The virtual laboratory: Radio-immunoassay of oestradiol
copyright © 1982 - 2006 David A Bender
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Measurement of hormones
Screening antisera and determining the antibody titre
Antiserum sensitivity
Antiserum specificity
Assay of oestradiol in plasma samples
In this program you will investigate a number of antisera raised against oestradiol, in order to determine which are likely to be useful for measurement of oestradiol in plasma samples.
Measurement of hormones
There are two problems in the measurement of hormones and their metabolites in plasma, saliva, urine and other biological samples which mean that conventional chemical assay methods are unlikely to provide adequate sensitivity or specificity:
The compounds to be analysed (the analytes) are present in extremely low concentrations, or the order of nmol or pmol /L. This means that an assay has to be sensitive enough to detect a few fmol of the analyte in a sample.
Many hormones with very different biological actions are chemically similar, raising problems of the specificity of the assay for the analyte in the presence of potentially interfering compounds.
The most widely used methods of hormone assay are ligand binding assays, in which the analyte is the ligand; the binding protein may be:
a naturally occurring binding protein - for example, the plasma cortisol binding globulin that transports cortisol in the bloodstream, or intrinsic factor, the vitamin B12 binding protein that is secreted in the gastric juice to permit absorption of the vitamin.
an antiserum raised against an immunogenic derivative of the analyte. Sometimes this may be a monoclonal antibody, but more commonly it is a polyclonal antiserum, containing a variety of different antibodies of varying specificity and sensitivity. Where the binding protein is an antiserum, the technique is known as immunoassay.
At low concentrations all or most of the analyte is bound to the protein; as the concentration of increases, so the protein becomes saturated and an increasing proportion is free rather than bound. The proportion of the analyte bound to the protein is determined using a very small amount of a labelled analyte as a tracer. This is often a radioactive tracer at high specific activity, but other labels, including fluorescent markers and enzymes covalently linked to the binding protein can also be used. When a radioactive tracer is used, the technique is known as radio-ligand binding assay, or (when the binding protein is an antiserum) radio-immunoassay.
In order to determine the proportion of the ligand bound to the protein, it is necessary to separate the protein-bound ligand from that which is free in solution. This can be achieved in a number of ways:
dialysis or ultrafiltration using a semi-permeable membrane that will permit the free ligand to pass through, but not that which is protein-bound
adsorption of the unbound ligand onto charcoal, activated alumina or some other suitable adsorbent
covalent binding of the protein to resin beads or the inside of the assay vessel, so that the unbound ligand can readily be washed off.
In this exercise you will investigate a number of antisera raised against oestradiol, to determine their sensitivity and specificity, then use one that you think will be useful to measure oestradiol in plasma samples.
The assay procedure
The
diluted antiserum is incubated overnight at 4C with varying concentrations of
oestradiol, using [2,4,6,7,16-triated] oestradiol as the tracer; the specific
activity of the tracer is 152 Ci /mmol.
A suspension of activated charcoal is then added to adsorb the unbound ligand, and the sample is centrifuged at 0C. The radioactivity in an aliquot of the supernatant (i.e .the protein-bound ligand) is then determined by liquid scintillation counting, and the results are shown as percentage of the radioactivity that is bound to protein.
Further details of the incubation conditions will be provided in the theory screens for each set of studies.
Screening antisera and determining the antibody titre
There are 8 antisera available. They have been raised in rabbits that were immunised with oestradiol covalently bound to serum albumin as the immunogen. Each animal will respond differently to the immunogen, and as a result some antisera will have a high titre (i.e. a high concentration of anti-oestrogen antibodies) and some will have a low titre. Obviously, it is desirable to have as high an antibody titre as possible, since this minimises the amount of antiserum that you have to use for each assay.
Determination of the antibody titre
The antibody titre is defined as the dilution of the antiserum that will bind 50% of the radioactivity when incubated with tracer alone (i.e. with no additional non-radioactive oestradiol). To determine the titre, you will incubate a range of dilutions of the antiserum with tracer, and plot the percentage bound against the dilution of the antiserum (on a semilogarithmic scale).
For future studies you should use a dilution of the antiserum that gives approximately 50% binding of the tracer, since this gives the best sensitivity for the assay.
Non-specific binding of tracer
Some of the antisera will contain proteins that bind the some of the tracer radioactive oestradiol non-specifically; this is useless if the aim is to use specific oestradiol binding as a means of measuring oestradiol in samples. It is easy to determine whether the binding is specific (i.e. to oestradiol binding sites) or non-specific (i.e. to sites that have a general affinity for lipophilic molecules), since if the binding is specific then the bound radioactive ligand will be displaced by an increasing concentration of oestradiol, while non-specifically bound radioactivity will not.
In the studies of antiserum dilution to determine the antibody titre of each antiserum, you will also carry out a series of incubations in the presence of a saturating amount (10 nmol) of non-radioactive oestradiol as well as the radioactive tracer. This should displace more or less all of the bound radioactivity if there is not much non-specific binding.
A good antiserum that is likely to be useful for radio-immunoassay will have a high titre, and low non-specific binding, as shown in the graph below. In this case the antibody titre (the dilution that gives 50% binding of tracer ligand) is around a dilution of 1:1000 (there are points at a dilution of 1:800 and 1:1600)
The graph below shows an antiserum that will not be useful. Although again it has an antibody titre around 1:1000, little of the radioactive tracer is displaced by the saturating non-radioactive oestradiol, showing that there is a great deal of non-specific binding.
Incubation conditions
For each of the antisera you will determine the percentage of radioactive oestradiol that is bound to protein as the antiserum is diluted over a range from 1:100 to 1:102,400.
All solutions are prepared in, and diluted with, phosphate buffered saline (pH 7.4) containing 1 g/L bovine serum albumin (PBS+BSA). The incubations at 4C overnight are prepared as:
100 µL tritiated oestradiol (50,000 dpm per assay)
100 µL PBS+BSA or 10 nmol oestradiol in 100 µL PBS+BSA
100 µL diluted antiserum
At the end of the incubation 200 µL of charcoal suspension in PBS+BSA is added and the mixture is centrifuged.
200 µL aliquots of the supernatant (= bound ligand) are used for liquid scintillation counting
Antiserum sensitivity
Two factors affect the sensitivity of an antiserum for use in radio-immunoassay:
The affinity of the antiserum for the ligand. This is usually expressed as the dissociation constant (Kdiss) of the antiserum-ligand complex
The concentration of ligand binding sites. This may be expressed in two different ways
As the antibody titre - the dilution of antiserum at which 50% of the tracer ligand is bound. You will determine (approximate) values for the antibody titre when you screen the antisera for their potential usefulness in radio-immunoassay.
As the maximum binding capacity (Bmax) in molar units; i.e. the molar concentration of binding sites in the solution.
For
the equilibrium between free [F] and bound [B] ligand, the dissociation constant
is defined by
Kdiss = (Bmax * [F]) / [B] = ((Bmax - [B]) * [F]) / [B]
This can be rearranged as:
[B] / [F] = (Bmax - [B]) / Kdiss
Plotting a graph of the ratio [B] / [F] against [B] gives a straight line with:
gradient = -1 / Kdiss
x intercept = Bmax
Hence, in the example shown on the right
Kdiss = 1.94 nmol /L
Bmax = 8.1 nmol /L
(This graph of the ratio [B] / [F] against [B] is known as the Scatchard plot; it was first described by Scatchard G. Annals of the New York Academy of Sciences 51: 660-72 1949.)
Incubation conditions
All solutions are prepared in, and diluted with, phosphate buffered saline (pH 7.4) containing 1 g/L bovine serum albumin (PBS+BSA). The incubations at 4C overnight are prepared as:
100 µL of oestradiol solution (a range of dilutions from 1280 to 40 fmol /mL)
100 µL tritiated oestradiol (50,000 dpm per assay)
100 µL diluted antiserum (using the dilution that you found from screening gave ~ 50% binding of the tracer)
At the end of the incubation 200 µL of charcoal suspension in PBS+BSA is added and the mixture is centrifuged.
200 µL aliquots of the supernatant (= bound ligand) are used for liquid scintillation counting
Antiserum specificity
Although
the antisera were raised against oestradiol covalently bound to serum albumin,
each antibody in the antiserum will be specific for a different epitope in the
oestradiol molecule (i.e., only a small region of the molecule). Other steroids
that are likely to be present in the plasma samples that you wish to assay may
also bind to one or more of the antibodies present, and so displace radioactive
oestradiol from protein binding. This will give a falsely high value for oestradiol.
The usual way of ensuring that the antiserum you plan to use has adequate specificity is to incubate with the tracer labelled ligand and a range of concentrations of not only the analyte of interest, but also other possibly interfering compounds that may be present in the samples you plan to analyse.
If we plan to measure oestradiol, then we have to be sure that our antiserum does not cross-react significantly with other physiological oestrogens that may be present in the plasma samples (e.g. oestrone and oestriol), as well as synthetic oestrogens that may be used on contraceptives or as menopausal hormone replacement therapy (e.g. ethynyl oestradiol).
In addition, a variety of non-steroidal compounds have oestrogenic activity because they have hydroxyl groups that (in the three dimensional structure) are in the same positions as those of oestradiol, so that they can bind to, and activate, oestrogen receptors. Many of these can also cross-react with anti-oestradiol antibodies. Some of these non-steroidal oestrogens are used therapeutically, others are found in a variety of plant foods - the phyto-oestrogens. An example of a non-steroidal oestrogen is equol.
In the graph below, the black line shows the binding of radioactive oestradiol with increasing amounts of non-radioactive oestradiol, and the coloured lines the effect of adding increasing amounts of the other steroids. This is an antiserum with good specificity; none of the other steroids that were added caused any significant displacement of bound radioactive oestradiol.
By contrast, the graph below shows the results for an antiserum that cross-reacts significantly with two of the other steroids added, so that it would not provide adequate specificity for use in measurement of oestradiol in plasma samples.
Incubation conditions
For each of the antisera that you think will be useful (based on your screening of the antisera), you will determine the percentage of radioactive oestradiol that is bound to protein in the presence of a range of concentrations of oestradiol, oestrone, oestriol, ethynyl oestradiol and equol.
All solutions are prepared in, and diluted with, phosphate buffered saline (pH 7.4) containing 1 g/L bovine serum albumin (PBS+BSA). The incubations at 4C overnight are prepared as:
100 µL of the steroid solution (a range of dilutions from 5120 to 10 fmol /mL)
100 µL tritiated oestradiol (50,000 dpm per assay)
100 µL diluted antiserum (using the dilution that you found from screening gave ~ 50% binding of the tracer)
At the end of the incubation 200 µL of charcoal suspension in PBS+BSA is added and the mixture is centrifuged.
200 µL aliquots of the supernatant (= bound ligand) are used for liquid scintillation counting
Assay of oestradiol in plasma samples
Having selected an antiserum that has adequate sensitivity and specificity, you are now ready to use that antiserum to determine the concentration of oestradiol in plasma samples. Using the dilution of your chosen antiserum that gives approximately 50% binding of the tracer radioactive oestradiol, you prepare a calibration curve by determining the percentage of label bound in the presence of a range of concentrations of oestradiol from 40 - 1280 fmol /mL. At the same time, you incubate the diluted antiserum with extracts of your plasma sample to determine the percentage of the tracer that is bound. You can then determine the amount of oestradiol in each sample by interpolation into the calibration curve.
The graph below shows the results of such an experiment; the red curve is drawn form the standard solutions of oestradiol (for clarity the points are not shown) and the blue points show the percent of label bound for 10 plasma samples; the amount of oestradiol in each can be read from the x axis.
In this example, several of the points (ringed in pencil) are in a shallow region of the curve, where a relatively large change in oestradiol has only a small effect on the percentage of tracer bound. The solution to this problem is to use a smaller volume of sample, or change the dilution of the antiserum used.
Incubation conditions
All solutions are prepared in, and diluted with, phosphate buffered saline (pH 7.4) containing 1 g/L bovine serum albumin (PBS+BSA). The incubations at 4C overnight are prepared as:
100 µL of the plasma extract or a standard solution of oestradiol in PBS+BSA
100 µL tritiated oestradiol (50,000 dpm per assay)
100 µL diluted antiserum (using the dilution that you found from screening gave ~ 50% binding of the tracer)
At the end of the incubation 200 µL of charcoal suspension in PBS+BSA is added and the mixture is centrifuged.
200 µL aliquots of the supernatant (= bound ligand) are used for liquid scintillation counting
The plasma extract is prepared as follows:
Between 0.5 - 2.0 mL of plasma is mixed with 50 µL of PBS+BSA containing 500 dpm radioactive oestradiol (at high specific activity) to permit calculation of recovery of oestradiol through the extraction procedure.
This is then extracted with 2 mL diethyl ether
The ether layer (containing steroids and other lipids) is evaporated to dryness, and the residue dissolved in 200 µL of PBS+BSA
The radioactivity in a 50 µL aliquot of this solution is determined by liquid scintillation counting to determine the recovery of oestradiol. At 100% recovery you would find 50 / 200 x 500 (=125) dpm in the sample. When you perform the assay, the percentage recovery for each sample is shown in a table. The apparent concentration of oestradiol in each sample should be corrected for the percentage recovery in that sample.