The production procedure

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The production procedure

• Step 1
• Hybridoma cell production
• Hybridoma cell(?????) 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.

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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

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• Monoclonal antibodies are typically made by
  fusing myeloma cells(?????) 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).

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• 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.
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• The hybridomas can be grown indefinitely in a
  suitable cell culture media, or they can be
  injected in mice (in the peritoneal cavity(???),
  the gut), they produce tumors containing an
  antibody-rich fluid called ascites fluid(??). 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.

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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(????? ). 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.

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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
   antibody-containing 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.

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To simplify

1. Affinity purification
2. Antibody precipitation
3. Using a chromatogram to identify the impurities

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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.