IMMUNOASSAY TESTS

DEFINITION
Immunoassays are chemical tests used to detect or quantify a specific substance, the
analyte, in a blood or body fluid sample, using an immunological reaction.
Immunoassays are highly sensitive and specific. Their high specificity results from
the use of antibodies and purified antigens as reagents. An antibody is a protein
(immunoglobulin) produced by B-lymphocytes (immune cells) in response to
stimulation by an antigen. Immunoassays measure the formation of antibody-
antigen complexes and detect them via an indicator reaction. High sensitivity is
achieved by using an indicator system (e.g., enzyme label) that results in
amplification of the measured product.

Immunoassays may be qualitative (positive or negative) or quantitative (amount

measured). An example of a qualitative assay is an immunoassay test for pregnancy.
Pregnancy tests detect the presence of human chorionic gonadotropin (hCG) in
urine or serum. Highly purified antibodies can detect pregnancy within two days of
fertilization. Quantitative immunoassays are performed by measuring the signal
produced by the indicator reaction. This same test for pregnancy can be made into a
quantitative assay of hCG by measuring the concentration of product formed.

PURPOSE
The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect)
an analyte. Immunoassay is the method of choice for measuring analytes normally
present at very low concentrations that cannot be determined accurately by other
less expensive tests. Common uses include measurement of drugs, hormones,
specific proteins, tumor markers, and markers of cardiac injury. Qualitative
immunoassays are often used to detect antigens on infectious agents and antibodies
that the body produces to fight them. For example, immunoassays are used to detect
antigens on Hemophilus, Cryptococcus, and Streptococcus organisms in the
cerebrospinal fluid (CSF) of meningitis patients. They are also used to detect
antigens associated with organisms that are difficult to culture, such as hepatitis B
virus and Chlamydia trichomatis. Immunoassays for antibodies produced in viral
hepatitis, HIV, and Lyme disease are commonly used to identify patients with these
diseases.
DESCRIPTION
There are several different methods used in immunoassay tests.

• IMMUNOPRECIPITATION. The simplest immunoassay method

measures the quantity of precipitate, which forms after the reagent antibody
(precipitin) has incubated with the sample and reacted with its respective
antigen to form an insoluble aggregate. Immunoprecipitation reactions may
be qualitative or quantitative.
• PARTICLE IMMUNOASSAYS. By linking several antibodies to the
particle, the particle is able to bind many antigen molecules simultaneously.
This greatly accelerates the speed of the visible reaction. This allows rapid
and sensitive detection of antibodies that are markers of such diseases, as
infectious mononucleosis and rheumatoid arthritis.
• IMMUNONEPHELOMETRY. The immediate union of antibody and
antigen forms immune complexes that are too small to precipitate. However,
these complexes will scatter incident light and can be measured using an
instrument called a nephelometer. The antigen concentration can be
determined within minutes of the reaction.
• RADIOIMMUNOASSAY (RIA) is a method employing radioactive
isotopes to label either the antigen or antibody. This isotope emits gamma
raysare, which are usually measured following removal of unbound (free)
radiolabel. The major advantages of RIA, compared with other
immunoassays, are higher sensitivity, easy signal detection, and well-
established, rapid assays. The major disadvantages are the health and safety
risks posed by the use of radiation and the time and expense associated with
maintaining a licensed radiation safety and disposal program. For this
reason, RIA has been largely replaced in routine clinical laboratory practice
by enzyme immunoassay.
• ENZYME (EIA) IMMUNOASSAY was developed as an alternative to
radioimmunoassay (RIA). These methods use an enzyme to label either the
antibody or antigen. The sensitivity of EIA approaches that for RIA, without
the danger posed by radioactive isotopes. One of the most widely used EIA
methods for detection of infectious diseases is the enzyme-linked
immunosorbent assay (ELISA).
• FLUORESCENT IMMUNOASSAY (FIA) refers to immunoassays
which utilize a fluorescent label or an enzyme label which acts on the
substrate to form a fluorescent product. Fluorescent measurements are
inherently more sensitive than colorimetric (spectrophotometric)
measurements. Therefore, FIA methods have greater analytical sensitivity
than EIA methods, which employ absorbance (optical density) measurement.
• CHEMILUMINESCENT IMMUNOASSAYS utilize a
chemiluminescent label. Chemiluminescent molecules produce light when
they are excited by chemical energy. These emissions are measured by a light
detector.
ASSAYS OF HORMONES AND RECEPTORS

HORMONAL ANALYSES. Most hormone assays performed today are of the

competitive-binding variety. For a competitive-binding assay to be of value it must
be practical and reliable.

RADIOIMMUNOASSAY
The RIA is the conventional prototype of a competitive-binding assay. There are
three fundamental components to the RIA - radioactive ("hot") hormone, unlabeled
("cold") hormone (standard or sample), and antibody. Radioisotopes of tritium (β
emitter) and iodine (high specific activity γ emitter) are incorporated into steroid
and protein (Tyr or His residues) hormones, respectively; this must be done without
significant damage to the immunoreactivity of the hormone. Tracer and standard or
unknown sample compete for a limited number of binding sites on the antibody.
Amounts of (excess) tracer and antibody for each reaction are held constant, while
quantities of standard hormone are increased step-wise. Reactions are allowed to
proceed to equilibrium, and free (unbound) hormone is segregated from antibody-
hormone complexes. Emission of energy from the bound complex is monitored by
radiation detection equipment. As content of standard is increased from 0 (ie., 100%
of antibody is bound by tracer), the amount of antibody-bound tracer declines
reciprocally - a standard curve is constructed from these data. Reaction tubes
containing sample in place of standard are assayed simultaneously. Estimates of
mass of hormone within a sample are interpolated from the standard curve (Figure
2-38).

Antibodies belong mainly to the gamma globulin (IgG) class of immunoglobulins.

Each Fab arm of the (bivalent) antibody can bind a molecule of ligand. Binding is
mediated by weak noncovalent forces (ionic interactions, hydrogen bonding,
hydrophobic attractions, van der Waals attraction); therefore, like that of enzyme-
substrate binding, the reaction is reversible.

Antisera can be generated by injecting purified hormone into a species of animal

that is capable of mounting an immunological reaction to that hormone (ie., do not
produce the hormone in a chemical form that is exactly similar). Some small
molecules (haptens) are not antigenic on their own (eg., steroid and peptide
hormones, prostaglandins) and must first be coupled (at a nonactive site) to an
immunogenic carrier (eg., albumin, keyhole limpet hemocyanin) before injection.

Even under the best of conditions of immunization, antisera can contain antibodies
(polyclonal) that cross-react with related substances - the development of technology
using monoclonal (homogenous) antibodies has helped in this respect. To obtain
monoclonal antibodies an animal (eg., mouse) is injected with purified antigen,
spleen cells capable of secreting a single type of antibody (clones) are screened and
isolated, and selected cells are fused with myeloma (immortal) cells to produce a
hybridoma. Cells maintained in culture provide a continuous source of antibody. A
single hybridoma can yield approximately 1000 specific molecules of antibody per
second.

A convenient method to separate antibody-hormone complexes from free hormone

is to adhere the antibody to a solid phase, such as to the walls of a test tube. The free
hormone can then be decanted (a centrifugation step is not required). Because
proteins attach nonspecifically to plastic (eg., polyvinyl chloride or polystyrene),
tubes can be coated by simply incubating with a solution containing antibody.
Remaining unoccupied sites are then filled with an irrelevant protein, such as serum
albumin or gelatin. One criticism of antibody-coated tubes is adsorption can mask
immunoreactive (Fab) sites: to overcome this problem, protein A, a molecule
derived from staphylococcus aureus that binds the Fc tail of IgG, can be coated to
the solid phase (this permits extraction of IgG from the fluid-phase reaction
mixture). Alternatively, precipitation of hormone-antibody complexes can be
achieved using ammonium sulfate, magnetically-activated antibody, or with a
second antibody generated against the first antibody (ie., anti-IgG). Adsorption of
free (low molecular weight ligand) can be achieved with dextran-coated charcoal.

Radioimmunoassay is a scientific method used to test hormone levels in the blood

without the need to use a bioassay. It involves mixing a radioactive antigen
(frequently labelled with isotopes of iodine attached to tyrosine) with antibody to
that antigen, then adding unlabeled or "cold" antigen in known quantities and
measuring the amount of labeled antigen displaced.

Initially, the radioactive antigen is bound to the antibodies. When cold antigen is
added, the two compete for antibody binding sites - and at higher concentrations of
cold antigen, more binds to the antibody, displacing the radioactive variant. The
bound antigens are separated from the unbound ones in solution and the
radioactivity of each used to plot a binding curve.

The technique is both extremely sensitive, and specific, if costly, and it is especially
useful in diagnosing and treating autoimmune diseases such as Hashimoto's
thyroiditis and Systemic Lupus Erythematosus.

The technique of radioimmunoassay has revolutionized research and clinical

practice in many areas, e.g.,

• blood banking
• diagnosis of allergies
• endocrinology
The technique was introduced in 1960 by Berson and Yalow as an assay for the
concentration of insulin in plasma. It represented the first time that hormone levels
in the blood could be detected by an in vitro assay.

THE TECHNIQUE
• A mixture is prepared of
o radioactive antigen
 Because of the ease with which iodine atoms can be introduced
into tyrosine residues in a protein, the radioactive isotopes 125I
or 131I are often used.
o antibodies against that antigen.
• Known amounts of unlabeled ("cold") antigen are added to samples of the
mixture. These compete for the binding sites of the antibodies.
• At increasing concentrations of unlabeled antigen, an increasing amount of
radioactive antigen is displaced from the antibody molecules.
• The antibody-bound antigen is separated from the free antigen in the
supernatant fluid, and
• the radioactivity of each is measured.
• From these data, a standard binding curve, like this one shown in red, can be
drawn.

• The samples to be assayed (the unknowns) are run in parallel.

• After determining the ratio of bound to free antigen in each unknown, the
antigen concentrations can be read directly from the standard curve (as
shown above).
SEPARATING BOUND FROM FREE ANTIGEN

There are several ways of doing this.

• Precipitate the antigen-antibody complexes by adding a "second" antibody

directed against the first. For example, if a rabbit IgG is used to bind the
antigen, the complex can be precipitated by adding an antirabbit-IgG
antiserum (e.g., raised by immunizing a goat with rabbit IgG). The antigen-
specific antibodies can be coupled to the inner walls of a test tube.After
incubation,
o the contents ("free") are removed;
o the tube is washed ("bound"), and
o the radioactive of both is measured.
• The antigen-specific antibodies can be coupled to particles, like Sephadex.
Centrifugation of the reaction mixture separates
o the bound counts (in the pellet) from
o the free counts in the supernatant fluid.
o Radioimmunoassay is widely-used because of its great sensitivity.
Using antibodies of high affinity (K0 = 108–1011 M−1), it is possible to
detect a few picograms (10−12 g) of antigen in the tube.

The greater the specificity of the antiserum, the greater the specificity of the assay.
The main drawbacks to radioimmunoassay are the expense and hazards if
preparing and handling the radioactive antigen.

• Both 125I or 131I emit gamma radiation that requires special counting
equipment;
• The body concentrates iodine atoms — radioactive or not — in the thyroid
gland where they are incorporated in thyroxine (T4).
• Despite these drawbacks, RIA has become a major tool in the clinical
laboratory where it is used to assay

• plasma levels of:

o most of our hormones;
o digitoxin or digoxin in patients receiving these drugs;
o certain abused drugs
• for the presence of hepatitis B surface antigen (HBsAg) in donated blood;
• anti-DNA antibodies in systemic lupus erythematosus (SLE).

In the RIA, IgG subclasses are quantified as immune complexes after binding of
radioactively labelled specific antibody. In case of a 'direct' technique, a
radioactively labelled anti-IgG subclass-specific antibody is used. In an 'indirect'
technique, an anti-IgG subclass-specific antibody directed against the first antibody.
Since working with radioactively labelled reagents requires special precautions and
is relatively costly, radio immuno assays have been largely replaced by enzyme-
linked immuno assays.
RADIOIMMUNOASSAY
ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA)

The Enzyme-Linked Immunosorbent Assay (ELISA or EIA for short) is a

biochemical technique used in immunology to detect the presence of an antibody or
an antigen in a sample. It utilizes two antibodies, one of which is specific to the
antigen and the other which is coupled to an enzyme. This second antigen gives the
assay its "enzyme-linked" name, and will cause a chromogenic or fluorogenic
substrate to produce a signal. Because the ELISA can be performed to evaluate
either the presence of antigen or the presence of antibody in a sample, it is a useful
tool both for determining serum antibody concentrations (such as with the HIV test
or West Nile Virus) and also for detecting the presence of antigen.

Because of its high sensitivity and specificity, this assay allows accurate
measurement of very low levels of IgG subclasses. In figure 10, a schematic outline
of the ELISA technique is shown. The sensitive ELISA comprises many incubation
and washing steps. Because of the need for high dilutions when measuring IgG
subclasses in sera, ELISA assays may be less reproducible in comparison with RID
and nephelometry. For measurement of IgG and its subclasses in large numbers of
samples, ELISA is increasingly being replaced by nephelometry. ELISA may be
advocated for measuring IgG subclasses in other body fluids than serum/plasma,
e.g. saliva, cerebrospinal fluid and broncho-alveolar lavage fluid.

Brief outline of the method (figure 10):

An ELISA is generally performed in wells of microtitre plates.
- Wells of the plates are coated with unlabelled monoclonal antihuman IgG subclass-
specific antibody and washed (figure 10A);
- Test samples, standard-and control sera are introduced in the respective wells and
incubated; The IgG subclass to be determined will bind to the solid phase and non-
bound IgG is removed by washing (figure 10B);
- Enzyme-labelled anti-human IgG antibodies are added to each well and non-
bound conjugate is removed by washing (figure 10C);
- Plates are incubated with substrate solution;
- After incubation, the coloured reaction product is measured photometrically
(figure 10D);

- The concentration of the IgG subclasses in the test samples is calculated relative to
the values of the calibration curve.
ELISA PROCEDURE

1.Indirect ELISA
2.Sandwich ELISA
3.Sompetitive binding ELISA

INDIRECT ELISA

The steps of the general, "indirect," ELISA for determining serum antibody
concentrations are:

1. Apply a sample of known antigen to a surface, often the well of a microtiter

plate. The antigen is fixed to the surface to render it immobile.
2. The plate wells or other surface are then coated with serum samples of
unknown antibody concentration, usually diluted in another species' serum.
The use of non-human serum prevents non-specific antibodies in the patient's
blood from binding to the antigen.
3. The plate is washed, so that unbound antibody is removed. After this wash,
only the antibody-antigen complexes remain attached to the well.
4. The second antibodies are added to the wells, which will bind to any
antibody-1 remaining. These second antibodies are coupled to the substrate-
modifying enzyme.
5. Wash the plate, so that excess unbound antibodies are removed.
6. Apply a substrate which is converted by the enzyme to elicit a chromogenic
or fluorescent signal.
7. View/quantify the result using a spectrophotometer or other optical device.

The enzyme acts as an amplifier: even if only few enzyme-linked antibodies remain
bound, the enzyme molecules will produce many signal molecules.

ELISA may be run in a qualitative or quantitative format. Qualitative results

provide a simple positive or negative result for a sample. The cutoff between positive
and negative is determined by the analyst and may be statistical. Two or three times
the standard deviation is often used to distinguish positive and negative samples. In
quantitative ELISA, the optical density or fluorescent units of the sample is
interpolated into a standard curve which is typically a serial dilution of the target.

A less-common variant of this technique, called "sandwich" ELISA, is used to detect

sample antigen.
SANDWICH ELISA ASSAYS
One of the most useful of the immunoassays is the two antibody
“sandwich” ELISA. This assay is used to determine the antigen
concentration in unknown samples. This ELISA is fast and accurate,
and if a purified antigen standard is available, the assay can determine
the absolute amount of antigen in an unknown sample. The sandwich
ELISA requires two antibodies that bind to epitopes that do not overlap
on the antigen. This can be accomplished with either two monoclonal
antibodies that recognize discrete sites or one batch of affinity-purified
polyclonal antibodies.

To utilize this assay, one antibody (the “capture” antibody) is purified

and bound to a solid phase typically attached to the bottom of a plate
well. Antigen is then added and allowed to complex with the bound
antibody. Unbound products are then removed with a wash, and a
labeled second antibody (the “detection” antibody) is allowed to bind to
the antigen, thus completing the “sandwich”. The assay is then
quantitated by measuring the amount of labeled second antibody bound
to the matrix, through the use of a colorimetric substrate. Major
advantages of this technique are that the antigen does not need to be
purified prior to use, and that these assays are very specific. However,
one disadvantage is that not all antibodies can be used. Monoclonal
antibody combinations must be qualified as “matched pairs”, meaning
that they can recognize separate epitopes on the antigen so they do not
hinder each other’s binding.

Unlike Western blots, which use precipitating substrates, ELISA

procedures utilize substrates that produce soluble products. Ideally the
enzyme substrates should be stable, safe and inexpensive. Popular
enzymes are those that convert a colorless substrate to a colored
product, e.g., pnitrophenylphosphate (pNPP), which is converted to the
yellow p-nitrophenol by alkaline phosphatase. Substrates used with
peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine
base (TMB), which yield green, orange and blue colors, respectively.
The   Sensitivity   of   the   Sandwich   ELISA   is   Dependent   on   Four 
Factors:

1. The number of molecules of the first antibody that are bound to the solid 
phase. 
2. The avidity of the first antibody for the antigen. 
3. The avidity of the second antibody for the antigen. 
4. The specific activity of the second antibody. 

The   amount   of   the   capture   antibody   that   is   bound   to   the   solid   phase   can   be 
adjusted easily by dilution or concentration of the antibody solution. The avidity of 
the   antibodies   for   the   antigen   can   only   be   altered   by   substitution   with   other 
antibodies.   The   specific   activity   of   the   second   antibody   is   determined   by   the 
number and type of labeled moieties it contains.

The steps are as follows:

1. Prepare a surface to which a known quantity of antibody is bound.
2. Apply the antigen-containing sample to the plate.
3. Wash the plate, so that unbound antigen is removed.
4. Apply the enzyme-linked antibodies which are also specific to the antigen.
5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
6. Apply a chemical which is converted by the enzyme into a fluorescent signal.
7. View the result: if it fluoresces, then the sample contained antigen.

COMPETITIVE ELISA ASSAYS
When two “matched pair” antibodies are not available for your target, another
option is the competitive ELISA. Another advantage to the competitive ELISA is
that non-purified primary antibodies may be used. Although there are several
different configurations for competitive ELISAs, below is an example for one such
configuration. In order to utilize a competitive ELISA, one reagent must be
conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may
be linked to either the immunogen or the primary antibody. The protocol below uses
a labeled immunogen as the competitor. Briefly, an unlabeled purified primary
antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody
is then incubated with unlabeled standards and unknowns. After this reaction is
allowed to go to equilibrium, conjugated immunogen is added. This conjugate will
bind to the primary antibody wherever its binding sites are not already occupied by
unlabeled immunogen. Thus, the more immunogen in the sample or standard, the
lower the amount of conjugated immunogen bound. The plate is then developed
with substrate and color change is measured
The steps for this ELISA are somewhat different than the first two examples:
 1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an antigen
coated well.
3. The plate is washed, so that unbound antibody is removed. (The more
antigen in the sample, the less antibody will be able to bind to the antigen in
the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added. This
second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker
the eventual signal

RADIO IMMUNOSORBENT TEST (RIST)

The antibody content of a patient’s serum can be assessed by the ability of

antibody to bind to Antiglobulin, which has been immobilized on a solid surface by
adsorption. The solid surface could be polycarbonate tube or nitrocellulose paper
discs. This test is usually done for the detection of IgE antibodies in severely allergic
patients.

Anti-IgE is raised in rabbits and used in labelled as well as unlabeled form.

Unlabeled anti-IgE is adsorbed on to a solid surface (polycarbonate tube) and to
this, patient’s serum to be tested for the presence of IgE is added and incubated.
After some time excess of serum is removed and the tube is washed with saline to
remove excess of serum proteins. To the washed tube, radiolabeled anti- IgE is
added. Radiolabeled anti IgE will bind to IgE, which has already complexed with
adsorbed anti- IgE. Geiger counter measures radioactivity in the tube and the
antibody quantity is detected.

A convenient method to separate antibody-hormone complexes from free hormone

is to adhere the antibody to a solid phase, such as to the walls of a test tube. The free
hormone can then be decanted (a centrifugation step is not required). Because
proteins attach nonspecifically to plastic (eg., polyvinyl chloride or polystyrene),
tubes can be coated by simply incubating with a solution containing antibody.
Remaining unoccupied sites are then filled with an irrelevant protein, such as serum
albumin or gelatin. One criticism of antibody-coated tubes is adsorption can mask
immunoreactive (Fab) sites: to overcome this problem, protein A, a molecule
derived from staphylococcus aureus that binds the Fc tail of IgG, can be coated to
the solid phase (this permits extraction of IgG from the fluid-phase reaction
mixture). Alternatively, precipitation of hormone-antibody complexes can be
achieved using ammonium sulfate, magnetically-activated antibody, or with a
second antibody generated against the first antibody (ie., anti-IgG). Adsorption of
free (low molecular weight ligand) can be achieved with dextran-coated charcoal.

Other analytical systems that exploit the same basic principle as the RIA
include the protein-binding assay, radioreceptor assay (RRA),
scintillation proximity assay (SPA), enzyme immunoassay (EIA),
fluoroimmunoassay (FIA), and chemiluminescent assay (CIA).

Protein-binding and radioreceptor assays are radioligand assays that utilize an

endogenous plasma protein (eg., for steroid hormones) or cellular receptor,
respectively - instead of an antibody. Protein-binding assays lack the specificity of an
immunoassay. The radioreceptor assay has an advantage over the RIA in that it only
detects bioactive hormone (ie., antibodies can interact with sites on the hormone
molecule not involved in receptor binding). Notwithstanding, it is difficult to isolate
abundant quantities of stable receptor for routine analyses. Fortunately, data
obtained from RIAs and RRAs are usually comparable.

RADIORECEPTOR ASSAY (RRA)

SCREENING FOR ANALYTES USING LABELED RECEPTORS. Introduction :-
Receptor binding assays have been commonly used for the assessment of the
pharmacological properties of New Chemical Entities (NCE). Due to the
introduction of combinatorial chemistry in the pharmaceutical industry, in an
attempt to find succesful drug candidates, an enormous increase in NCE requires a
concomitant demand for high through-put screening systems. Due to their specific
properties receptor assays have been considered valuable analytical tools for the
quantitation of highly potent drugs that exert their pharmacological action via a
receptor interaction.

The term receptor is exclusively used for proteins which can interact with
hormones, neurotransmitters and drugs or poisons yielding or blocking a
pharmacological response. Thus therefore antibodies, circulating or membrane-
bound proteins e. g. enzymes cannot be considered receptors even if they should
have ligand binding properties.

The principle of receptor binding assays is based on the competition between a

ligand and an analyte for binding to a certain receptor. After incubation of ligand,
analyte and receptor followed by separation of the receptor bound and the free
fraction of the ligand by either filtration, centrifugation or dialysis, subsequently
one or both resulting fractions are quantitated. The subsequently acquired data can
be used for assessment of the affinity of a NCE for the receptor or for quantitation
of a particular receptor binding analyte.
Up till now, all receptor assays have been construed around the use of a (radio)
labeled ligand. Furthermore generally receptors present in animal tissue or
cultivated cell lines have been used after having undergone only little purification.

Typical receptor densities in commonly used receptor assays range from 10-100
picomole per gram tissue. This subsequently implies that the amount of displaceble
labeled ligand in such assays is limited. The use of radioactive ligands in such cases
is notwithstanding this attractive because radioactivity can be detected with good
sensitivity and limited back-ground signals from such receptor material. Another
important reason in favor of use of radioactive labels is that development is easy
once a compound has been identified that binds to a particular receptor with high
affinity. Replacing 1-6 hydrogen atoms by the same number of tritium atoms yields
a product that has a receptor affinity similar to that of the unlabeled ligand.
However disadavantages such as the limited shelf-life and the problems encountered
with the use of radioactive tracers has stimulated the search for alternatively labeled
ligands. Another motivation for such search was also the expectation that alternative
labels might improve the sensitivity of the assay with regard to limits of
quantitation. Taken into consideration the physical half-life and the counting time of
each sample it can be calculated for tritium by way of example that only 1 out of
each million labeled molecules is detected.

Almost all approaches with non-radioactive labeled ligands have been with
fluorescent labels. The development of fluorescent ligands with a high receptor
affinity, if the ligands itself does not have sufficient native fluorescence, is quite
difficult and a compromise between affinity and fluorescence properties has always
been required. The aforementioned low receptor density implies that the maximal
signal is limited and lies close to the limitations of available instrumentation. High
amounts of receptor containing material needs to be used per assay in order to
sufficiently increase the signal.

Furthermore the currently used receptor containing materials in receptor assays

contain large amounts of non-receptor proteins which cause a high fluorescence
background.

Traditional receptor assays using radioactive or non-radioactive ligands require a

separation step enabling quantitation of bound and or free fractions of the labeled
ligand. Procedures used for the separation are dialysis, centrifugation and filtration.
The selection depends on available instrumentation and equilibrium dissociation
constants of the labeled ligand and of the analyte. It is a requirement that the
separation step may not alter the amount of receptor bound ligand.

A newly-developed methodology, SPA, does not require separation of

bound from free ligand. Competitive binding of labeled ligand in
proximity to antibody- or receptor-coated fluoromicrospheres allows the
energy emitted to excite the fluor and produce detectable light that can
be measured in a scintillation counter without liquid cocktail. Unbound
tracer is too far from the microsphere to enable energy transfer before it
is absorbed by the aqueous solution.

In the EIA, FIA ,and CIA, radioactive hormone is replaced by an

enzyme-, fluorescein- or luminol-tagged ligand, respectively.
Quantification is accomplished with a fluorometer in FIA and a
luminometer in CIA. In EIA an extra step is required first - addition of
substrate. An example of an enzyme commonly used in enzyme
immunoassays is horseradish peroxidase: hydrogen peroxide (substrate)
is reduced by this enzyme, and in the process an appropriate hydrogen
donor (eg., o-phenylenediamine) is oxidized, causing a change in color of
solution - appearance of product is measured by spectrophometric
analysis of color reactions (ie., absorbance) to graded concentrations of
hormone.

Antibody-excess immunoassays include the immunoradiometric assay

(IRMA) and enzyme-linked immunosorbent assay (ELISA). In the
IRMA cold ligand is "sandwiched" between an antibody coated to a
solid phase and a second radiolabeled antibody raised against a
different hormonal epitope (this works best with macromolecular
hormones); sensitivity is not mandated by competition, and therefore,
reactions can be carried out expeditiously over a wide range of
detection. In a sandwich ELISA, hormone is bound to an antibody
attached to a solid phase, and then an antibody-enzyme conjugate and
substrate are added (Figure 2-39). These methods engender a direct
relationship between radioactivity measured in the final complex and
concentration of standard or analyte (in contrast to the inverse
correlation between bound radioactivity and standard or sample
concentrations in an RIA).

Nonradioisotopic procedures, such as ELISAs, are becoming popular

because of lowered equipment costs, reduced hazard to users and the
environment (ie., associated with handling and disposal of
radionuclides), and can be adapted (subjective appraisal of color-
change) for in-the-home or on-the-farm/ranch diagnostics. However,
ELISAs tend to be less sensitive than the RIA (Table 2-8).
A reverse hemolytic plaque assay is used to detect secretion of hormone
from individual cells (eg., gonadotropes) contained within a
heterogeneous population. The concept is that a secretory product of a
cell can be measured by specific antibodies in the presence of
erythrocytes coated with protein A and added complement. Interaction
of hormone with binding sites on the antibody causes stearic alterations
in the antibody allowing for fixation of complement by juxtaposed Fc.
Complement-induced hemolysis leads to the formation of a clear zone of
erythrocyte membrane "ghosts" (ie., a plaque) surrounding the
secretory cell (Figure 2-40). The plaque technique is sensitive and areas
of lysis can be quantitated.

Receptor analyses. It is technically more difficult to monitor changes in

populations of hormonal receptors than to evaluate alterations in
patterns of secretion of hormones; yet, knowledge of dynamics of
cellular receptors is no less important (eg., in diseases of endocrine
resistance). The task of receptor measurement can be accomplished by
exposing a constant amount of receptor (eg., tissue homogenate) to
increasing concentrations of radioactive hormone. Receptor bound with
hormone is separated from free radiolabel and each fraction is counted -
the Scatchard plot is a common method of data assessment (Figure 2-
41). Receptors not occupied by hormones are generally characterized
unless special methods are first used to elute endogenous ligand from its
binding site.

PRECAUTIONS
Blood samples are collected by vein puncture with a needle. It is not necessary to
restrict fluids or food prior to collection. Blood should be collected in tubes
containing no additive. Risks of vein puncture include bruising of the skin or
bleeding into the skin. Random urine samples are acceptable for drug assays;
however, 24-hour urine samples are preferred for hormones and other substances
which show diurnal or pulse variation.

Special safety precautions must be observed when performing RIA methods.

Radioactive isotopes are used by RIA tests to label antigens or antibodies. Pregnant
females should not work in an area where RIA tests are being performed. Personnel
handling isotope reagents must wear badges which monitor their exposure to
radiation. Special sinks and waste disposal containers are required for disposal of
radioactive waste. The amount of radioisotope discarded must be documented for
both liquid and solid waste. Leakage or spills of radioactive reagents must be
measured for radioactivity; the amount of radiation and containment and disposal
processes must be documented.

NORMAL RESULTS
Immunoassays which are qualitative are reported as positive or
negative. Quantitative immunoassays are reported in mass units, along
with reference intervals (normal ranges) for the test. Normal ranges
may be age- and gender-dependent. Positive immunoassay test results
for HIV and drugs of abuse generally require confirmatory testing.

Although immunoassays are both highly sensitive and specific, false

positive and negative results may occur. False-negative results may be
caused by improper sample storage or treatment, reagent deterioration,
or improper washing technique.

False-positive results are sometimes seen in persons who have certain

antibodies, especially to mouse immunoglobulins (immune cells) that
may be used in the test. False-positive results have been reported for
samples containing small fibrin strands that adhere to the solid phase
matrix. False-positives may also be caused by substances in the blood or
urine that cross-react or bind to the antibody used in the test.

PREPARATION
Generally, no special instructions need be given to patients for immunoassay testing.
Some assays require a timed specimen collection, while others may have special
dietary restrictions.

AFTERCARE
When blood testing is used for the immunoassay, the vein puncture site will require
a bandage or light dressing to accomplish blood clotting.

RISKS
Immunoassay is an in vitro procedure, and is therefore not associated with
complications. When blood is collected, slight bleeding into the skin and subsequent
bruising may occur. The patient may become lightheaded or queasy from the sight
of blood.