 Immunodiagnostic assays are procedures that utilize products of the immune response as integral

parts of the test. Basically, immunodiagnostic assays use antibodies generated either against a
single antigen or antigens associated with a specific analyte, pathogen, or disease condition.

 These antibodies are labeled with enzymes to aid in detection of their interaction with antigens in
biological samples to be tested

 Assay systems involving use of antigens, haptens, or antibodies labeled with an enzyme have y
been applied to the measurement of biological substances for diagnosis known as Enzyme
immunoassays (EIA)

 Labeled component of an antibody/antigen reaction binds to its complementary binding site. The
amount bound depends on the concentrations of the other components of the system and if one of
these is varied this causes a change in the distribution of the labeled component between the
bound and unbound fractions
 .
 Used in ELISA ,Enzyme Histochemistry, Enzyme cytochemistry, Western blotting
1. Specific and sensitive assays of wide applicability
2. Equipment required is relatively cheap and is widely available
3. Reagents are relatively cheap and have a long shelf-life
4. Manipulations are simple
5. Assays may be very rapid
6. A separation step may not be required
7. The variety of labels available may allow multiple,
simultaneous assays to be performed
8. Potential for automation
9. No radiation hazards
10. In general, the sensitivities of EIAs are high and many
antigens can be detected in the 1-10 gig/liter range. However, many EIAs are
not as sensitive as the corresponding RIAs.
The solid-phase methods used which includes :

(a)attachment by physical adsorption to polystyrene tubes, polystyrene

hemagglutination plates (Microtiter), glass microscope slides and cellulose-
ester filter disks (Millipore)

(b) covalent coupling to cellulose fibres and to polyacrylamide, dextran, and

agarose beads

(c) cross-linked protein.

(d) excess second antibody and solid-phase second antibody (in the
competitive EIA for antigen).
1. Competitive EIA for antigen- Labeled antigen competes with unlabeled antigen for
binding to a limited quantity of antibody. The antibody-bound antigen
 is separated from the free antigen by the use of solidphase antibody or a second antibody
with specificity for the first. The enzyme activity in either the bound or free fraction is
determined and related to the concentration of the unlabeled antigen

2) “Immunoenzymometric” assay for antigen- Antigen reacts with excess labeled

antibody and, after incubation, excess solid-phase antigen is added. The
solid-phase antigen reacts with the free labeled antibody remaining and, after separation of
the solid-phase, the enzyme activity associated with soluble antigen is measured and
related to the concentration of antigen

3)“Sandwich” EJA for antigen.-This procedure requires the antigen to have at least two
binding sites. Antigen reacts with excess solid-phase antibody, and after incubation
followed by washing, the bound antigen is treated with excess labeled antibody. After
further washing the bound label is assayed, and this provides a direct measure of the
amount of antigen present .
4) EIA for antibody-. Antibody binds to excess solid-phase antigen and, after
incubation followed by washing, labeled second antibody with specificity for
the first antibody is added. The bound label is assayed after further washing and
it provides a direct measurement of the amount of specific antibody present.

5) Homogeneous EIA for haptens- This type of EIA does not require
separation of free and bound label, because the assay depends on inhibition or
activation of the enzyme label by antibody binding.
Enzyme Labels
Enzymes make suitable labels because their catalytic properties allow them
to act as amplifiers, and many enzyme molecules can catalyze the formation of
more than 105 product molecules per minute
Horse-radish peroxidase and alkaline phosphatase have been the most
commonly used while lysozyme and glucose-6- phosphate dehydrogenase are
used in the Homogeneous EIAs that are commercially available.
A suitable cross-linking reaction should produce, in good yield, a stable conjugate
of enzyme and antibody, antigen, or hapten with adequate labeling and with
minimal impairment of enzyme activity and immunoreactivity.
The conjugates formed are usually separated from unreacted enzyme and
antigen or antibody by gel filtration.

1) One-step glutaraldehyde linkage - The dialdehyde, glutaraldehyde, can link

proteins through their free amino groups .
In this method, conjugates are made by simply mixing the enzyme and the second
protein in the presence of glutaraldehyde. These conjugates have a high molecular
weight and are heterogeneous
Self-linkage of antibodies in the reaction appears to be extensive
Most widely used
Two-step glutaraldehyde linkage –
Horse-radish peroxidase is unusual in that it normally reacts with only one
glutaraldehyde molecule and the second aldehyde group of the cross-linker
is unable to react with the same or other peroxidase molecules (94). This
feature is basic to the two-step glutaraldehyde method, and
it minimizes self-coupling of the second protein. The peroxidase is first
treated with glutaraldehyde, the excess of the latter is removed and the
“activated” peroxidase is allowed to react with the free amino groups of the
antigen or antibody.

Dimaleimide linkage -. Dimaleimide derivatives can cross-link proteins

by reacting with their suithydryl groups and this approach has been used to
label IgG with /3-galactosidase, which has several sulfhydryl
groups. IgG is reduced with 2-mercaptoethylamine and is treated with N,N’-
o-phenylenedimaleimide, excess reagent removed, and the dimaleimide-
containing IgG is allowed to react with 3-galactosidase.
Oxidation of saccharide residues and Schiff base formation - Horse-
radish peroxidase has several oligosaccharide groups, and their oxidation to
aldehyde groups that can react with amino groups is the basis of this method
of linkage. The peroxidase is first treated with 1-fluoro-2,4-dinitrobenzene to
block its free amino groups and prevent self-linking. The saccharides are
oxidized with sodium periodate, the “activated” peroxidase is then allowed to
react with the free amino groups of the second protein, and the Schiff bases
formed are reduced with sodium borohydride to give
stable linkages.

M-maleimidobenzoyl N-hydroxysuccinimide ester- was used in a similar

two-step reaction to couple insulin and /3-galactosidase (44). In the first step,
the amino groups of insulin are acetylated by the active ester to yield an
insulin carrying a maleimide group, and this group, in the second step, reacts
with the sulfhydryl groups of the enzyme.
ELISA- Enzyme Linked Immuno sorbent assay – developed by sceintist Engvall
and Perlman in 1981
It is most commonly used immuno diagnostic assay for detection of antigen or
antibody in biological samples.
Basic steps involoved in ELISA Assay are-
1.Coating: Polystyrene plate is treated with a
solution of either antigen or antibody
2. Blocking: An unrelated protein-based solution
is used to cover all unbound sites on the plate
3.Detection: Enzyme-conjugated antibody or
antigen binds specifically to the target antigen or
antibody
4.Read result: Substrate is added and the signal
produced by the enzyme- substrate reaction is
measured
Types of ELISA
1. Direct ELISA:
An antigen coated to a multiwell plate is detected by an antibody that has
been directly conjugated to an enzyme. This can also be reversed, with an
antibody coated to the plate and a labeled antigen used for detection, but the
second option is less common.

2) Indirect ELISA:
Antigen coated to a polystyrene multiwell plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is
applied. Next, an enzyme-labeled secondary antibody is bound to the first
antibody. The secondary antibody is usually an anti-species antibody and is
often polyclonal.
Sandwich ELISA:
Require the use of matched antibody pairs, where each antibody is specific for a
different, non-overlapping part (epitope) of the antigen molecule.

The first antibody, termed the capture antibody, is coated to the polystyrene plate.
Next, the analyte or sample solution is added to the well.

A second antibody layer, the detection antibody, follows this step in order to
measure the concentration of the analyte. If the detection antibody is conjugated to
an enzyme, then the assay is called a direct sandwich ELISA. If the detection
antibody is unlabeled, then a second detection antibody will be needed resulting in
an indirect sandwich ELISA
Competitive ELISA
1.Primary antibody (unlabeled) is incubated with sample antigen.
2.Antibody-antigen complexes are then added to 96-well plates which are pre-coated
with the same antigen.
3.Unbound antibody is removed by washing the plate. (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 that is specific to the primary antibody and conjugated with
an enzyme is added.
5.A substrate is added, and remaining enzymes elicit a chromogenic signal
For competitive ELISA, the higher the sample antigen concentration, the weaker the
eventual signal. The major advantage of a competitive ELISA is the ability to use
crude or impure samples and still selectively bind any antigen that may be present.
Avidin is a protein derived from both avians and amphibians that shows
considerable affinity for biotin, a co-factor that plays a role in multiple
eukaryotic biological processes.
Avidin and other biotin-binding proteins, including streptavidin and
NeutrAvidin Protein, have the ability to bind up to four biotin molecules.
The avidin-biotin system can be incorporated into virtually every
immunoassay, whereby an antibody is conjugated to biotin and then
detected with avidin or streptavidin conjugated to variety of enzymes.
This makes biotinylated antibodies advantageous to signal amplification
and increased sensitivity
To detect and quantify proteins in complex mixtures.
Western blotting starts with gel electrophoresis of the sample. Once the
proteins are separated in the gel, they are transferred or blotted to a
membrane via electrophoresis.
The gel and membrane are sandwiched between blotting papers soaked
in buffer, and the set is compressed between two parallel electrodes in a
cassette. Current is passed at right angles to the gel, causing the proteins
in the gel to migrate towards the membrane. 
 The detection step usually involves probing the blot using an antibody to
detect a specific protein.
.The blot is next incubated in a dilution of antiserum (the primary
antibody), that is directed against the protein of interest. After several
washing steps, the blot is incubated with a labeled secondary antibody,
which is directed against the species that provided the primary antibody.
The Enzyme label on the secondary antibody provides a way to visualize
the interaction of the secondary antibody with the primary antibody on the
blot
Immunohistochemistry (IHC) is the most common
application of immunostaining.
It involves the process of selectively
identifying antigens(proteins) in cells of a tissue
section by exploiting the principle of antibodies binding
specifically to antigens in biological tissues
Visualising an antibody-antigen interaction can be
accomplished  by an antibody is conjugated to an
enzyme, such as peroxidase, that can catalyse a
colour-producing reaction (immunoperoxidase
staining).
localization of Diagnostic  biomarkers antigens and
differentially expressed proteins in different parts of a
biological tissue.Cancer antigen detction
Two Methods – Direct method
Indirect method
Molecules that have been measured include macromolecular
hormones, other serum proteins, bacterial toxins, and steroids. Most of the
assays have been applied to serum, but there also are assays for
components of cerebrospinal fluid and urine
The EIA for antibodies may also be used to measure antigens and
haptens.
EIAs may also be used to identify antigenic determinants.e.g investigation
of the source of the tumor-associated antigenic determinants of
carcinoembryonic antigen
EIAs for Antibodies Antibodies against macromolecules, viruses, bacterial
products, and eukaryotic parasites have been identified.
Determining class & subclass of antibody- IgG, IgA, and 1gM
determination of antibody avidity by measuring the binding rate of the
antibody to antigen immobilized.
Enzyme-labeled antigens and antibodies have been used for the specific
staining of cells and tissues for light and electron microscopy.