Immunofluorescence in Clinical Immunology: A Primer and Atlas

231 Pages·2000·1.09 MB·English

by  Professor Dr. med. Wulf B. Storch (auth.)

Immunofluorescence
in Clinical Immunology
A Primer and Atlas
by
Wulf B. Storch
translated by
R.A. Klein
Springer Basel AG
Author: Translated by:
Professor Dr. med. Wulf B. Storch Dr. R.A. Klein
Netztal 32 Institut für Physiologische Chemie
D-69469 Weinheim Universität Bonn
Germany Nussallee 11
D-53115Bonn
Germany
Library of Congress Cataloging-in-Publication Data
Storch, Wulf.
[Immunofluoreszenzfibel. English]
Immunofluorescence in clinical immunology : a primer and atlas / by Wulf B. Storch ; trans
lated by R.A. Klein,
p. ; cm.
Includes bibliographical references and index.
ISBN 978-3-0348-9540-8
1. Fluorescent antibody technique. 2. Fluorescent antibody technique-Atlases. I. Title.
[DNLM: 1. Fluorescent Antibody Technique. 2. Antibodies-analysis. QW 525.5.F6 S884i 2000]
RB46.7.S7613 2000
616.07'98-dc21
99-059889
Die Deutsche Bibliothek - CIP-Einheitsaufnahme
Storch, Wulf:
Immunofluorescence in clinical immunology : a primer and atlas / by Wulf B. Storch. Transl. by
R. A. Klein. - Basel; Boston ; Berlin : Birkhäuser, 2000
Einheitssacht.: Immunofluoreszenzfibel <engl.>
ISBN 978-3-0348-9540-8 ISBN 978-3-0348-8376-4 (eBook)
DOI 10.1007/978-3-0348-8376-4
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The original edition entitled "Immunfluoreszenzfibel - Grundlagen und neue Anwendungen in
der klinischen Immunologie" was published in 1997 by Blackwell Wissenschafts-Verlag, Berlin -
Wien.
© 2000 Springer Basel AG
Originally published by Birkhäuser Verlag in 2000
Softcover reprint of the hardcover 1st edition 2000
ISBN 978-3-0348-9540-8
98765432 1
v
Preface
The release ofthe English translation of of the current state of knowledge. The
WulfStorch's primer on immunofluores fact that the clinical significance ofmany
cence is especially to be welcomed, since antibodies has not, or not yet, been de
this painstaking and clearly organised scribedfully is no argument against their
survey will thus be accessible to the existence. It must be left for the future to
widest possible readership. The author, show whether these possess a defined
whom Ihave known for more than three role in the pathogenesis of disease, or
decades, has worked tirelessly on im not.
proving the methods employed in immu This Primer is an illustration ofthe rele
nofluorescence and has pursued consis vance of immunofluorescence in the
tently the broad spectrum ofpossibilities spectrum of modern diagnostic techni
fortheir use indiagnosis. ques. Itis to be hoped that it will enjoy a
The abundance of proven antibodies, widecirculation.
many observed for the first time by Wulf
Storch, is an expression of the devotion
with which the author has applied to his Prof. Dr. med. G. Geiler
goal ofbringing about a complete survey Leipzig, February2000
Table of contents
Preface. . . .. .. . .. .. . .. .. . .. .. . .. . .. .. . .. ... .. .. .. . . .. . .. . .. . .. . V
ForewordtotheEnglishedition VII
Forewordtothe 2ndGermanedition IX
Abbreviations XV
1 Introductiontothebasicprinciplesandtechniquesofimmunofluor-
escence 1
1.1 Fluorescence, primaryandsecondaryfluorescence 1
1.2 Principles ofimmunofluorescence 1
1.3 Nomenclature used inimmunofluorescencestudies 3
1.4 Shortdescriptionofthe mostimportantproceduresfor directimmuno-
fluorescence 4
2 Productionofantibodiesandlabellingwithfluorochromes 5
2.1 Structure andfunction ofantibodies(immunoglobulins) . . .. . .. .. . . 5
2.2 Productionofantisera 6
2.3 Isolationofimmunoglobulinsfrom serum 8
2.4 Labellingofantiserawithfluorochromes 9
2.4.1 Fluorescentdyes (fluorochromes) . . .. ... .. .. . .. . .. . .. .. . .. .. . 9
2.4.1.1 Requirementsfor fluorochromes 9
2.4.1.2 Fluoresceiniso-thiocyanate(FITC) 9
2.4.1.3 Tetramethyl-rhodamine-iso-thiocyanate(TRITC) 10
2.4.1.4 Furtherfluorochromes 10
2.4.2 Couplingwith FITC .. . . .. .. . .. . .. .. ... . .. . .. . .. .. . .. .. .. .. 11
2.4.3 CouplingwithTRITC 12
2.5 Purificationofconjugates 13
2.5.1 Gel-filtrationwithSephadexG25 andG50 13
2.5.2 Gel-filtrationwith BiogelP6 15
2.5.3 FractionationonDEAE-SephadexA50 15
2.5.4 Adsorptionandimmunologicalpurification 15
2.6 Concentrationofdiluteproteinsolutions 16
3 Characterisationandqualitycontrolofantiseraandconjugates .. 19
3.1 Identificationdatafor conjugates 19
3.2 Methodsfor characterisingantisera 19
3.2.1 Testingfor free dye . .. .. . .. . .. .. . .. ... . .. . .. .. . .. . .. .. . .. . 19
3.2.2 Determinationofthefluorochrome-protein ratio(F/P quotient) 20
3.2.2.1 Determinationofthe FIP ratiofor FITC-conjugates 20
3.2.2.2 Determinationofthe molarfluorochrome-protein ratioforTRITe-la-
belled conjugates 23
XII
3.2.2.3 Determinationofthe F/P ratiofor conjugateslabelledwithotherdyes 23
3.2.3 Cellulose acetatethin-layerelectrophoresis 24
3.2.4 Testingthe activityofantiserausingdouble radialimmunodiffusionin
agargels 25
3.2.5 Determinationofthe antibodyconcentrationbyreversed radialimmu-
nodiffusion 26
3.2.6 Determinationofthe antibodyprotein contentusingstandard double
geldiffusion .. .. . .. . .. .. . .. . .. ... .. . .. .. .. .. .. .. . .. .. .. . 27
3.2.7 Specificitycontrolbycouplingantigens to artificialsubstrates,theso-
called DASSmethod 27
3.2.8 Specificitycontrolusingbiologicalsystems 28
3.2.8.1 Testingusingbonemarrowcells 28
3.2.8.2 Specialassaysfor membraneimmunofluorescence 29
3.3 Suitabilitytestinginspecialassaysystemswithdeterminationofthe
optimalworkingdilutionfor the conjugate 29
4 Actualopticalimmunofluorescencetechniques 35
4.1 Storage ofsera . .. . .. .. . .. . .. .. .. .. .. .. .. .. . .. .. .. . .. .. . . 35
4.2 Preparationand productionofsectionedspecimens 36
4.2.1 Sectioningtechnique 36
4.2.1.1 Cryotome-sectioningtechnique (cryomicrotomy) . .. .. .. .. .. .. . .. 36
4.2.1.2 Paraffinembedding 37
4.2.1.3 Freeze-drying 38
4.2.2 Immunohistologicalstaining 38
4.2.2.1 Carryingoutthe directmethod 39
4.2.2.2 Carryingoutthe simpleindirectmethodwithoutfixation atroomtem-
perature 40
4.2.2.3 Carryingoutthe "double-indirect" methodforthe detectionofcomple-
ment-bindingantibodies 40
4.2.2.4 Specialmethods 40
4.2.3 Background- and post- orcounter-staining 42
4.2.4 Embedding 42
4.3 Preparationand productionofspecimensfor membrane immunofluor-
escence 43
4.3.1 Membraneimmunofluorescenceforlymphocytes 43
4.3.1.1 Isolationoflymphocytes 43
4.3.1.2 Incubation 43
4.3.1.3 Embeddingand preparationofpermanent(longterm) specimens .. 43
4.3.2 Membraneimmunofluorescencefor livercells 44
4.3.2.1 Preparationofhepatocytes 44
4.3.2.2 Incubation 44
4.4 Undesirablefluorescence andspecificitycontrolsduringimmunofluor-
escenceinvestigations 44
4.4.1 Controls duringindirectimmunofluorescencefor the detectionofauto-
antibodies 46
4.4.2 Controlsin directimmunofluorescence 46
4.4.3 Controlsinthe detectionofcomplement-fixingantibodies . . .. .. . .. 46
5 Instrumentaltechniqueandthedocumentationoffindings 47
5.1 Microscope 47
5.2 Additionalequipment 51
XIII
5.2.1 Settingupfor photomicrography 51
5.2.2 Confocallaser-scanningfluorescence microscopy 53
6 Gener~advice 57
6.1 Sera, antibodies, conjugatesandantigenicsubstrates 57
6.2 Analysis ofresults .. .. .. .. . .. . .. .. . ... .. .. .. . .. .. . . . .. .. .. 57
7 Applicationsofimmunofluorescence .. .. . .. . .. . .. . ... 59
7.1 Overviewofthefield ofapplication .. . .. .. . . .. . ... 59
7.2 Detectionofimmunoglobulins, immunecomplexes, auto-, allo-, xeno-
and neo-antigensinbiopsymaterial . . ... .. .. .. . .. . .. . .. . .. ... 59
7.2.1 Kidneybiopsy. . .. .. .. . .. . .. .. . .. ... ... . . .. .. .. . .. . . .. . .. 59
7.2.2 Liverbiopsy 65
7.2.3 Skinbiopsy. . .. . .. .. .. . .. . .. ... .. ... ... . .. . .. . .. . .. . .. .. 70
7.3 Detectionofhumoralantibodies 72
7.3.1 Antibodies againstcellmembrane antigens 75
7.3.1.1 Antibodiesagainstlivercellmembranes 75
7.3.1.2 Antibodiesagainstthyroidcellmembranes 75
7.3.1.3 Antibodiesagainstmembranesofpancreaticisletcells 75
7.3.1.4 Antibodies againstlymphocytemembranes 75
7.3.1.5 Antibodies againsttumourcellmembrane antigens 76
7.3.2 Antibodies againstcomponents ofthe cytoplasm 76
7.3.2.1 Antibodies againstthyroidantigens 76
7.3.2.2 Antibodies againstthe cells ofthe stomach. .. . .. .. . . .. .. . ... . .. 79
7.3.2.2.1 Antibodiesagainstthe parietalcells ofthe stomach 79
7.3.2.2.2 Antibodies againstthe gastrin-producingcellsofthe stomach 80
7.3.2.2.3 Antibodiesagainstthe chiefcells ofthe stomach 80
7.3.2.3 Antibodiesagainstpancreas. . . .. .. . ... .. .. . .. . .. . .. .. . .. ... 81
7.3.2.3.1 Antibodiesagainstthe pancreaticisletcells (Langerhans) 81
7.3.2.3.2 Antibodiesagainstthe pancreaticacinuscells 82
7.3.2.4 Antibodies againststeroid-producingcellsinthe adrenal, testis, ovary
and placenta . .. .. .. . .. . .. .. . .. .. .. .. .. .. . .. . .. .. . .. .. ... 83
7.3.2.5 Antibodies againstcells ofthe parathyroid 83
7.3.2.6 Antibodiesagainstcellsofthe hypophysis 85
7.3.2.7 Antibodiesagainstcellsofthe hypothalamus 86
7.3.2.8 Antibodiesagainstthe efferentductsofthe salivaryglandexcretory
ducts 86
7.3.2.9 Antibodiesagainstkidneyantigens 87
7.3.2.9.1 Antibodiesagainstthe glomerular podocytes 87
7.3.2.9.2 Antibodies againstthe cells ofHenle'sloop. . .. .. . .. ... .. .. . .. .. 88
7.3.2.9.3 Antibodies againstcollectingtubules 88
7.3.2.9.4 Antibodiesagainstthe brush-borderofthe proximaltubulesin
ratkidney 91
7.3.2.9.5 Antibodies againstvariousproximaland distaltubules 92
7.3.2.10 Antibodies againstcellsofthegut. . . .. .. .. .. .. . .. . .. .. . .. . . .. 93
7.3.2.11 Antibodies againstthe endometrium 93
7.3.2.12 Antibodies againstnervous tissue. .. .. .. .. .. . .. . .. . .. .. . .. ... 97
7.3.2.12.1 Antibodies againstperipheralnerves 97
7.3.2.12.2 Antibodies againstAuerbach's plexus 100
7.3.2.12.3 Antibodiesagainstneurone cellnuclei 100
7.3.2.12.4AntibodiesagainstPurkinje cells 100
XIV
7.3.2.13 Antibodiesagainstthe retina . 101
7.3.2.14 Antibodiesagainstgranulocytesandmonocytes . 101
7.3.2.15 Antibodiesagainstthrombocytes . 105
7.3.2.16 Antibodiesagainstthe cytoplasmoflivercells . 106
7.3.2.17 Antibodiesagainstthe endoplasmicreticulum(so-calledliverand kid-
neymicrosomesLKM) . 108
7.3.2.18 Antibodiesagainstmitochondria . 112
7.3.2.19 Antibodiesagainstribosomes . 118
7.3.3 Antibodiesagainstcellnuclei . 118
7.3.3.1 Antibodiesshowingnuclearmembranefluorescence . 124
7.3.3.2 Antibodiesshowinghomogeneous nuclearfluorescence . 125
7.3.3.3 Antibodiesshowingspecklednuclearfluorescence . 127
7.3.3.4 Antibodiesshowingpunctatenuclearfluorescence . 130
7.3.3.5 Antibodiesshowingnucleolarnuclearfluorescence . 133
7.3.3.6 Antibodiesshowingmitosis-associatedfluorescence . 136
7.3.4 Antibodiesagainstcytoplasmicantigensincultured cells . 141
7.3.4.1 Antibodiesshowinggranularcytoplasmicfluorescence . 141
7.3.4.2 Antibodiesshowingfilamentous cytoplasmicfluorescence . 149
7.3.5 Antibodies againstspecialcomponents . 149
7.3.5.1 Antibodiesagainstspermatozoa . 149
7.3.5.2 Antibodiesagainstepidermis . 150
7.3.5.2.1 Antibodiesagainstthesurfaceofthe stratumcorneum . 152
7.3.5.2.2 Antibodiesagainstthe stratumcorneum . 152
7.3.5.2.3 Antibodiesagainstthe cellsurface(inter-cellularsubstance) especially
ofthe stratumspinosum("Pemphigus"-antibodies) . 153
7.3.5.2.4 Antibodiesagainstthe basalcellsofthe epidermis . 155
7.3.5.2.5 Antibodiesagainstthe basementmembrane betweenthe epidermis
and coriumofsquamousepithelium("Pemphigoid"-antibodies) . 156
7.3.5.2.6 Antibodiesagainstperinuclearantigens(perinuclearfactors) . 159
7.3.5.3 Antibodiesagainstsmoothmuscle . 160
7.3.5.4 Antibodiesagainstskeletalmuscle . 167
7.3.5.5 Antibodiesagainstcardiacmuscle . 171
7.3.5.6 Antibodies againstvascularendothelium . 173
7.3.5.7 Antibodiesagainstconnectivetissue . 177
7.3.5.8 Antibodiesagainstrenalglomerularbasementmembranes . 181
7.3.5.9 Antibodiesagainstrenaltubule basementmembranes . 182
7.3.5.10 Antibodiesagainstbileducts, bile ductepitheliumandbile canaliculi 184
7.4 Detectionofimmunoglobulinreceptorsonlymphocytes . 187
7.5 Immunofluorescenceusingartificialsphericalsubstrates . 190
7.5.1 DetectionofantibodiesagainstD-penicillamine . 191
7.6 DetectionofcomplementactivationbyC-reactiveprotein(CRP) . 193
Bibliography 197
Index 221
xv
Abbreviations
Ab antibody
Ab/P ratio ofantibodyprotein to totalprotein
AbIF ratio ofantibodyproteintofluorochrome content
ACA anti-centromere antibodies
ADC adrenalcortex
AER antibodiesagainstendoplasmicreticulum
Ag antigen(antigensubstrate)
AMA anti-mitochondrialantibodies
AMCA aminomethyl-coumarinyl-aceticacid
ANCA anti-neutrophilcytoplasmicantibodies
ANF anti-nuclearfactors (antibodies)
APA anti-podocyteantibodies
ARA anti-ribosomalantibodies
B "bursa-derived", thymus-independent
BBA brush-borderantibodies
BCA bile canaliculiantibodies
BM basementmembrane
BSA bovine serumalbumin
C1-C9 componentsofthe complementcascade
CAH chronicaggressive hepatitis
CBR complement-bindingreaction
CENP centromere nuclearprotein
CF complementfixation
CFR complement-fixingreaction
CTA-3 connectivetissueantibodies type 3
DAB DeutschesArzneibuch(GermanPharmacopoeia)
DASS definedantigenicsubstratespheres
DLE discoid Lupus erythematosus
DMSO dimethylsulphoxide
DNA desoxy-ribonucleicacid
DNP desoxy-ribonucleoprotein
ds-(RNA, DNA) double-stranded RNA orDNA
E extinction
EDTA ethylene-diamine-tetraaceticacid
FlAb ratio offluorochrome to antibodyprotein
F fluorochrome
F/P ratio offluorochrome to totalprotein
Fab, F(ab'h fragment antigen bindingfrom immunoglobulin
Fc fragment crystallisablefrom immunoglobulin
FDA fluorescein diacetate
FITC fluorescein iso-thiocyanate
XVI
gay average accelerationalconstantwhen centrifuging
GBM glomerularbasementmembrane
HBcAg hepatitisBcore antigen
HBeAg hepatitisBe-antigen
HBsAg hepatitisBsurfaceantigen
HLA histocompatibilityleukocyte antigens
IF immunofluorescence
Ig(G, A, M, D, E)immunoglobulinsG, A, M, Dand E
i.m. intra-muscular
Lv. intra-venous
kDa kilodaltons
LE Lupus erythematosus
LKM-Ab liver-kidneymicrosomalantibodies
LMA livercellmembrane antibodies
M.m. muscularismucosae
OD opticaldensity
PI, P2, P3 first, secondand third segments ofthe proximaltubulesin ratkidney
PBC primarybiliarycirrhosis
PBS phosphate-bufferedsaline
PCA parietalcellantibodies
PEP plateauend-point
PT plateautitre
RA rheumatoidarthritis
RNA ribonucleicacid
RNP ribonucleoprotein
RPM revolutions perminute
s second
s.c. subcutaneous
SLE systemic(disseminated) Lupus erythematosus
SMA smoothmuscleantibodies
ss-(RNA, DNA) single-strandedRNAorDNA
T "thymus-derived", thymus-dependent
TRITC tetramethy1-rhodamine-isothiocyanate
v/v compositionbyvolume
w/v compositioninpartsweightperunitvolume
w/w compositionbyweight
WHO World HealthOrganisation