• An immunoassay is a test that uses antibody and antigen
complexes as a means of generating a measurable result.
• An antibody:antigen complex is also known as an immuno-complex.
• “Immuno” refers to an immune response that causes the
body to generate antibodies, and “assay” refers to a test.
Thus, an immunoassay is a test that utilizes
immunocomplexing when antibodies and antigens are
• In competitive formats, unlabelled analyte (usually antigen) in the
test sample is measured by its ability to compete with labeled
antigen in the immunoassay.
• The unlabeled antigen blocks the ability of the labeled antigen to
bind because that binding site on the antibody is already
• Thus, in a competitive immunoassay, less label measured in the assay
means more of the unlabeled (test sample) antigen is present. The
amount of antigen in the test sample is inversely related to the amount
of label measured in the competitive format.
One step competitive format
• In the one step competitive format (see Figure 1-8), both the labeled
antigen reagent (Ag*) and the unlabeled specimen (or test sample
analyte) compete for a limited amount of antibody.
Two step competitive format
• In the two step competitive format, the antibody concentration of
the reaction solution is present in excess in comparison to the
concentration of antigen.
• Antibody reagent is first incubated with specimen containing
antigens of interest; then in the second step, labeled antigen is
• Remember that in the competitive format, less bound labeled
antigen indicates more antigen present in the test sample. assay
formats provide several fold improved assay sensitivity compared to
one step assay formats.
Noncompetitive (Sandwich) Method
• Noncompetitive assay formats generally provide the highest level of
assay sensitivity and specificity and are applied to the measurement of
critical analytes such as cardiac and hepatitis markers. This format is
referred to as a “sandwich” assay because analyte is bound
(sandwiched) between two highly specific antibody reagents.
• Noncompetitive assay formats can also utilize either one step or two
step methods, as with the competitive assay.
• The two step assay format employs wash steps in which the sandwich
binding complex is isolated and washed to remove excess unbound
labeled reagent and any other interfering substances.
• The two step noncompetitive format usually offers the highest
specificity and sensitivity of all the assay formats discussed here.
Homogeneous and Heterogeneous Immunoassay
• Immunoassay methods that require separation of bound Ab-Ag*
complex are referred to as heterogeneous immunoassays. Those that do
not require separation are referred to as homogeneous immunoassays.
• Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and therapeutic drugs.
Since homogeneous methods do not require the separation of the bound
Ab-Ag* from the free Ag*, they are generally much easier and faster
Non specific adsorbents Low molecular weight ligands are adsorbed
by particles such as charcoal and removed by
Specific adorbents Antibodies to the ligand or to the ligand
binding antibody are immobilized on the
surface of a Solid matrix such as glass
fibres,latex micro particles.
The immobilized antibody ligand complex are
separated from unbound ligand by
chromatography The protein bound ligand moves at a rate
through the chromatographic medium
different from that of free ligand
Precipitation by ammonium sulfate The antibody bound ligand is ppt. by
• It can measure both small and large molecules.
• Heterogeneous immunoassay such as the popular EMIT assay
on the other hand measures only small molecules such as drugs.
As [Test antigen] is
increased it occupies more
of the antibody molecules,
so fewer of the enzyme
labels are blocked.
Substrate ---> product
• The ELISA (Enzyme-Linked ImmunoSorbent Assay) can be
used both qualitatively and quantitatively to measure antigen-antibody
binding. Depending on what variation you use, it will
detect antigen (hormones, enzymes, microbial antigens, illicit
drugs) or antibody (anti-HIV in the screening test for HIV
infection) in body fluids or tissue culture supernatents.
• Purified antigen (to detect or quantify antibody).
• Purified antibody (detect or quantify antigen).
• Standard solutions (positive and negative controls).
• Sample to be tested.
• Microtiter dishes: plastic trays with small wells in which the
assay is done.
• Wash fluid (buffer).
• Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative
To detect antibody
• Coat the microtiter plate with purified antigen by letting an
antigen solution sit in the wells for 30-60 minutes. Wash
away unbound antigen with buffer and cover any sites that
might nonspecifically bind antibody with unrelated protein
(such as solution of powdered milk), again washing away
• Add serum sample to be tested for specific antibody to plate
and allow specific antibody to bind to the antigen. Wash off
• Add anti-Ig that will bind to Fc region of specific antibody (for
example, anti-human gamma chain that will bind human IgG).
The Fc region of the anti-Ig is covalently linked with enzyme.
Wash off unbound antibody-enzyme complex.
• Add chromogenic substrate: colorless substrate that the
enzyme will convert to a colored product. Incubate until color
develops; measure color in a spectrophotometer.
• The more color that is detected, the more specific antibody is
present in the unknown sample.
• Negative controls include
• omit the antigen
• omit the test antiserum or substitute with an antibody that
will not bind the antigen.
• Positive control substitutes known positive serum for unknown
TO DETECT ANTIGEN
• Coat the microtiter plate with purified antibody to the
antigen. Wash away unbound antibody and cover any sites
that might nonspecifically bind with unrelated protein.
• Add sample to be tested for antigen to plate and allow
antigen to bind antibody.Wash off unbound antigen.
• Add enzyme-labeled specific antibody to a different epitope
of the antigen to make a "sandwich"; wash away unbound
• Add chromogenic substrate for enzyme that will be
converted to a colored product.
• Negative control omits unknown antigen; positive control
uses known antigen.
How to interpret the results
• The amount of colored product is proportional to the amount
of enzyme-linked antibody that binds, which is directly
related to the amount of antibody that was present to bind
antigen or antigen that was present to bind antibody.
• If known amounts of antigen or antibody are added, a
standard curve can be constructed which will allow the
amount of unknown antigen or antibody to be determined.
• In fluorescence, a photon of an appropriate energy excites the
molecule from its ground state to a higher electronic state.
• When the molecule returns to the ground state, energy is
released as light emitted at a longer wavelength.
• The difference between the excitation wavelength and the
emission wavelength is the stocks shift.
• A large stokes shift in nanometer means that there is a large
difference between the excitation and emission wavelengths.
• For example, fluorescein, a common flurophore has small
stocks shift at 30 min. It has a maximal absorption at 490nm
and emission at 520 nm.
• In contrast a rare earth chelate such as europium has a large
stocks shift of 270 nm.
• It has a maximal absorption at 340 nm and an emission at
• In principle, a fluoroimmunoassay using flurometry has an
advantage over an enzyme immunoassay using colorimetry.
• In colorimetry, the light absorbed by a sample is related directly to
the concentration of the absorbing molecule and is independent of the
• In fluorometry the intensity of the fluorescence emission is directly
proportional to the intensity of the incident light.
• There are problems associated with fluorescence measurements.
1) Endogenous fluorophores , such as bilirubin and proteins, can
increase the non specific background fluorescence and reduce the
sensitivity of FIA.
2) Light scattering by high concentrations of protein, lipid and other
particles in serum will reduce the fluorescence signal.
3) The inner filter effect of hemoglobin and albumin will absorb
part of the excitation or emission beam.
4) Quenching duo to the non specific binding of albumin and
interaction with other specific quenching species may change the
quantum yield of the fluorescence.
• An important function of this step is the removal of
endogenous fluorescent compounds and interfering
substances from the sample prior to the detection step.
• This also allows large sample sizes to be used and improves
sensitivity as well as sensitivity.
• Solid phase FIA is a convenient approach either as
competitive FIA or sandwich immunometric assay.
Fluorescence Polarization Assay
• Fluorescence Polarization (FP) assays are homogeneous,
single-step assays ideally suited for high-throughput screening
(HTS) of large numbers of samples.
• All FP assays employ a large molecular species, or binding
partner (BP), in conjunction with a small, low molecular
weight fluorophore-labeled analyte (FA).
• When the large BP molecule is an antibody, the assay is
referred to as a fluorescence polarization immunoassay (FPIA).
• FPIAs are based on the competition of FA with free (i.e.
unlabeled) analyte in the samples or standards for the high
affinity binding site an antibody.
• A microplate well filled with the FA:antibody complex will
give a high FP reading.
• Addition of a increasing amounts of unlabeled analyte will
result in a competition between the unlabeled FA for the
• As the competition happens, some of the FA will be released
from the antibody, and will resume its intrinsic, rapid rate of
rotation. This will cause a detectable loss of FP in the well.
• The addition of large amount of analyte will result in a much
larger reduction in the mP of the well (well Plotting mP versus
analyte concentration allows the construction of a standard
curve with a broad dynamic range. This is similar to, but not
strictly analagous to, the sigmoidal dose-response curve in a
traditional solid phase EIA.
• A mixture is prepared of 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.
• Known amounts of unlabeled ("cold") antigen are added to
samples of the mixture. These compete for the binding sites of
• 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.
• The samples to be assayed
(the unknowns) are run in
• After determining the ratio of
bound to free antigen in each
unknown, the antigen
concentrations can be read
directly from the standard
• Immunoassays can be either competitive or noncompetitive.
• In competitive immunoassays, the amount of antigen is indirectly
proportional to the amount of signal.
• In noncompetitive immunoassays, the amount of antigen is directly
proportional to the amount of signal.
• Homogeneous immunoassays do not require separation of
unbound complexes from the bound complexes, and thus are
faster and easier to perform then heterogeneous immunoassays.
• Heterogeneous immunoassays require the separation of
unbound complexes, often utilizing a solid phase reagent such
as a magnetic particle or plastic bead.
• Wild, David (Ed.). (2005).The Immunoassay Handbook.
Kidlington, Oxford: Elsevier.
• Evans, Susan (2004, June 15). Retrieved January 19, 2008,
from SACB OnlineWeb site: http://sacb.org.sg/
• Bell, Suzanne (2006). Forensic Chemistry. Upper Saddle
River, New Jersey: Pearson Prentice Hall.
• Moody, David E. (2006).Immunoassay in Forensic Toxicology.
Encyclopedia of Analytical Chemistry.