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The problems : How can I make large amounts of monoclonal antibodies in a safe, reproducible and efficient manner? How can I circumvent the immunization of animals, i.e. how can I create new antibody specificities in vitro ? How can I select the desired antibody specificities from large synthetic repertoires? Is it always necessary to make a full size antibody or can I use small fragments for certain applications , like !"I#$?
%hich e&pression systems are suitable for the various antibody formats?
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 ,
Antibody Engineering
5lassical antibody production .unctions and $pplications of antibodies #tructure function relationships of antibodies 'odern techni6ues for the generation and selection of new antibodies $lternatives to antibodies engineered binding proteins
Antibody Applications
8H!/$9: 2. ;I#!$#!# I'$<I=< 2. ;I#!$#!# !=>:'! "I=3!; I''4=2#2/(!=8 $##$: ?!"I#$@ 9/28!I= ?%!#8!/=@ ("288I=< I''4=2HI#825H!'I#8/: 1 I''4=25:825H!'I#8/: I''4=29/!5I9I8$8I2= '$=: '2/! ...
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 A
Ig<
How can I engineer this molecule...
8he antigen binding site of an antibody is formed by C 5;/ regions in the variable domain
http:11www.clunet.edu1(io;ev1omm1gallery.htm
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 C
num na
$ntibody diversity
8he human genome is presently estimated to contain ca. ,-.--- 7-.--thousand ) genes 8he number of different kinds of antibody molecules is probably about ,.B & +-
different antibody
molecules ?
8he answer: each antibody chain is encoded by several different gene segments 8he genome contains a pool of gene segments for each type of chain. /andom assortment of these segments and mutations make the largest contribution to receptor diversity.
%e can learn
framework +
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framework ,
framework 7
5;/, 5;/7
"D
4nrearranged DH genes
5h H +
5H,
5H+
,-)CB7 ca. AG +-
B+ DH
,B ;
CE
rearrangement
;
5H+
heavy and light chain combinations G +- C plus Functional diversity plus somatic mutations G +- H
http:11www.bio.davidson.edu1courses1Immunology1.lash1somaticrecomb.htmlc
HereIs an animation
of D" rearrangement
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 +,
<ene segments that contribute to antibody diversity .or the heavy ?H@ chains of human antibodies, the gene segments are:
J B+ D segments . !ach of these encodes most of the = terminal of
H
the antibody, including the first two ?but not the third@ hypervariable region.
J ,B ; ?KLdiversityL@ gene segments . 8hese encode part of the third
H
hypervariable region.
J C E ?KLFoiningL@ gene segments . 8hese encode the remainder of the
H
$ntibodies
DM
EM
DN
<ene #egments AB 7+
A B+ ,B C
5ombinations
,-- M chains
+,A N chains
EN
D ;
H H
),CB- H chains
$ny H chain ?)CB-@ with any " chain ?7,A@: ,.B & +- C possibilities (ut the true number is probably virtually limitless because of J variation in the e&act splice point and J the introduction of = nucleotides
monoclonal antibodies
by immortalization of antibody producing ( cells
Making Hybridomas
'yeloma cells have to #pleen cells are primary cells thus they are not able to grow in culture and: they supply an intact H<9/8 gene to the
7 types of cells in the mi&ture after cell fusion: unfused spleen cells: will die naturally unfused myeloma cells: cannot survive in H$8 medium ?because they are H<9/8 negative@ hybridoma cells: will survive in H$8 medium ?H<9/8 positive, transformed cells@
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 ,,
5ell fusion
>immermann 4, Dienken E, Halfmann E, !meis 55. !lectrofusion: a novel hybridization techni6ue. $dv (iotechnol 9rocesses. +*HBOA:)* +B-. <abriFel ', 3reft ', >orec /. 'onitoring lysosomal fusion in electrofused hybridoma cells. (iochim (iophys $cta. ,--) 2ct ,7O .
'ethods in 'olecular (iology )*+.++) %# ,--) .lorian /0ker I$' 1 (234 11 ,B