Transcript The production procedure

The production procedure
Step 1
Hybridoma cell production
•Hybridoma cell(杂交瘤细胞): the cell which
made by fusing a specific 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.
Details of production
• (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
• Monoclonal antibodies are typically made by fusing
myeloma cells(骨髓瘤细胞) with the spleen cells from a
mouse that has been immunized with the desired antigen.
• The selective culture medium is called HAT medium
because it contains hypoxanthine, aminopterin, and
thymidine. This medium is selective for fused (hybridoma)
cells. Unfused myeloma cells cannot grow because they
lack HGPRT (an enzyme necessary for the salvage synthesis--a
pathway in which nucleotides are synthesized from intermediates in
the degradative pathway for nucleotides of nucleic acids ), and
thus cannot replicate their DNA. Unfused spleen cells
cannot grow indefinitely because of their limited life span.
Only fused hybrid cells, referred to as hybridomas, are
able to grow indefinitely in the media because the spleen
cell partner supplies HGPRT and the myeloma partner
has traits that make it immortal (as it is a cancer cell).
• This mixture of cells is then diluted and clones
are grown from single parent cells on microtitre
wells. The antibodies secreted by the different
clones are then assayed for their ability to bind
to the antigen (with a test such as ELISA or
Antigen Microarray Assay) or immuno-dot blot.
The most productive and stable clone is then
selected for future use.
A Dot blot (or Slot blot) is a
technique in molecular biology
used to detect biomolecules.
Schematic of the use of two ASO probes on duplicate Dot-blot filters.
• The hybridomas can be grown indefinitely in a
suitable cell culture media, or they can be
injected in mice (in the peritoneal cavity(腹膜腔),
the gut), they produce tumors containing an
antibody-rich fluid called ascites fluid(腹水). The
medium must be enriched during selection to
further favour hybridoma growth. This can be
achieved by the use of a layer of feeder fibrocyte
cells or supplement medium such as briclone (a
kind of medium for use in the stages following
fusion in hybridoma production. ). Production in
cell culture is usually preferred as the ascites
technique is painful to the animal and if
replacement techniques exist, this method is
considered unethical.
Step2
Purification of monoclonal antibodies
• WHY?
The contaminants in the cell culture sample would
consist primarily of media components such as
growth factors, hormones, and transferrins(铁传递
蛋白 ). In contrast, the in vivo sample is likely to
have host antibodies, proteases, nucleases,
nucleic acids, and viruses. In both cases, other
secretions by the hybridomas such as cytokines
may be present. There may also be bacterial
contamination and, as a result, endotoxins which
are secreted by the bacteria.
HOW?
1. To achieve maximum purity in a single step, affinity purification can be
performed, using the antigen to provide exquisite specificity for the
antibody. In this method, the antigen used to generate the antibody is
covalently attached to an agarose support. If the antigen is a peptide, it
is commonly synthesized with a terminal cysteine which allows
selective attachment to a carrier protein, such as KLH during
development and to the support for purification. The antibodycontaining media is then incubated with the immobilized antigen,
either in batch or as the antibody is passed through a column, where it
selectively binds and can be retained while impurities are washed
away. An elution with a low pH buffer or a more gentle, high salt
elution buffer is then used to recover purified antibody from the
support.
2. To further select for antibodies, the antibodies can be precipitated out
using sodium sulfate or ammonium sulfate. Antibodies precipitate at
low concentrations of the salt, while most other proteins precipitate at
higher concentrations. The appropriate level of salt is added in order
to achieve the best separation. Excess salt must then be removed by a
desalting method such as dialysis.
3. The final purity can be analyzed using a chromatogram. Any impurities
will produce peaks, and the volume under the peak indicates the
amount of the impurity. Alternatively, gel electrophoresis and capillary
electrophoresis can be carried out. Impurities will produce bands of
varying intensity, depending on how much of the impurity is present.
To simplify:
1. Affinity purification
2. Antibody precipitation
3. Using a chromatogram to identify the
impurities
Step3
Recombinant
• The production of recombinant monoclonal antibodies
involves technologies, referred to as repertoire cloning or
phage display/yeast display. Recombinant antibody
engineering involves the use of viruses or yeast to create
antibodies, rather than mice. These techniques rely on
rapid cloning of immunoglobulin gene segments to create
libraries of antibodies with slightly different amino acid
sequences from which antibodies with desired specificities
can be selected. These techniques can be used to
enhance the specificity with which antibodies recognize
antigens, their stability in various environmental conditions,
their therapeutic efficacy, and their detectability in
diagnostic applications. Fermentation chambers have
been used to produce these antibodies on a large scale.