Diagnosis is a decision point. The decision is the intention
to treat. It is the point at which sufficient evidence has
been accumulated to state, beyond reasonable doubt, that
the patient is or is not suffering from a particular disease.
Laboratory tests remain one of the mainstays on which
the clinicians rely for diagnosis and management of the
patient. Laboratory tests are indicated for:
The presence or absence of a particular substance, e.g.
testing for infectious diseases like Venereal Diseases
Research Laboratory (VDRL), hepatitis B surface antigen
Accurately determining the concentration of a particular
substance as an aid to diagnosis or differential diagnosis
(e.g. concentration of CRP in differential diagnosis of viral
and bacterial infections) and for establishing the extent
The course of clinical condition or response to therapy.
For predicting the probability of occurrence of a disease/
disorder (e.g. microalbuminuria for predicting diabetic
nephropathy) or predicting the outcome of a disease/
Microscopy, biochemical assays, microbiology
procedures, and immunoassays are various techniques
that fulfil the requirements of routine laboratory tests to
meet the needs of the clinicians.
Certain clinical analytes can be measured by specific
techniques only, whereas for the measurement of certain
analytes options exist for selecting the techniques of
measurement. For example, urinary albumin can be
measured by biochemical methods such as pyrogallol
albumin is in the range of 30–300 mg/L, the accuracy
of the measurements by biochemical methods is
questionable because these methods also react with other
proteins in addition to albumin which are frequently
found to be present in urine. Immunochemical methods
(immunoassays) which are more sensitive and specific
have a distinct advantage and, hence, are preferred.
Immunoassays are assays that detect the presence of an
antigen in the human body with the help of an antibody
or detect the presence of an antibody with the help of an
antigen. In this text for simplicity, all further information
provided is based on considering antibody as a reagent to
detect antigen in the human body fluids.
The first reported immunoassays were homogeneous.
They are attributed to Kraus (1897), who coined the term
‘precipitin’ for the precipitate formed upon mixing an
Meyer in 1922, employed sheep erythrocytes to serve
as a label and conjugated human immunoglobulin to
them. Anti-immunoglobulin antibodies appearing in
rheumatoid arthritis patients were shown to cause visible
clumping of these erythrocytes. This method was known
Singer and Plotz replaced the erythrocytes with latex
particles, which were easier to standardize, and these
assays are popularly known as latex agglutination
tests. High degree of sensitivity for a wide variety
of antigens/antibodies, which can be detected by
these latex agglutination assays, has promoted their
usage worldwide for screening since, 1956 in clinical
laboratories. The simplicity of performance and
obviating the need for equipments, have made these
The need for quantitative estimation, and higher
sensitivity led to the development of radioimmunoassays
(RIA) first in 1959 by Berson and Rosalyn Yalow. The first
RIA developed was used to detect and quantify insulin.
Since then immunoassays have been used to detect and
quantify a variety of molecules native to humans such as
proteins, hormones as well as foreign molecules such as
bacteria, viruses and parasites.
Qualitative immunoassay techniques provide test results,
which only help to identify or indicate the presence of
analytes. Various techniques for qualitative detection of
These techniques at the best can give a semiquantitative
or comparative information about analytes under assay.
Single immunodiffusion technique uses the diffusion
of an antigen into agar impregnated with antibody.
Double immunodiffusion technique allows the direct
comparison of two or more test materials providing a
simple and direct method for determining whether the
antigens in the test specimens are identical, cross-reactive,
or non-identical. Immunoelectrophoresis has been used
over the years for detection of several different antigens
present in a common solution. The latex agglutination
assays though simple to use are subject to variations in
results as the interpretation pattern between negative and
weakly reactive samples may vary between laboratory to
laboratory and person to person.
Lower sensitivity for many analytes and the need for
correct quantification of analytes for:
¾ Effective monitoring of disease
¾ For differential diagnosis to aid correct therapy,
have created the need for more sensitive and precise
Quantitative results of immunoassays are extremely useful
¾ Establishing the extent of severity of a disease
¾ Assessing the course and stage of clinical condition
¾ Differential diagnosis of many diseases
¾ Monitoring response to therapy
¾ Accurate prognosis of disease.
Various techniques have been used to develop
quantitative methods that include radial immunodiffusion
(RID) and electroimmunoassays, turbidimetric and
nephelometric assays and labeled immunochemical assays.
The RID and electroimmunoassay (rocket electrophoresis)
though reliable, are slow, relatively involvement intensive,
and expensive. This limits their usage in routine laboratories.
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