Friday, December 4, 2009

But what do you do with the spit after you get it?

Last night, before bed, I collected some saliva from my dog Jack and my roommate’s dog Casey. Jack has, by this point, become inured to the idea that occasionally I am going to grab his muzzle and stick a sponge on a stick inside his lip and swab around for two minutes. He was ready for bed when I did it last night and decided he might as well just doze through it.

Casey is another matter. When I cornered him and grabbed his muzzle, he freaked out.


Me: Casey, I have done this to you five times. Why do you always act like it is going to hurt?


Me: I have done it to myself. I know it doesn’t hurt.


Me: I have done it to about 30 dogs and it didn’t seem to hurt any of them much.

Casey: Oh.

Most dogs are annoyed when I try to insert the swab, but then discover it doesn’t hurt and just deal with it. Casey inevitably squeezes his eyes shut, hyperventilates, and sometimes even collapses to the ground. (You might at this point ask “how do you know there isn’t something special about Casey?”, to which I would reply that, while there are certainly many special things about Casey, he also reacts this way to having his nails clipped, so I am pretty sure he is not experiencing any unusual physical sensations when I collect his saliva.)

I imagined Casey asking me “Why are you doing this to me?” and what my answer might be: “To find out how stressed you are.” As bizarre as that might be from a dog’s perspective, in fact my plan was to analyze his saliva to find out how much cortisol was in it. Cortisol has been called the “stress hormone,” and is an indicator of how much stress an animal is under.

Because I’ve never worked in a lab before, I wanted to do a few sample assays with unimportant data (i.e., saliva from my own dog that is easy to collect) before using the irreplaceable data collected from hospital dogs. It has taken me four months to successfully enroll 24 dogs; if I waste those samples, I am SOL. (Six dogs a month? Well, I’m a lot better at it than I used to be, and I had to take some time off in the middle. I am actually averaging 3-4 dogs a week at this point.) I performed my test assay this morning.

The cortisol assay is a kit costing a couple of hundred dollars which I purchased from the ever-helpful Salimetrics. It is an ELISA, or enzyme-linked immunoabsorbent, assay. The main equipment in the kit is a little plate (I was surprised by how tiny it was) made up of 96 wells, into each of which one might pour a very small amount of liquid. Each of the wells has some cortisol antibodies in the bottom (or so I’m told, since obviously antibodies are too small to see). These will grab on to any cortisol they see and hold on to it.

When I got into the lab this morning, I took the kit out of the fridge, and some older samples out of the freezer, so that they could warm up to room temperature. Then, while last night’s samples from Casey and Jack centrifuged, I sat down with the lab tech’s computer and figured out where everything was going to go on the plate. Aside from all my samples, I had to have room for the standards, which are samples given to you with the kit containing a known amount of cortisol. If your assay doesn’t tell you that the well with the 3.000 standard has about 3.000 µg/dL of cortisol in it, you know you’ve done something wrong. There are also the controls, which contain an arbitrary “high” and “low” amount of cortisol, and are also useful for telling you when you’ve messed up.

Then there are the “zero” and “blank” wells. The zero wells contain nothing but the diluent, or the liquid used to dilute some of the chemical agents in the kit. When the calculations are done at the end, you need to have this well to be able to tell the difference between a real effect and an effect caused by the diluent. And, finally, there are the blank wells, which don’t have any cortisol binding antibodies in them, and therefore will behave differently than the zero wells when the plate is read.

For me, the hardest part of the process is making sure that everything goes where it’s supposed to. This is more complicated than it sounds, involving lots of finding the right well in a small plate that is packed with small wells; remembering to leave space for a sample that I’m going to add later, after it has been diluted; getting the standards and controls in the right places; etc. Doing something that doesn’t require a lot of brainpower but does require a lot of precision, over and over and over, is hard for me.

When all the standards, controls, and samples were in place, and diluent was in the zero wells and the blank wells, I added conjugate to everything. The “conjugate” is cortisol which is attached to an enzyme. Of course, the whole point of the exercise is that you are putting in your saliva samples which contain cortisol, and now you’re adding this new source of cortisol as well. The two sources of cortisol are going to compete for the chance to bind to the cortisol-binding antibodies in the bottom of each well. When there is more cortisol in the sample, less of the conjugated cortisol (with its attached enzyme) can bind, and vice versa.

I put the plate with its samples and conjugate on to a rotator. This is a small device with a flat top which basically waves the plate around so that its contents mix. Every time I use it I am terrified that the plate is going to go flying off and spread my samples across the lab floor. It has not happened yet.

After 55 minutes for the whole competition thing to happen, I washed the plate off (four times), which includes blotting: picking the plate up, turning it upside down, and slamming it on to an absorbent pad. This is also a scary procedure, especially as sometimes strips of wells break off and you have to figure out in which direction to reattach them. Attach them upside-down, and you won’t know which sample is which.

At this point, in theory, the wells were mostly full of bound cortisol. Some of the cortisol, which came from the conjugate I added, had enzyme bound to it. Some of the cortisol, which came from my samples, did not. The wells which contained samples high in cortisol had less of the bound enzyme, and the wells which contained samples low in cortisol had more of the bound enzyme. Next I added TMB (tetramethylbenzidine), which reacts with the bound enzyme to change its color. Obviously, the wells with more bound enzyme will change to a different color than the wells with less.

More plate rotating is next, and then a brief time (sequestered in the dark) for the plate to let the reaction run its course. When I took the plate out of its light-proof bag at the end of this waiting period, the wells were all full of a lovely blue liquid, and different wells were visibly different shades. I added stop solution to stop the reaction, which immediately changed the color to yellow. And then I put the plate in the plate reader, which is a small machine attached to a Windows computer on the lab tech’s desk. It made some thumping noises as it determined the optical density of each well — its color, expressed as a number. It relayed these numbers to the waiting computer, which did the calculations to convert the numbers into concentrations, and I exported the results into Excel.

So how did I do? This was actually my second attempt at doing this assay. Both times, the results were fairly close to what I expected, based on the standards. However, part of what I wanted to figure out today was whether I could dilute small samples and still calculate values which were close to undiluted values. (In other words, if I dilute one sample by 50%, and multiple the calculated concentration by 2, will the result be similar to what I get when I put an undiluted version of that sample in a different well?) My dilutions were way off, so that’s something I need to figure out. I’m hoping to find a lab on campus which does these assays frequently, and ask if I can work with them for a little while, to get some experience.

By the way, the above implies that I competently performed this entire operation on my own, which is completely untrue: I worked under the close guidance of the extremely talented hospital lab technician. I am very lucky that she enjoys teaching; she told me today that she once considered being a school teacher. Her patience and good nature made the day much more enjoyable than it might have been.

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