Further notes on RIAs
In radio immunoassay (RIA), a fixed concentration of I125-labelled drug (tracer antigen) and
unlabelled drug (analyte antigen) compete for a limited number of antigen binding sites on the
antibody. The amount of tracer antigen that binds to antibody decreases as the concentration of
analyte antigen increases. The amount of analyte antigen can be determined, after separating
antibody-bound tracer from free tracer, by measuring the radioactivity of either the bound fraction,
the free fraction or both and by comparing the result against a calibration curve obtained for
increasing amounts of known analyte antigen. The method relies on accurate measurement of
Radioactive materials are unstable and emit energetic particles and rays from their nuclei. I125 in the
labelled antigen emits gamma rays (photons). The number of nuclei that decay each second is called
the activity and is measured in units of Curies (Ci). One microcurie equals 3.7 x 104 disintegrations
per second (DPSs) and 2.22 x 106 disintegrations per minute (DPMs). Photons emitted from I125 are
detected using a solid sodium iodide crystal gamma counter (scintillator), which gives off a flash of
yellow light when it absorbs gamma radiation and a signal pulse is produced by an optically
coupled photomultiplier every time it detects scintillation. Each signal pulse characteristic of the
I125 isotope is amplified and counted.
In the case where the radioactive count of the precipitated solids (the bound antigen-antibody
conjugate) in the RIA system is measured, five configurations of labelled antigen-analyte antigen-
antibody are examined in the assay:
Maximum radioactive count using maximum binding tubes
Total radioactive count using total count tubes
Background radioactive count using non-specific binding tubes
Calibration radioactive counts using calibration assay tubes
Analyte radioactivity counts for tubes containing the “unknown”
100%, or Maximum Binding Radioactive Count
The maximum binding count is measured for tubes to which tracer antigen and limiting antibody
but no analyte antigen have been added and after free tracer has been removed from the tubes.
These tubes containing the antigen-antibody mixture are called 100% tubes and will exhibit the
highest possible CPM because no analyte antibody was present to compete for binding sites on the
Total Radioactive Count
The total radioactive count is measured for tubes to which tracer antigen, analyte antigen and
limiting antibody have been added but where, after antigen sites on antibody have bound to
antibody, no attempt is made to separate bound from free tracer antigen. These tubes containing the
antigen-antibody mixture are called total-count tubes and the measured count therefore represents
the total amount of radioactivity added in an RIA tube. This assay produces the highest possible
radioactive DPM and is used as a quality-control standard for comparison against tubes entirely free
from analyte antigen (the 100% tubes).
Since the amount of antibody is limited and tracer is added in excess, total count tubes are used to
precisely demonstrate this excess, which is expressed by:
Bound Count (100% tubes) x 100
Excess tracer (%) = .
Total Count (Total count tubes)
Non-Specific Background Radioactive Count
The background count is measured for tubes to which tracer antigen but no antibody has been added
and after the tracer antigen has been removed. These tubes are called non-specific binding tubes or
background tubes. The assay produces the lowest CPM's in an RIA and the count is subtracted from
the CPM for all other tubes counted.
Calibration Radioactive Count
The calibration radioactive count, also called a reference or standard or known count is measured
for tubes to which tracer antigen, antibody and a precisely known concentration of a standard
sample of the analyte antigen have been added and after free tracer has been removed. This assay
produces a radioactive count that falls between the background and the 100% binding counts and
generates a calibration curve against which comparison of the “unknown” is made. RIA calibration
curves are typically obtained for between five and seven different concentrations of standard
unlabelled antigen, the measurements being made at least in duplicate for each concentration.
“Unknown” Radioactive Count
The radioactive count for the “unknown” is measured for tubes to which have been added a known
volume of the unknown analyte antigen, the tracer antigen and antibody and after free tracer has
been removed. This assay produces a radioactive count that again falls between the background and
the 100% binding counts and it is compared against the calibration curve so that the concentration
of “unknown” in the sample can be determined. Unknowns include samples submitted in blood,
urine and saliva, faeces and in some cases cerebrospinal fluid.
The RIA can be carried out in solution or on a solid support. The following discussion assumes an
homogeneous, double-antibody approach in which the tracer antigen, analyte antigen and limiting
antibody are all initially mixed in a buffer solution containing EDTA. After incubation, the bound
antigen-antibody conjugate is precipitated either by the addition of a second antibody (which binds
to the immunoglobulin protein of the first antibody), or a mixture of polyethylene glycol and second
antibody, and the precipitate, obtained as a pellet using a centrifuge, is washed free from unbound
species. The radioactive count of the pellet or, if the pelleted conjugate is re-dissolved, of the
solution is measured.
Concentration of first antibody in the RIA
The storage concentration of the first antibody is the concentration at which the 1st antibody is kept
in the freezer and is most often expressed as a ratio between a ml of whole undiluted antibody and b
ml of assay buffer. For example, if 1 ml of whole first antibody were added to 399 ml of assay
buffer, the storage concentration of first antibody is 1:400.
In practice, aliquots (450 l) of the freezer-stored solution are made so that one aliquot taken from
the freezer is of sufficient strength when diluted, to conduct one assay. The use of single-assay
aliquots avoids the need to thaw and refreeze bulk solutions, which if repeated a number of times
would reduce the immuno-potency of the first antibody.
The working concentration of the first antibody is the concentration of antibody after mixing with
assay buffer. For example, when a 450µl aliquot of first antibody is thawed and diluted with assay
buffer up to 67.5 ml, the working concentration of first antibody is 1:60,000.
The incubating concentration of the first antibody is the concentration of antibody in an RIA assay
tube containing assay buffer, sample (or standard) analyte antigen, EDTA, and tracer antigen. For
example, when 200 µl of the working solution (1:60,000) of first antibody is added to an assay tube
together with 400 µl of assay buffer, 100 µl of EDTA, 100 µl of analyte antigen solution, and 200
µl of tracer antigen, the incubating concentration of first antibody is 1:300,000.
Determining the required first antibody concentration
A preliminary set of RIAs is conducted to determine the concentration of first antibody needed to
best determine analyte antigen. In these preliminary RIAs, the radioactive count is measured in
triplicate for total count tubes (T), maximum-binding tubes (B) and non-specific background tubes
(N). The concentration of first antibody is varied in the maximum binding tubes and the amounts of
tracer antigen and second antibody are held constant in both background and maximum-binding
tubes. The plot below shows how the radioactive count varies with the working concentration of
The concentration selected for the analyte antigen depends on the sensitivity desired in the RIA.
The less of the first antibody used, the lower the standard curve doses can be reliably used.
Determining the concentration of second antibody in the assay
In a double-antibody RIA, a second set of preliminary immunoassays is conducted to determine the
minimum concentration of second antibody needed to completely precipitate all bound antigen-first
antibody conjugate. In these RIAs, the radioactive count is again measured in triplicate for total
count tubes, maximum-binding tubes and non-specific background tubes. In this assay, the amount
of tracer antigen and first antibody are held constant while the concentration of second antibody is
varied in the background tubes and maximum-binding tubes.
With both the required first antibody and second antibody concentrations having been determined,
the analyst proceeds to determination of the amount of analyte antigen (unknown) in the sample.
However, the analyst must be aware of sources of error which can, and do, effect the validity of the
results. Errors can arise through:
Inter-assay drift Specific activity Cross reaction
Inter-assay drift (also called end of assay variation) refers to the situation where RIA tubes known
to contain identical concentrations of the various components give different radioactive counts. The
phenomenon is generally observed because of the time difference between the first and the last
tubes in an assay. To determine the extent of drift in the analysis, the first and final triplicate assays
are usually duplicated.
Errors in specific activity of the tracer antigen arise through variations in the degree of radio-
iodination of antigen with I125. Generally, the more I125 incorporated into the antigen the better,
although excessive radio-iodination can reduce the binding efficiency of labelled antigen to first
The occurrence of cross-reactions is observed when a substance other than the target analyte
antigen or tracer antigen either binds to antibody or interferes with the binding of antigen to
antibody. Interfering agents can arise in the buffer, tracer, second antibody or the analytical sample