2. To aid in the diagnosis of disease caused by infectious microorganisms, immunoassays have
been developed.
These biochemical and serological techniques are based on the detection and quantitation of
antibodies generated against an infectious agent, a microbe, or non-microbial antigen.
Because antibodies can be produced against any type of macromolecule, antibody-based
techniques are useful in identifying molecules in solution or in cells.
A blood sample is collected from the patient during the acute phase of the disease when
antibody levels are high.
Serum is then isolated and the concentration of antibodies is measured through various
methods.
Most assays rely on the formation of large immune complexes when an antibody binds to a
specific antigen which can be detected in solution or in gels.
Recent methods employ pure antibodies or antigens that have been immobilized on a platform
and that can be measured using an indicator molecule.
These methods provide high sensitivity and specificity and have become standard techniques in
diagnostic immunology.
3.
4. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is a solid-phase enzyme immunoassay used to
detect the presence of a substance in solution.
ELISA is a quantitative technique that measures serum concentration of antigens, antibodies,
and allergens.
Standard ELISA uses antibody-antigen-antibody trapping principle with the second antibody
coupled to an enzyme. If the complex is formed, the enzyme converts a clear solution into a
coloured one that can be measured with a spectrophotometer.
ELISA is performed in a multi-well microtiter plate. In addition to the test solution, standard
solutions are added with known antigen concentration. These solutions will be used to infer the
concentration of the antigen being tested.
Spectrophotometrically: By using spectrophotometry.
Epitope: That part of a biomolecule (such as a protein) that is the target of an immune
response.
5. Several variations of ELISA, seen in, exist but the most commonly used method is the sandwich ELISA.
The sandwich assay uses two different antibodies that are reactive with different epitopes on the antigen
with a concentration that needs to be determined.
A fixed quantity of one antibody is attached to a series of replicate solid supports, such as plastic microtiter
multi-well plate.
Test solutions containing antigen at an unknown concentration are added to the wells and allowed to bind.
Unbound antigen is removed by washing, and a second antibody which is linked to an enzyme is allowed
to bind.
This second antibody-enzyme complex constitutes the indicator system of the test.
The antigen serves as bridge, so the more antigen in the test solution, the more enzyme-linked antibody
will bind.
The test solution is used in parallel with a series of standard solutions with known concentrations of
antigen that serve as control and reference.
The results obtained from the standard solutions are used to construct a binding curve of the second
antibody as a function of antigen concentration.
The concentration of antigens can be inferred from absorbance readings of standard solutions.
