these samples, I have to fill two wells.

So, math.  6 time points x 3 conditions x 3 samples x 2 samples x 2 wells = 216

But I have to do a standard curve so I have something to compare the sample concentrations to!  There are a bunch of different concentrations (2000 pg, 1000 pg, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.2 pg, 15.6 pg, 0 pg) that I need for the standard.  Additionally, since my samples are all conditioned medium (medium that was used to grow cells--it's not fresh and clean), I need non-conditioned medium so I can show that the VEGF didn't just come from the medium itself.  So that's 10 more samples:

10 standards/zeroes x 2 wells = 20

That leaves me with 20 + 216 = 236 wells.  But wait!  In order for me to be sure that my results are good, I need to make sure that I take into account plate-to-plate variation.  Since we use 96-well plates and I've got more than twice that number of wells, I'm going to need at least 3 plates, which means at least 3 standards/zeroes.  So I need to see how many wells that'll take up, so I know whether I need 3 plates or 4 (4 plates also means 4 standards/zeroes!).

3 plates x 20 standards/zeroes wells = 60 wells.  60 wells + 216 wells = 276, which fits on 3 plates (12 wells to spare!).

So, there you have it.  276 wells.  Monday, I'm coating the first plate, and I'll stagger them all throughout the week.  It's going to be interesting AND ridiculous!

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