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IMMUNODIAGNOSTICS

ANTIGEN-ANTIBODY reactions
HISTORY
• The first comprehensive theories of antigen-antibody interaction were due to Ehrlich
and to Bordet
...

• At the time the Ehrlich and Bordet theories were formulated, and for many years
afterward, little was known of the chemical nature of either antigens or antibodies
...


• Immunodiagnostics is the diagnostic procedure using antigen-antibody
reactions as the primary means of detection
...

• Diagnosis based on the detection of antibodies is very sensitive and relatively
repeatable, tractable and time-saving compared with traditional direct
parasitological methods
...

• Immunodiagnostic testing can be performed on serum, and there are a variety
of commercially available tests
...


• It is the fundamental reaction in the body by which the body is protected from
complex foreign molecules, such as pathogens and their chemical toxins
...

• The immune complex is then transported to cellular systems where it can be
destroyed or deactivated
...
Lock and Key Concept
The combining site of an antibody is located in the Fab portion of the molecule
and is constructed from the hypervariable regions of the heavy and light chains
...

• Thus, our concept of antigen-antibody reactions is one of a key (i
...
the antigen)
which fits into a lock (i
...
the antibody)
...
These include hydrogen bonds, electrostatic bonds, Van der
Waals forces and hydrophobic bonds
...


Types of antigen antibody reactions used in vitro
• 1
...
Precipitation
• 3
...
Complement fixation
• 5
...
ELISA- Enzyme linked immunosorbent assay
• 7
...
ImmunochromatographY (ICT)
Applications / use in vitro
• Diagnosis of many diseases
• Severity or stage of diseases
• Respond to treatment
• Epidemiology

How antigen – antibody reactions in vitro helps in Dx?
• Infectious disease
• By determining whether an individual has developed antibodies in response to
infection
...

• Metabolic diseases
• Physiological conditions

Which diseases can be diagnosed by antigen- antibody reactions?
• Infectious diseases
• Bacterial
• Viral
• Protozoa
• Fungal
• Parasitic
• Autoimmune diseases
• Tumors

How antigen – antibody reactions in vitro helps in Diagnosis of infectious
disease?
• By determining whether an individual has developed antibodies in response to
infection
• IgM antibodies are usually a reflection of a recent infection
...

Why is Conjugation Necessary?
• B Lymphocytes produce antibodies against foreign antigens that they
encounter
...

• Thus, in order to produce an antibody to a specific antigen, the researcher
must ensure that the antigen is part of a molecular complex that is sufficiently
large to activate a humoral response
...

• These two molecules are highly immunogenic, meaning they are good at
activating humoral response that creates antibodies directed against the
antigen
...

• The term “antibody production” has both general and specific meanings
...


• The more restricted sense, antibody production refers to the steps leading up to
antibody generation but does not include various forms of purifying and
labeling the antibody for particular uses
...

• Polyclonal antibodies are recovered directly from serum (bleeds)
...


• Successful antibody production depends upon careful planning and
implementation with respect to several important steps and considerations:
• Synthesize or purify the target antigen (e
...
, peptide or hapten)
• Choose an appropriate immunogenic carrier protein
• Conjugate the antigen and carrier protein to create the immunogen
• Immunize animals using appropriate schedule and adjuvant formula
• Screen serum (or hybridoma) for antibody titer and isotype (also called
antibody characterization
...

• Examples include the use of branched DNA probes that contain a reporter
group or enzyme amplification
...

• Inside the cell, the signal must be amplified so that the response is carried out
multiple times rather than just be a single molecule
...

• Any molecule that catalyzes a reaction can do so multiple times producing
more than one product molecule
...

• The enzyme turns over multiple copies of a fluorogenic or chromogenic
substrate (Enzyme Substrates and Assay), resulting in much higher targetassociated signal levels
• than are obtainable by dye-labeled affinity reagents
...

• Major applications of enzyme labeling include immunocytochemical and
immunohistochemical detection and enzyme-linked immunosorbent assays
(ELISAs)
...

• Examples include the use of branched DNA probes that contain a reporter
group or enzyme amplification
...

• Inside the cell, the signal must be amplified so that the response is carried out
multiple times rather than just be a single molecule
...

• Any molecule that catalyzes a reaction can do so multiple times producing
more than one product molecule
...

• If a signaling chain is several steps long then there is a great potential for
amplification of the signal
...


• The number of target molecules per unit volume of sample is a key variable in
all biological detection applications
...

• There is therefore always some degree of need to detect molecules at their
native abundance levels, which can vary by many orders of magnitude
...

• For example, proteins in mammalian cells have abundances varying by at least
seven orders of magnitude (~101–108 copies per cell)
...

• Since the reaction between the ligand and its specific antibody does not
produce a precipitate, it is necessary to effect a physical separation of the two
forms before either or both forms can be quantitated
...

• The homogeneous EIAs, in contrast, are separationfree systems; they do not
require separation of the free and bound forms
...

• Numerous techniques have been developed to separate labeled antigenantibody complexes from the unbound labeled fraction
...

• Regardless of methodology, either competitive or immunometric; or of the
label, radioisotopic, enzymic, or fluorometric, the more efficient the separation
of the bound from the unbound fraction, the more the sensitivity of the assay
will be enhanced
...

• This in turn increases the technical requirements for performing the assay

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