Trends in Biotechnology
150415 TB 12 Antibodies
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Acquired or Adaptive Immunity.
A. Recognizes foreign invaders and responds to
the invader, called an “antigen”.
B. Can also recognize the body as self and the
tissues of others as non-self.
C. Antigens can be protein, glycoprotein,
polysaccharides, and nucleic acids, and small parts
of the antigen can trigger a response (called the
“antigenic determinant”).
D. Based on the complex interactions of different
types of cells and other parts.
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• T cells and B cells are activated, some become
“memory” cells.
• The next time that an individual encounters
that same antigen, the immune system is
primed to destroy it quickly.
• This is active immunity because the body’s
immune system prepares itself for future
challenges.
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• Long-term active immunity can be naturally
acquired by infection or artificially acquired by
vaccines made from infectious agents that
have been inactivated or, more commonly,
from minute portions of the microbe.
• Short-term passive immunity can be
transferred artificially from one individual to
another via antibody-rich serum.
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• Any non-self substance capable of triggering
an immune response is known as an antigen.
• The distinctive markers on antigens that
trigger an immune response are called
epitopes.
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• Very important to the immune response is the
ability to distinguish between “self” and “nonself.”
• Every cell in your body carries the same set of
distinctive surface proteins that distinguish
you as “self.”
• Normally your immune cells do not attack
your own body tissues, which all carry the
same pattern of self-markers.
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• This set of unique markers on human cells is
called the major histocompatibility complex
(MHC) proteins.
• There are two classes: MHC Class I proteins,
which are on all cells, and MHC Class II
proteins, which are only on certain specialized
cells.
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• Cells work chiefly by secreting soluble
substances known as antibodies.
• They wait around a lymph node, waiting for a
macrophage to bring an antigen or for an
invader such as a bacteria to arrive.
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When an antigen-specific antibody on a B cell
matches up with an antigen:
• The antigen binds to the antibody receptor,
the B cell eats it, and, after a special helper T
cell joins the action, the B cell becomes a large
plasma cell factory that produces identical
copies of specific antibody molecules very
quickly -up to 10 million copies an hour.
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An antigen may have one or more of the same antigenic
determinants or different antigenic determinants.
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An antibody molecular binds to an antigenic determinant
(epitope) of a cell that has different antigens on its
surface
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• Antigenic Determinants (Epitopes)
• http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch
apter32/animation_quiz_5.html
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Structure of an antibody
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The generation of a functional antibody L-chain by
recombination of DNA regions
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• Antibody Diversity
• http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch
apter32/animation_quiz_2.html
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Monoclonal antibodies
A general representation of the method used to produce monoclonal
antibodies.
http://en.wikipedia.org/wiki/Monoclonal_antibody#/media/File:Monoclonals.png Adenosine CC BY-SA 3.0
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• Monoclonal antibodies (mAb or moAb) are
monospecific antibodies that are made by
identical immune cells that are all clones of a
unique parent cell, in contrast to polyclonal
antibodies which are made from several
different immune cells.
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• Monoclonal antibodies have monovalent
affinity, in that they bind to the same epitope.
• Given almost any substance, it is possible to
produce monoclonal antibodies that
specifically bind to that substance; they can
then serve to detect or purify that substance.
• This has become an important tool in
biochemistry, molecular biology and medicine.
• When used as medications, the nonproprietary drug name ends in –mab
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Monoclonal Antibody Production
http://highered.mcgrawhill.com/sites/0072556781/student_view0/chap
ter32/animation_quiz_3.html
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• Hybridoma technology is a technology of
forming hybrid cell lines (called hybridomas)
by fusing an antibody-producing B cell with a
myeloma (B cell cancer) cell that is selected
for its ability to grow in tissue culture and for
an absence of antibody chain synthesis.
• The antibodies produced by the hybridoma
are all of a single specificity and are therefore
monoclonal antibodies (in contrast to
polyclonal antibodies).
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(1) Immunisation of a
mouse
(2) Isolation of B cells
from the spleen
(3) Cultivation of
myeloma cells
(4) Fusion of myeloma
and B cells
(5) Separation of cell
lines
(6) Screening of suitable
cell lines
(7) in vitro (a) or in vivo
(b) multiplication
(8) Harvesting
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• Laboratory animals (mammals, e.g. mice)
are first exposed to the antigen that an
antibody is to be generated against.
• Usually this is done by a series of
injections of the antigen.
• Splenocytes are removed from the
mammal's spleen, the B cells are fused
with immortalized myeloma cells.
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• The myeloma cells are selected to ensure
they are not secreting antibody
themselves and that they lack the
hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) gene,
making them sensitive to HAT medium.
• Electrofusion causes the B cells and
Myeloma cells to align and fuse with the
application of an electric field.
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• Fused cells are incubated in HAT medium
(hypoxanthine-aminopterin-thymidine
medium) for roughly 10 to 14 days.
• Aminopterin blocks the pathway that
allows for nucleotide synthesis.
• Unfused myeloma cells die, as they
cannot produce nucleotides by alternate
pathways because they lack HGPRT.
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• Unfused B cells die as they have a short
life span.
• Only the B cell-myeloma hybrids survive,
since the HGPRT gene coming from the B
cells is functional.
• These cells produce antibodies (a
property of B cells) and are immortal (a
property of myeloma cells).
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• The incubated medium is then diluted into
multi-well plates to such an extent that each
well contains only one cell.
• Since the antibodies in a well are produced by
the same B cell, they will be directed towards
the same epitope, and are thus monoclonal
antibodies.
• The next stage is a rapid primary screening
process, which identifies and selects only
those hybridomas that produce antibodies of
appropriate specificity.
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The B cell that produces the desired
antibodies can be cloned to produce
many identical daughter clones.
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Western Blotting
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Immunoblotting (Western Blot)
proteins are separated by electrophoresis,
blotted to nitrocellulose sheets, then
treated with solution containing enzymetagged antibodies
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Western blotting for antibodies to HIV proteins.
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• The western blot (sometimes called the
protein immunoblot).
• Widely used analytical technique used to
detect specific proteins in a sample of tissue
homogenate or extract.
• Gel electrophoresis seperates proteins by 3-D
structure or denatured proteins by the length
of the polypeptide.
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• The proteins are then transferred to a
membrane (typically nitrocellulose or PVDF),
where they are stained with antibodies
specific to the target protein.
• The gel electrophoresis step is included in
western blot analysis to resolve the issue of
the cross-reactivity of antibodies.
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• This method is used in the fields of molecular
biology, immunogenetics and other molecular
biology disciplines.
• A number of search engines, such as CiteAb,
Antibodypedia, and SeekProducts, are
available that can help researchers find
suitable antibodies for use in western blotting.
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Other related techniques include dot blot
analysis, immunohistochemistry and
immunocytochemistry where antibodies are
used to detect proteins in tissues and cells by
immunostaining, and enzyme-linked
immunosorbent assay (ELISA).
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Immunofluorescence
• Dyes coupled to antibody molecules will
fluoresce (emit visible light) when irradiated with
ultraviolet light
• Direct-used to detect antigen-bearing organisms
fixed on a microscope slide
• Indirect-used to detect the presence of serum
antibodies
• Used for light microscopy with a fluorescence
microscope.
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• This technique uses the specificity of
antibodies to their antigen to target
fluorescent dyes to specific biomolecule
targets within a cell, and therefore allows
visualisation of the distribution of the target
molecule through the sample.
• Immunofluorescence is a widely used example
of immunostaining and is a specific example of
immunohistochemistry that makes use of
fluorophores to visualise the location of the
antibodies.
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Immunofluorescence can be used on
tissue sections, cultured cell lines, or
individual cells, and may be used to
analyze the distribution of proteins,
glycans, and small biological and nonbiological molecules.
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• Immunofluorescence can be used in
combination with other, non-antibody
methods of fluorescent staining.
• Several microscope designs can be used for
analysis of immunofluorescence samples; the
simplest is the epifluorescence microscope,
and the confocal microscope is also widely
used.
• Various super-resolution microscope designs
that are capable of much higher resolution
can also be used.
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The direct antibody assay where tagged
antibody interacts directly with antigens on a
cell surface.
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Enzyme-linked immunosorbent assay
The ELISA method. (a) The sandwich of substrate-bound
antibody, specific antigen, and a second antibody with a
bound enzyme, with the subsequent conversion of a
substrate to a colored product.
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• A test that uses antibodies and color change
to identify a substance.
• Uses a solid-phase enzyme immunoassay
(EIA) to detect the presence of a substance,
usually an antigen, in a liquid sample or wet
sample.
• Used as a diagnostic tool in medicine and
plant pathology, as well as a quality-control
check.
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• Antigens from the sample are attached to a
surface.
• A further specific antibody is applied over the
surface so it can bind to the antigen.
• This antibody is linked to an enzyme, and, in
the final step, a substance containing the
enzyme's substrate is added.
• The subsequent reaction produces a
detectable signal, most commonly a color
change in the substrate.
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Performing an ELISA involves at least one
antibody with specificity for a particular antigen.
The sample with an unknown amount of antigen
is immobilized on a solid support (usually a
polystyrene microtiter plate) either nonspecifically (via adsorption to the surface) or
specifically (via capture by another antibody
specific to the same antigen, in a "sandwich"
ELISA).
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After the antigen is immobilized, the detection
antibody is added, forming a complex with the
antigen. The detection antibody can be
covalently linked to an enzyme, or can itself be
detected by a secondary antibody that is linked
to an enzyme through bioconjugation.
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Between each step, the plate is typically washed
with a mild detergent solution to remove any
proteins or antibodies that are non-specifically
bound. After the final wash step, the plate is
developed by adding an enzymatic substrate to
produce a visible signal, which indicates the
quantity of antigen in the sample.
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The steps in
conducting an ELISA
assay.
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• ELISA Enzyme-Linked Immunosorbent Assay
• http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch
apter33/animation_quiz_1.html
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ELISA can perform other forms of ligand binding
assays instead of strictly "immuno" assays. The
technique essentially requires any ligating
reagent that can be immobilized on the solid
phase along with a detection reagent that will
bind specifically and use an enzyme to generate
a signal that can be properly quantified.
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Looking back:
Using antibodies means using molecules which
can bind to other molecules.
Look for similarities with other binding
techniques.
Eg. Nucleic acid hybridization, hydroxyapatite
column,
Also, think about different labeling techniques.
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