Antibodies and hybridomas. Presented by Nis giladi. Antibodies. Antibodies are the antigen binding protein present on the B-cell membrane and secreted by plasma cells. Secreted antibodies circulate in the blood, where they serve as the
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Presented by Nis giladi
membrane and secreted by plasma cells.
effectors of humoral immunity by searching out and neutralizing
antigens or marking them for elimination.
participate in a limited number of effecter functions.
25,000 molecular weight, and two identical heavy (H) chains, larger polypeptides
of molecular weight 50,000 or more.
noncovalent interactions as salt linkages, hydrogen bonds, and hydrophobic
bonds, to form a heterodimer (H-L).
disulfide bridges link the two identical heavy
and light (H-L) chain combinations to each
other to form the basic four-chain (H-L)2
antibody structure, a dimer of dimers.
of a light or heavy chain varies greatly among antibodies of different
specificity. These segments of highly variable sequence are called V
regions:VL in light chains and VH in heavy.
can be traced to differences in the amino acid sequences of V regions. In fact,
most of the differences among antibodies fall within areas of the V regions
called CDR, and it is these CDRs, on both light
and heavy chains, that constitute the antigen
binding site of the antibody molecule.
far fewer differences are seen when one
compares sequences throughout the rest of
the molecule. The regions of relatively
constant sequence beyond the variable
regions have been dubbed C regions.
chains There are four human IgG subclasses, distinguished by differences in –
chain sequence and numbered according to their decreasing average serum
concentrations: IgG1, IgG2, IgG3, and IgG4.
-IgG1, IgG3, and IgG4 readily cross the placenta
and play an important role in protecting the
-IgG3 is the most effective complement activator,
-IgG1 and IgG3 bind with high affinity to Fc receptors
on phagocytic cells and thus mediate opsonization.
Monomeric IgM, with a molecular weight of 180,000, is expressed
as membrane-bound antibody on B cells.
units are held together by disulfide bonds that link their carboxyl-terminal heavy chain
chain, which is disulfide-bonded to the carboxyl-terminal cysteine residue of two of
the ten chains.
response to an antigen, and it is also the first immunoglobulin to be synthesized by the
concentrations in the intercellular tissue fluids.
in serum, it is the predominant immunoglobulin class in external secretions such
as breast milk, saliva, tears, and mucus of the bronchial, genitourinary,and
digestive tracts. In serum,IgA exists primarily as a monomer.
Symptoms of hay fever, asthma, hives, and anaphylactic shock.
and kill pathogens are:
1.opsonization-which promotes antigen phagocytosis by macrophages
2. complement activation, which activates a pathway that leads to the
generation of a collection of proteins that can perforate cell membranes.
3. Antibody dependent cell-mediated cytotoxicity (ADCC), which can
kill antibody-bound target cells.
heavy chains, which are linked by disulfide bonds.
region..In any given antibody molecule, the constant region contains
one of five basic heavy-chain sequences (m,d ,a ,e , or c) called isotypes and one
of two basic light-chain sequences ( or ) called types.
concentrations, and half-lives.
produced by plasma cells, and membrane-bound antibody.
heterogeneous collection of binding sites, a monoclonal antibody is derived
from a single B cell clone and is a homogeneous collection of binding sites.
cytokines or the extracellular portions of transmembrane proteins such as
growth factor receptors (GFRs) and adhesion molecules.
variety of diseases. Examples include ErbB family members and single-pass
receptor tyrosine kinases (cancer);tumor necrosis factor (TNF)-a and other
cytokines (inflammatory diseases such as ulcerative colitis, rheumatoid
arthritis, juvenile rheumatoid arthritis, ankylosis spondylitis,psoriatic arthritis,
mAbs there are major effort to reduce inherent mAb immunogenicity.
by production of chimeric and Humanized mAbs .
associated with markedly fewer AARs compared with murine mAbs.
Nevertheless, the development of the human anti-chimeric antibody (HACA)
response remains a potentially significant problem that requires close
monitoring and. HACA response rates of up to 61% have been reported with
infliximab and have been associated with shorter duration of effect and
increased risk for infusion reactions.
of immunogenicity, the successful use of many murine mAbs has been
restricted to acute indications (eg, acute graft rejection, imaging applications).
intended to replace all rodent sequences except the CDRs regions with
1.Mice are immunized with antigen A and given an intravenous booster immunization three days before they are killed, in order to produce a large population of spleen cells secreting specific antibody. Spleen cells die after a few days in culture. In order to produce a continuous source of antibody.
2. Spleen cells are fused with immortal myeloma cells by using polyethylene glycol (PEG) to produce a hybrid cell line called a hybridoma.
3.The myeloma cells are selected by HAT medium because they lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT). The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture because of the malignant potential contributed by the myeloma cells. Therefore, unfused myeloma cells and unfused spleen cells die in the HAT medium.
4.Individual hybridomas are then screened for antibody production, and cells that make antibody of the desired specificity are cloned by growing them up from a single antibody-producing cell.
5. The cloned hybridoma cells are grown in bulk culture to produce large amounts of antibody. As each hybridoma is descended from a single cell, all the cells of a hybridoma cell line make the same antibody molecule, which is thus called a monoclonal antibody.
the diversity of the human immune system within an animal that can be
immunized and killed for retrieval of splenocytes for subsequent fusion.
the germ line, necessitating regeneration of xenograft mice for
development of each specific mAb.
mAb production largely circumvent the need for human
donors and are contributing numerous therapeutic
molecules and candidates.
isolate genes based on their protein products Phage display synthesis of
human mAbs. Variable (VH and VL) domains from hybridomas or pools of B
cells are cloned by reverse transcriptase polymerase chain reaction (PCR). The
PCR fragments are inserted into gene III offilamentous phage. The domains
are joined by a short peptide linker and associate via framework region-
mediated interactions,generating a scFv.
Display of the pIII-scFv fusion on the phage coat allows it to interact with
antigen. High-affinity scFvs bind phage to a immobilized antigen during
multiple highstringency washes, whereas low-affinity phage are removed
(‘‘panning’’). Adherent phage are then eluted and used to
infect Escherichia coli. The scFv can then be altered through
directed or random mutagenesis, creating subpools of phage
with novel binding characteristics.
When optimized, the VH and VL domains can then be cloned into heavy and
light chain expression vectors
and transfected into hybridoma
cells for expression of
have been disrupted and replaced with human immunoglobulin gene clusters.
immunoglobulin genes are arranged in large clusters, rendering their cloning
quite arduous:the human heavy, l-light, and k-light chain loci,located on
chromosomes 14, 22, and 2, respectively, span more than 1 megabase each.
D end to interact with