2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 30
Chatper 3: Antibodies
Antibodies 101
An antibody is an Immunoglobulin (Ig) produced in response to the recognition of foreign molecules in the
body. Structurally, antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide
chains. Each molecule contains two identical copies of a heavy chain, and two identical copies of a light chain,
named according to their relative molecular weights. The N-terminal region of the light and heavy chains each
have a domain of approximately 110 amino acids known as the variable region and serve as the antigen binding
site of the molecule. The specificity of an antibody is based on the antibody epitope affinity, the
antibody/antigen valency, and the structural arrangement of the interacting parts.
Sorbent Assay (ELISA), Western Blot, Immunohistochemistry, and Flow
Cytometry rely on the specificity of antibodies. It is important to note that
when designing experimental procedures, the goal is to choose an antibody
system that provides the greatest sensitivity and the least amount of cross-
reactivity and background. It is also important to choose between monoclonal
and polyclonal antibodies as they both have their advantages and limitations
of their use in immunochemical techniques.
Enzyme-Linked Immuno-Sorbent Assay (ELISA)
The basic principle for ELISA makes use of immobilized antibodies to
specifically “capture” soluble antigens in samples. The fixed antibody/antigen
complex, once washed free of excess reagents, reacts with a substrate to yield
a colored product that can be easily visualized and quantified
Polyclonal antibodies are obtained from serum of animals immunized with a
spectrophotometrically. The method chosen for each ELISA depends on factors
particular antigen. The antibody mixture obtained from serum is the result of
such as solid phase antibody coating condition, antibodies specificity and
many B-cell clones, each producing one specific antibody. This mixture of
incubation time, as well as washing conditions.
antibodies often can recognize multiple epitopes (region of an antigen that
binds to an antibody), making them more tolerant to small changes in the
Flow Cytometry
structure of the antigen. It is often a better choice for detection of denatured
Flow Cytometry is a technology that allows the measurements of a variety of
proteins. Also, polyclonal antibodies may be generated in a variety of species
characteristics of microscopic particles such as cells in a stream of fluid.
such as rabbit, sheep, and donkey. This provides a greater number of
Instruments used for this method can gather information about cells by
experimental options to researchers.
measuring visible and fluorescent light emissions. Measurable parameters can
Monoclonal antibodies are produced by fusing myeloma cells with antibody
be based on physical, biochemical and antigenic traits. Researchers frequently
secreting B-cells. The resulting hybridoma cells produce large quantities of
used flow cytometry to determine the types of markers and receptors on the
homogenous antibodies that recognize a single epitope. Because of their
surface of a cell by using labeled antibodies or receptor ligands.
specificity, monoclonal antibodies are excellent as the primary antibody in an
assay, or detecting antigen in tissue, and will often give significantly less
IFN Neutralization
background staining than polyclonal antibodies. However, due to its specificity
The neutralizing antibody (NAb) prevents formation of an active IFN receptor
to only one type of epitope, a monoclonal antibody will not bind to its specific
complex thus inhibiting the immunomodulatory, antiproliferative, or antiviral
antigen if it is degraded. In contrast, a polyclonal antibodies mixture may still
mediated response induced by interferon. In a neutralization assay, interferon
retain some of its binding ability even if certain molecules within the mixture
is pre-incubated with a specific neutralizing antibody. The IFN-NAb mixture is
are structurally compromised.
added to the cells plus virus or agonist. For optimal conditions, the
The interaction between an antibody and an antigen forms the basis of all
concentrations of interferon, neutralizing antibody dilution and incubation
immunochemical techniques. Methods such as Enzyme-Linked Immuno-
times need to be determined empirically.
PBLInterferonSource Toll Free: 1 877- PBL-8881
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