so, how would you analyze for something like this this is tetrahydrocannabinol
or something like this... the primary metabolite of cocaine is benzoylecgonine.. this is benzoylecgonine
or how about this one ... this is lysergic acid diethyl amide or LSD
or this is a little bit out of the realm of what we think of as analytical chemistry
but is a very important one..How would we analyze for this guy this is the HIV virus
this one looks like a picture of the goat it is a picture of a goat,
but I'm not changing subject.
If this goat is subjected to THC, for example, the metabolism of the goat will produce antibodies against the THC
but the goats body will recognize this as a foreign substance and built an antibody against it
we can use this antibody as a basis for analysis for THC
the antibody is.. it's like the glove and the hand scenario or the lock and key
the antibody conforms to the LSD (THC) according to it's shape
it's a protein substance and it fits the THC like a hand fits into a glove
analytical chemistry is usually ....maybe prep the sample somehow and then just do it
an immunoassay is kind of this drama that goes on
it's like a play and it has these characters
let me introduce the characters to you. The first character is the antibody
the antibody comes from the goat.. it's a proteins structure it has a specific shape
the second character is the THC remember now we're trying to analyze for THC in the specimen
it is typically urine and so we're trying to analyze for THC the THC fits into a slot in the antibody
like a key fits into a lock
there's a third character... character number one is antibody ....character number two
this is a play.... character number two is the THC ...the unknown variable in sample
character three is.....I want to call it an "antibody/THC complex"
it's a THC complex ...it's THC on one side... they say it's analog of THC on one side
but it's got something else attached to it which in this case is an enzyme
the antibody will react with the THC complex or the antibody will react with THC
So, how we do the analysis? well...
we take the antibody from the goat, the THC complex... which we've manufactured
and when they're mixed together, the THC complex will combine with the antibody
and it's a pretty stable material so that's fine
so we put a sample from the patient with the unknown THC in it in a reaction vessel
and we add to it, the antibody THC complex
if the patient ...if the sample ...has THC, the THC competitively binds with the antibody
this is a common theme in immunoassays
the THC basically kicks this.. this guy... out leaves him free and the antibody takes over
it competitively binds, its like the THC combines stronger with the antibody than the THC complex
and the result of this is that the amount of THC (in the,.. in the compound the amount of THC)
in the sample is now related to the amount of this complex floating around in the reaction vessel
there's a fourth character... let me introduce the fourth character to you
the fourth character is this red guy the red guy is a compound that will break down with this enzyme
and form a blue guy so the enzyme on the THC complex breaks the red guy down into a blue guy
we can detect the blue guy spectrophotometrically.
the reaction cell is spectrophotometric cell
so (to repeat) the THC complex is added to the sample THC antibody complex is added to the sample
if there's THC the THC displaces this complex and these guys become free
so now the presence of this enzyme is indicative of THC in the original sample
this enzyme changes this red guy into a blue guy and we can see it spectrophotometrically
I'm looking at this reaction and I'm thinking that this is a kinetic situation
the red enzymaticly changed to blue is a kinetic situation so the timing will be very critical
this analysis is done with a robot .... complex instrumental analysis using swinging arms
and timing and computers and every thing is with a robot robots are good at timing
in analytical chemistry, in general, we're not real concerned about the specifics of the situation
but it's got to be reproducible... robots are good at
that so how does the quantitation work? well it's just like any other analytical chemistry application
you run the standards..the standards give you a response
from the standards and the response you make a line and then you run unknowns...
everything under the same circumstances and, under the same circumstances,
the response of the samples against the line will give you a quantitation
is a little bit different in chromatography because in chromatography
you will always have a zero--zero.........zero response is zero quantitation
in an immunoassay you really don't have a good zero point so you have to run at least two points
it's common to run five points but you can't even pretend to do it without two
The antibody is specific to the THC according to its shape
so, what if the molecule comes along that is the same shape as THC but is not THC ?
if that happens then this analysis will give a false positive they call it in the business 'a false positive'
it will say "there's THC in this" when in fact there really isn't any THC at all
when the USDA approved this 'screen' in the 1970s they said "ehhhh..we really don't like this
but if you want to do this that's fine but if you ever find a positive sample you have to confirm it with a
secondary test that's more definitive"
that secondary tests is GC mass spec which is another story
so, this is a screen.. it is automated ...there's thousands and thousands probably done every day
in America for a lot of different things
I want to go back to take another look at the goat
we inject the goat with THC or maybe the goat goes out behind the barn with his friends.. I don't know
but anyway, if the goat is subjected to THC , the goat makes antibodies for THC
how does it do that? how does it know? the obvious answer, I think
would be that there is, like, a genetic trigger The THC finds the THC promoter
the THC promoter says "transcribe the THC antibody piece of RNA"
the THC RNA is translated into the THC peptide the peptide folds into an antibody
and that is the end of the story
but that's not the way it works
I don't think anybody really understands it very well .... I certainly don't
but the goat ....it's like it customizes every antibody for the job
so it looks as the THC .....figures out its shape and makes an antibody for it
the significance of this is that the goat can make a antibody for anything
not anything anything but (the goat) can make an antibody for a very large number of things
which means that we can make an immunoassay for a very large number of things with this same technology
they're expensive to develop but there's ..really ..really .. there's limits of what you could do
but there certainly a wide variety of things you can do with this
and then the last point is that I think the take-home lesson is that this is a biological specificity
in analytical chemistry we're always looking at the physical characteristics
of what it is we're looking at
does it have a color? can it react it with something? What's its boiling point?
in an immunoassay you're looking at a biological specificity I think it's cool!
I think it is cool!