All
You Ever Wanted to Know about Radioimmunoassays
I. INTRODUCTION
Okay, it is time to get
serious about radioimmunoassays (RIAs)
and try to figure out what we have been doing the past several weeks. As you
know, you extracted prostaglandin E2 (PGE2), along with
progesterone (P4), from several ovaries and then performed a number
of other steps on the extracts. What we want to do here is help you understand
why you performed each of the additional steps in order to complete the
competitive-binding assay known as an RIA. The PGE2 RIA method that
we used is more typical than the P4 RIA, and therefore the following
description is based on the PGE2 method. First though, let's start
with a few definitions:
1. antigen:
The word antigen is derived from antibody generator.
So, we see quite obviously that an antigen is a molecule (usually a protein)
that generates an antibody. In our study, the antigen is PGE2.
However, you may recall that prostaglandins are derivatives of the fatty acid arachadonic acid. Therefore, you might wonder how a lipid
substance can generate an antibody. We do not need to worry about the details,
but they somehow couple the PGE2 to a specific carrier protein and
this complex acts as an antigen that can be injected into an animal such as a
rabbit to generate antibodies that will bind quite specifically to PGE2.
(You might occasionally find the term "ligand"
used in RIA reports, and this term is synonymous with the term antigen.
Literally, ligand is a Latin word meaning "that
which is bound".) So, in an RIA, an antigen such as PGE2 is the
target molecule that will be bound during this "binding assay". And,
the binding molecule in this case is antibody specific for the PGE2.
2. antibody: But,
what is an antibody? Well, as most of you know, generally it is a gamma
globulin protein that possesses a molecular configuration that allows it to
react with (i.e., bind to) an antigen. There is usually a high degree of
specificity in the antigen-antibody reaction. That is to say, a given antibody
will usually react with only one specific antigen. Note that, in some
instances, an antibody may also react with several homologous antigens. For
example, the antibody that you are using for PGE2 will also react
(albeit to a lesser extent) with PGE1. However, this antibody
exhibits negligible "cross-reactivity" with a dozen or so other
members of the prostaglandin family.
In the next sections,
we will discuss the components of a common RIA system in the context of the
several steps that you carried out last week. Also, we will consider the standard
curve that must always be carried out as a part of an RIA.
II. COMPONENTS OF
THE RIA SYSTEM
1. 100 mL
of Ovarian Extract: You will recall
that after you homogenized and centrifuged your ovary, you siphoned 800 ml
of the supernatant fluid into a blue-capped tube and diluted this extract with
800 ml of water. Then, you placed 100 ml
of this diluted extract into each of two tubes. Thus, these tubes contain the
aliquots of extract with an unknown amount of PGE2.
Your objective was to determine the amount of PGE2 in each of your
tubes.
2. 100 mL
of PGE2 Antibody: Next,
you pipetted 100 ml of PGE2
antibody (or, antiserum, if you wish to call it that, which was
harvested from rabbits) into the reaction tube along with the aliquot of
diluted extract. After vortexing, you incubated this
mixture for 24 hours at 4° C. This incubation allowed the unknown amount of antigen
and the fixed amount of antibody an ample amount of time to get to know one
another (i.e., to bind to one another).
3. 100 mL
of Radioactively-labeled PGE2:
Just as the unknown amount of PGE2 and the antibody were forming a
nice relationship, you came along and added a competitor for the
PGE2. To each of your tubes you added 100 ml
of radioactively-labeled PGE2 to the reaction mixture. Since the
binding of an antigen to an antibody is a reversible reaction, the
radioactively-labeled PGE2 begins competing with the unlabeled (and
unknown amount of) PGE2 for the binding sites on the antibody
molecule. Therefore, rather than "playing cupid", you left your
samples overnight so that the radioactively-labeled PGE2 and the
unlabeled PGE2 could fight it out for the binding sites on the
antibody. (Actually, by morning, they all came to an
equilibrium.)
4. 500 mL
of Second Antibody: In the next step
of the RIA, what you needed was a method of separating the antibody from the
rest of the components of the reaction mixture (including separation from any
unbound PGE2). This separation step was achieved in a surprisingly
simply way. You may recall from introductory biology that antibodies are
actually proteins. And, as we stated above, proteins have antigenic
properties. That is to say, antibodies are,
themselves, capable of generating antibodies. Thus, when an antibody is used
(as an antigen) to generate another antibody, this second antibody is called
just that--a second antibody. In the RIA system that we used, the
manufacturer injected the PGE2 antibody (originally generated
in rabbits) into goats. The goats' immune system responded by producing the
second antibody, which readily binds to the PGE2-antibody complex.
(This step was performed by your professor on the Wednesday after you prepared
your RIA mixture.)
5. Centrifugation
Step: Now, the important point to the antibody-antibody reaction
described in the previous step is that the resulting molecule is quite large.
In fact, it is so large that it precipitates out of solution. Consequently, if
we now centrifuge our reaction tube, we will draw down as a pellet the antibody
with essentially all of the bound PGE2 (regardless of whether it is
radioactively labeled, or not). (This step was also performed by your
professor.)
6. Decantation
Step: Next, getting rid of the unbound radioactive PGE2 is
quite simple. All we had to do was decant (i.e., pour off) the supernatant
fluid from the top of the pellet that was obtained during centrifugation of our
reaction tubes in the previous step of the procedure. (This step was also
performed by your professor.)
7. Understanding
the amount of radioactivity in the pellet: If you think about it a
little (in terms of a "competitive-binding assay") if there was a
relatively small amount of PGE2 in your ovarian extracts, then there
was not much competition for the radioactive PGE2 in its effort to
bind to the antibody. In other words, if you measured and found a relatively
large amount of radioactivity in the pellet, this means that your ovarian
extract contained very little PGE2. Conversely, if there was very
little radioactivity in the pellet, then their had to
have been a relatively large amount of PGE2 in your ovarian
extract(s). (In the next section of this synopsis, we will describe the
standard curve that was constructed in order for you to determine the actual
amount of PGE2 in each of your ovarian samples.)
III. THE STANDARD
CURVE
In order to determine
the actual amount of PGE2 in each of your ovarian extracts, it was
necessary to construct a standard curve. Several of us carried out this part of
the RIA. The procedure was exactly the same as for your ovarian extracts,
except that known amounts of PGE2 were placed in the
reaction mixture with the set amount of radioactive PGE2 and the
antibody. In this case, the standards contained 1000, 500, 100, 50, 25, 10, 5,
2.5 and 1.0 picograms of PGE2/0.1
milliliter. (Note that, according to the comments in the previous paragraph,
the standard tube with 1000 pg/ml would end up with a pellet containing the
least amount of radioactivity, while the tube with 1.0 pg/0.1 ml would have the
most radioactivity.) With this information, it was possible to construct a
standard curve in which the CPM (i.e., the counts per minute of radioactivity)
is plotted on one axis and the concentration of PGE2 (in pg/0.1 ml)
is plotted on the other axis. For our experiment, next Tuesday afternoon
the computer will actually plot the curve for you.
IV. OTHER
NECESSARY INFORMATION TO COMPLETE THE RIA
In order to complete
the RIA with accuracy, it is necessary to have several other tubes that provide
the computer with information that allow it to complete the calculations:
1. Total Count
(TC): First, there must be a tube (actually duplicate tubes) that
contains only the 100 mL of radioactive PGE2, and nothing else.
These tubes represent the absolute total amount of radioactivity that was
originally placed in each tube.
2. Reference:
(Bo): This tube contains the 100 mL of radioactive PGE2
plus 100 mL of the antibody. This tube is also commonly referred to
as the "maximum binding" tube, because it tells you the
maximum amount of binding that can occur between the radioactive PGE2
and the antibody when there is no competition from a non-radioactive source of
PGE2. When starting from "scratch", and working out all
the concentrations and amounts of reagents to carry out an RIA, this
"Reference" data is important because it lets you know whether you have
enough antibody in your reaction mixture. Generally
speaking, you want enough antibody in the mixture to
bind up 30-40% of the set amount of radioactive PGE2 (or any other ligand you are attempting to measure by RIA). When you
obtain the numerical data from the gamma counter on Tuesday, you can calculate
the % binding by dividing the Reference value by the TC value.
3. Non-Specific
Binding (NSB): Lastly, you need a control tube (or, "blank")
which will tell you (or, tell the computer that is actually doing the
calculations) to what extent the radioactive PGE2 is binding to the
walls of the reaction tube, or to any components of the reaction buffer that
might end up in the pellet. In other words, you must know the amount of non-specific
binding that might be occurring above and beyond the binding to the
antibody.
V. WHAT YOU WILL
DO IN LAB NEXT TUESDAY
Next Tuesday, you will
simply program the computer to tell it which of the tubes (that you will be
placing in the wells of the gamma counter) are your TC tubes, which are
your Reference tubes, which are your NSB tubes, which are your standards
(and the amounts of each standard), and which are your ovarian extracts that
contain unknown amounts of PGE2.
