insights into weak affinity antibody-antigen interactions

59 Pages·2000·0.66 MB·English

I W A
OT NSITHGISN KAE FFINITY
A - A I
YDOBITN NEGITN SNOITCARETN

yhpargotamor hyctinif fgani ssueidutS
rosneso ilbacit pdona

MaSgtnruasn dh

tnemty rrfatopseiDmehC Department of Immunotech ygolon
and Biomedical Sciences ytisrev idnnUuL
r ay mtflioasKrevinU ne d,edwnSuL
ne d,erwaSmlaK

2000
© Magnus Strandh, Kalmar 2000
ISBN 91 - 628 - 4481 - 4
Printed in Sweden at the University of Kalmar
Till mamma, pappa och Chanti

I do not know what I may appear to the world, but to myself
I seem to have been only like a boy playing on the seashore,
and diverting myself in now and then finding a smoother pebble
or a prettier shell than ordinary, whilst the great ocean of truth
lay all undiscovered before me.

Sir Isaac Newton, 1642-1726

P
OPULÄRVETENSKAPLIG S GNINTTAFNAMMA

Livets finurlighet är fascinerande att st .aredu räN nam rekösrednu aksigoloibmas -
dnab i jlated rasiv ted gis tta tlla sryts va sanrelykelomoib agåmröf llit gninnäknegih co
nakrevlexäv dem .ardnarav sanrelykeloM alleiceps repaksnege reggyb ppu snepporka lla
edarecilpmok ,renoitknuf llit lepmexe i ,ravsröfnumm gninledllec hco . etebraleksum
Inter anrenoitka nallem anrelykelomoib nak arav va akilo ;rätkarak ed svirkseb m os
starka eller svaga (affinitet), långsamma eller snabba (kinetik). Det har visat sig att
agnåm aksigoloib remsinakem sryts va agavs renoitkaretni ,mos arelf , snammasllit
skapar igenkänning och stringens. Tack vare att de enskilda interaktionerna är svaga
sapaks tgiditmas ne kimanyd hco agåmröf röf nemetsys tta tbbans assapna gis llitmo -
givande förhållanden. Detta har samtidigt medför t att de varit har svåra att studera, just
på grund av att de är så svaga.

anneD gnildnahva raldnah mo assed agavs arälykelomoib .renoitkaretni n eD
raretuksid nekliv llor ed rah i aksimekoib renoitakinummok hco ruh nam naka reduts
.med netkivnoT reggild kco åp ruh nam nak ajttyntu tgavs ednakrevlexävr elykelomoib
mos snegaer i aksitylana .redotem moS snegaer rednävna nam åskco .rapporkitnaa sseD
proteiner är en viktig del i kroppens immunförsvar och kan på konstgjord väg fås att
annäk negi ed alleicepsom relykel )negitna( nam rä daressertni .va anrapporkitnAna -
sdnäv nades i resylana va sivlepmexe ,ledemekäl kitsongaid relle i aksinketoib .ressecorp
I ed netebra mos saretneserp räh rahgål - aniffa rapporkitna stnävna i akilo .metsyslledom
Artikel I visar at t svaga interaktioner lämpar sig väl för kromatografisk separation av
tllerutkurts edatkälsebrän .relykelom agirvÖ netebra raldnah mo ruh agavsr enoitkaretni
nak sapmällit i .rerosnesoib I anralkitra II hco III saksroftu anretehgiljöm llit gnintämv a
verkl tgi agavs .renoitkaretnilykelomoib neD kimanyd hco teticificeps mosr ankcetennäk
dessa gör att man kan använda dem till att övervaka föränderliga processer. Artiklarna
VI hco V rarecudortni ned agilreunitnok nrosnesnummi mos rah arots retehgiljöm llitt ta
sadnävna i nedårmo räd nam lliv atäm ednareutkulf renoitartnecnok va a tnassertni
.relykelom

C
STNETNO

ACKNOWLEDGEMENTS 8
SYMBOFL IST OLS AND ABBREVIATION S 9
PAPERSORIGINAL 10
1. INTRODUCTION 11
2. MOLECULAROF THEORY SNOITCA RETNI 12
2.1. M ATHEMATICAL MUSINGS 12
ytiniff A.1.1.2 12
2.1.2. Kinetics 14
2.1.3. Thermodynamics 14
2.2. M INOOFLB EACSUILSA R TERACTIONS : THE ANTIBODY – ANTIGEN RECO GNITION 15
2.S3. PECIFICITY AND SELEC TIVITY 18
AFBFWIIEONAIKT 3Y. MOLECULAR INTERACTIO SN 19
3.1. C ELL - ADHESION MOLECULES 19
3.2T. - CELL RECEPTOR – /CHM PEPTIDE INTERACTION 20
3.W3. YTINIFF AKAE ANTIBODIES IN VIVO 21
3.4. C ETARDYHOBRA - CARBOHYDRATE INTERAC TIONS 22
3.A4. SNOITACILPP FOITLUM VALENT AGENTS 22
HM4TOE.OWA SUWREE INTEBIOMOLECULAR AK SNOITCAR 23
4.A1. NALYTICAL AFFINITY C HROMATOGRAPHY 23
stnatsno cytiniff af onoitanimrete D.1.1.4 24
stnatsno cciteni kf onoitanimrete D.2.1.4 24
4.O2. PTICAL REAL - EMIT BIOSENSORS BASED ON SURFACE PLASMON RES ONANCE 26
4.2.1. Detector basics 26
4.2.2. Evaluation of SPR biosensor data 27
snoitacilpp arosnesoi bRP S.3.2.4 28
4.3. M ISCELLANEOUS BIOMOLE CULAR INTERACTION AS SAYS 30
5. HOW TO UTILIZE WE AK BIOMOLECULAR INTE RACTIONS - THE PRESE NT
NOITAG ITSEVNI 33
5.W1. TAMOR HYCTINIF FKAAE OGRAPHY 33
5.W2. ECAFR UYSTINIF FKAAE PLASMON RESONANCE B IOSENSOR 36
5.2.1. Experimental design of weak affinity SPR biosensor experiments 36
gnisnesonumm isuounitno C.2.2.5 41
G N.I6KDRUALMCENROC S 46
REFERENCES 47
PAPERS I - V
A
CKNOWLEDGEMENTS

tuohtiW eht pleh dna gnidnatsrednu morf eht elpoep dnuora ,em eht yaw ots iht
siseht dluow evah neeb .elbassapmi I lliw syawla rebmemer eht sraey sa aDhP - student
htiw .yoj fO ,esruoc ti sah neeb drah ;krow etal thgin ,stfihs gniydc ells and leaking
,snmuloc tub I eveileb taht I evah neeb tsom .etanutrof I evah yllaer deyojne !flesymo T
esu ruoy sniarb dna sdnah ot erolpxe eht nwonknu dna ot eb dnuora trams elpoep nia
norivn egnitalumits .egelivir p adeedn is itnem
I ma yllaicepseg lufetar ot ym rosivrepus netS noslhO rof gnicudortni em ote ht
dlrow fo yzzuf .snoitcaretni netS sah neeb a ’dilos‘ troppus dna na yranidroartxee cruos
of in noitarips tuohguorht eht .sraey tI sah neeb gniticxe ot eb a trap fo ehtreve - growing
re search ac seitivit ta eht tnemtraped dna I knaht netS dna eht ytisrevinU fo ramlaKr of
.ytinutropp osiht
ehT tnemtraped sah syawla neeb a ecin ecalp ot eb ni sa ti si detibahni ybt aerg
elpoep dna I dluow ekil ot ekat siht ytinutroppo ot knaht uoy .lla I dluow osla like to
express special appreciation to:
ehT ™eTALeR :maet ,asiL rof gnieb eht tsecinmoor - etam ereht ;si ,airaM rof ruoy
ssenluferac dna ruo gnitalumits ;snoissucsid dna annaeJ dna ,ttolesiL rof ruoyt aerg
.stcejor py mo tsnoitubirtnoc
Stina Jungar, Carl - Fredrik Mandenius and Peter Påhlsson a t gnipökniLy tisrevinU
.shgua lyna mdn asnoitaroballo cluftiur frof
Mats Ohlin, Carl Borrebaeck and the rest of the staff at the Department of
.tnemegaruocn edn atroppu sro f,ytisrevin Udnu L,ygolonhcetonummI
llA eht tsap dna tneserp srebmem fo ehto lanigir :murdnarotkod ,nakåH Katarina,
Mikael , Sofie, Torleif and Ulf for giving me a great start.
Anne, Agnetha, Annika, Eva, Leif, Peter K., Sara, Stefan, Gleffe for solving all
kinds of problems.
The staff of the bioorganic chemistry lab for sauna a .stabmo cyellovhcae bdn
.n otnuo cna cuo yelpoe pgnie bro fsalki Ndn aeniloraC
yM stnerap attigriB dna essoB dna ym srehtorb srednA dna leakiM rof gniveilebn i
.em
.efi ly mf oevo leh tdn aflesre hgnie bro fitnah C,yllaniF

The revision of the manusc .detaicerpp ahcu ms iitnah Cdn asta M,net Sy btpir

000 2rebotc O,ramlaK

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L
S DLTNO SABFIAMOYS BBREVIATIONS

Ab eti sgnidni bydobitna IS acigolonummi espany sl
[Ab] total Ab concntration, see ITC noitarti tlamrehtosi
t
also Q yrtemirolac
xam
Ag antigen K association constant
A
ACE yrallipa cytiniffa k association rate constant
ass
electrophoresis K dissociation constant
D
MFA i mecro fcimota ypocsorc k dissociation rate constant
diss
CPA antigen - presenting cell Le x Lewis X antigen
MAC elucelo mnoisehd allec (Gal b 1 - 4[Fuc a 1 - cANclG]3 - b 1 - R)
CD cluster of differentiation wml thgie wralucelo mwol
RDC gninimrete dytiratnemelpmoc CL uqil yhpargotamorh cdi
region CHM ytilibitapmocotsi hrojam
EC siserohportcel eyrallipac xelpmoc
Da )lom/g (notlaD SM yrtemortcep sssam
ELISA onumm idekni lemyzne - Mw thgie wralucelom
yass atnebros RMN ecnanose rcitenga mraelcun
Fab fragment antigen gnidnib pnp p - lynehportin
Fuc fucose PEEK enotekrehterehteylop
vF fragment variable Q ,yticapa cgnidni bmumixam
xam
Gal galactose equivalent to [Ab]
t
Glc glucose RI refractive index
CLPH high performance LC R U resonance unit
HPLAC high performance liquid RPS surface plasmon resonance
yhpargotamorh cytiniffa scFv single chain Fv
HSA nimubl amure snamuh t half - life
½
ICAM intercellular adhesion RCT T - cell receptor
elucelom CAW otamorh cytiniff akaew yhparg
Ig nilubolgonummi AGW ninitulgg amre gtaehw

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O
RIGINAL PAPERS

The present thesis is based on the following papers, which will be referred to in the text
.slaremu nnamo Rrieh tyb

I. Strandh, M., Ohlin, M., Borrebaeck, C. A. K., and Ohlson, S. (1998).
weN hcaorppa ot diorets noitarapes desab no a wol ytiniffaMgI
.ydobitna
Journal of Immunological Methods 412 , 73 - 79.

II. Ohlson , S., Strandh, M., and Nilshans, H. (1997).
Detection and characterization of weak affinity antibody - antigen
recognition with biomolecular interaction analysis.
Journal of Molecular Recognition 01 , 135 - 138.

III. Strandh, M., Persson, B., Roos, H., a nd Ohlson, S. (1998).
Studies of interactions with weak affinities and low molecular weight
compounds using surface plasmon resonance technology.
Journal of Molecular Recognition 11 , 188 - 190.

IV. Ohlson, S., Jungar, C., Strandh, M., and Mandenius, C . - F. (2000).
suouknaietwnoC - affinity immunosensing.
sdneryTgo lnoinhcetoiB 81 , 55 - 58.

V. Jungar, C., Strandh, M., Ohlson, S., and Mandenius, C. - F. (2000).
sisylanA f osetardyho bgrnaicsu dyihupqairlgotamorhc - surface
plasmon resonance immunosensin g systems.
yrt sliamceihtcyoliaBnA 182 , 151 - 158.

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