Acquired Specific Immunity

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Acquired Specific Immunity
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Fig. 1-5 Adaptive Immunity
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RBC
monocyte
Fig. 1-3 Macrophage attacking E. coli
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Fig. 1-7 Overview of Immune System
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Antibodies make great antigens!
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One antigen, many epitopes, many Ab classes
One antigen, one epitope, one Ab class
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Monoclonal antibodies are great! Why?
Specificity! Each one recognizes only one site of
the antigen structure. Why else? Immortality!
Mass cultures can be generated from a single
clone. Mice are small! They require less antigen
than a rabbit to get an antibody reaction. The
antigen travels through the circulation system.
Blood is channeled through the spleen where B
lymphocyte cells recognize the foreign bodies and
produce antibodies.
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Spleen cells, however, cannot survive alone in
tissue culture media. Myeloma cells can! Why not
fuse them? We can select for fused cells by
using a HAT (hypoxanthine, aminopterin,
thymidine) supplemented media. The mutant myeloma
cell line cannot survive in HAT media because it
lacks HGPRT. The aminopterin in the HAT
supplement blocks DNA synthesis. The enzyme HGPRT
can overcome this block. Spleen cells do have
HGPRT. Fuse the spleen and myeloma cells and
you'll find survivors in HAT tissue culture media.
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HAT Medium (Hypoxanthine Aminopterin Thymidine
medium) is a selection medium for mammalian cell
culture, which relies on the combination of
aminopterin, a drug that acts as a folate
metabolism inhibitor by inhibiting dihydrofolate
reductase, with hypoxanthine and thymidine, which
are intermediates in DNA synthesis - a purine
derivative and a deoxynucleoside, respectively.
The trick is that aminopterin blocks DNA "de
novo" synthesis, which is absolutely required for
cell division to proceed, but the other
components provide cells with the raw material to
evade the blockage (the "salvage pathway") -
provided that they have the right enzymes, which
means having functioning copies of the genes that
encode them.
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Figure 28-2 The metabolic pathway for the de novo
biosynthesis of IMP.
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The enzyme dihydrofolate reductase which produces
tetrahydrofolate (THF) by the reduction of
dihydrofolate,is specifically blocked by
aminopterin. THF, acting in association with
specific proteins, can receive single carbon
units that are then transferred to specific
targets.
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Figure 28-21 Regeneration of N5,N10-methylenetetra
hydrofolate.
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One of the important targets for cellular
reproduction is thymidylate synthase, which
creates thymidine monophosphate (TMP) from
uridine monophosphate (dUMP). By additional
phosphorylation reactions TMP can be used to make
thymidine triphosphate (TTP), one of the four
nucleotide precursors that are used by DNA
polymerases to create DNA. Without the THF
required to convert dUMP, there can be no TTP,
and DNA synthesis cannot proceed --- unless TMP
can be produced from another source. The
alternative source is that thymidine present in
HAT medium can be absorbed by the cells and
phosphorylated by thymidine kinase (TK) into TMP.
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The synthesis of IMP, (precursor to GMP and GTP)
also requires THF, and also can be bypassed. In
this case hypoxanthine-guanine phosphoribosyltrans
ferase (HGPRT) reacts hypoxanthine absorbed from
the medium with PRPP, liberating pyrophosphate,
to produce IMP by a salvage pathway.
Hypoxanthine PRPP ? IMP PPi
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Enzyme Linked Immunosorbent Assay (ELISA)
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Immunotherapeutics
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