Download Hybridomas - sources of antibodies

Hybridomas - sources of
antibodies
Chapter 8 from ‘The Basics’
Objectives
• What are monoclonal antibodies (Mabs)?
• What are polyclonal antibodies (Pabs)?
• What are hybridomas?
• How hybridomas produce Mabs?
Antibody production in vivo
• Found in specific protein fraction of blood –
gammaglobulin or immunoglobulin fraction
• Synthesized by subset of white blood cells – the Blymphocytes
• Abs have two heavy + two light chains
• Heavy chain – variation in length, number of
domains and glycan structures.
Antibodies are valuable
• Abs are found in all body fluids including
blood, milk and mucous secretions and serve
an essential role in immune system that
protects animals for infection or cytotoxic
effects of foreign compounds
• Bind antigens at their epitopes
• Valuable – specific recognition and affinity
for antigen
Structure of Antibodies
• IgG – major class of immunoglobulin found
in blood serum – molecular mass of 150KDa
• The heavy chain of IgG has four domains –
VH-CH1-CH2-CH3 and light chain has two
domains VL-CL
• Constant C region of any Ig class varies with
species
Structure of Antibodies
• Digestion of molecule with papain cleaves the heavy
chain in hinge region and forms three fragments (2
Fab + Fc)
• Two fab (antibody-binding fragments) contain Nterminal end of a heavy chain with disulfide linked
light chain
- Variable sequences of amino acids – allows to bind
to antigen with high affinity
• Fc consists of C-terminal end of two heavy chains
Antibody production in vivo
• Each B-lymphocyte – produce one type of
antibody in response to particular antigen –
Monoclonal antibody
• Immunoglobulin fraction of blood will contain
numerous other antibodies – Polyclonal
antibody
• The variety of antibodies present in any animal
reflect the population of B-lymphocytes –
exposure to previous range of antigens
Glycosylation of antibodies
• Ab are glycoproteins containing variable
glycan structures
• Glycosylation of Fc region is essential for
effector functions of antibody such as
complement binding, binding to Fc
receptors, and induction of ADCC
• 20% glycosylation is present on Fab fragment
in human ab
• Glycan structures – role in immune response
Hybridomas- Kohler and Milstein1975
• Hybridomas are
hybrid cells capable of
continuous production
of monoclonal ab
• Hybrid of BLymphocytes and
myelomas
• Can be grown in
bioreactors- produc. of
kilograms of Mabs.
Hybridomas
• Produce monoclonal antibodies for diagnosis
and testing in applications such as blood
typing, detection of virus, pregnancy testing or
for detection of contaminants in food
• Kohler and Milstein work – four stages –
Immunization, Cell Fusion, Genetic
Selection and cell selection
Immunization in vivo
• Injection of a chosen antigen into mice/rat
• Synthesis of antibodies will depend upon
antigen (3-4 weeks for good response)
- Large molecules – produce stronger response
in short period of time
- Small molecules - multiple injections spaced
over several days
- Spleen is homogenized - B-lymphocytes
isolated by centrifugation
Immunization in vitro
• Obtain spleen from nonimmunized mouse
• Cells suspended in medium containing selected
antigen + factors stimulating growth and
differentiation + incubation with mixed
lymphocytes = conditioned media
• Growth promoting factors – cytokines like
interleukins, B-cell growth factor and B-cell
differentiation factor
• Antigens at lower conc + activation of Blymphocytes takes 3-4 days rather than few weeks in
vivo
Cell Fusion/Hybridization
• Fusion between B-lymphocyte (ability to synthesize
antibodies) + Myeloma (ability for infinite growth)
• Suitable myeloma fusion partners selected for two important
characteristics
- Nonproduction of antibodies – resulting hybridoma produces
not more than one antibody
- Myelomas deficient in HGPRT (hypoxanthine guanine
phosphoribosyl transferase) are used. Allows selection in
HAT (hypoxanthine, aminopterin, thymidine
Cell Fusion/Hybridization
• Cells can be induced to fuse if two cell populations
are brought close
• Destabilization of adjacent cell membranes
• Two distinct nuclei fuse to form heterokaryon –
produce a stable hybrid cell
Methods of Cell Fusion
• Fusion by PEG at 4000-6000 KDa is suitable
for cell fusion – within 1-2 minutes
• Swelling accompanies fusion
• Allows adjacent cells to approach closely
• Plasma membrane becomes permeable to
small ions
Methods of Cell Fusion
• Electrofusion – two populations are
introduced into a small sterile chamber
• Electric current is applied in high-voltage
pulses (200 V/cell pellet) for short time
periods
• Allows cells to orient along the line of
current and fuse
• Produces high % of viable hybrid cells
Selectable gene markers for cell
selection
• Heterogeneous population of cells – unfused
parental cells, lysed cells and hybrid cells
• Two stages of selection
- Isolation of hybrid cells from parental cells
- Selection of antibody-secreting cells within
hybrid cell population
Selectable gene markers for cell
selection
• Selective medium contains HAT –
hypoxanthine, aminopterin and thymidine
• Allows selection and growth of hybridomas
which are HGPRT+
• Unable to support growth of HGPRTmyelomas because denovo pathway is
inhibited and salvage pathway cannot
function because of defective enzyme
Clonal selection of Mab-secreting
hybridomas
• Only some hybridomas (10%) from HAT culture will secrete
antibodies
• To select Mab-secreting hybridomas – dispense suspension
into 96-well plate so that each well has one cell
• Growth supported by feeder layer of cells (thymocytes,
macrophages or splenocytes treated to prevent growth/DNA
synthesis)
• 1-2 weeks for growth – medium of each well tested for
antibody by suitable assay
• Promising ones cultured in suspension to produce 1-2 x106
cells/ml and Mab conc. Of 100-200 μg/ml
Assay of Mabs Detection
• ELISA – enzyme-linked immunosorbent assay
- Most commonly used, done in multi-well plates
for analyzing multiple samples
• RIA-radioimmunoassay
- More time consuming and expensive
• Affinity chromatography
- Is ideal if an HPLC is available and
hybridomas – grown in serum-free media
Open book exam questions
• Why do murine-derived monoclonal antibodies
have limited success in human therapy?
• What is the disadvantage of using microbial
fermentation for production of antibodies?
How do usage of plant cells win over them?
• Mention one limitation of plant-derived
antibodies.
• What is glycosylation? What is its importance
to therapeutic antibodies?
What are the major difficulties in
producing human hybridoma cells?
• Source of antibody-secreting lymphocytes Spleen of immunized mouse is used – not
possible with humans. Can be taken from
patients – acquired immunity against particular
compound or disease
What are the major difficulties in
producing human hybridoma cells?
• Immortalization and chromosome
instability –
Human myeloma cell lines are difficult to
grow in culture.
Human lymphoblastoid cell lines – used as
fusion partners – cell fusion and genetic
stability is low
What are the major difficulties in
producing human hybridoma cells?
• Antibody secretion of human parental
fusion partners
• Mouse myeloma used in fusion are
nonantibody secretors
• Human myeloma or lymphoblastoid cells –
immunoglobulin secretors
- Will secrete two kinds of antibodies –
associated with parental cells
Why is antibody production in plants
very successful?
• Mabs in plants – absence of animal pathogens,
ease of genetic manipulation, ability of post
translational modification and potential for
scale up to an economic production level.
• Low cost of large scale produc. – plantibodies
• Directly eat plants without purification
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