Immunofluorescence in Clinical Immunology

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ISBN: 9783764361822 | 3764361824
Cover: Hardcover
Copyright: 8/1/2000

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Immunofluorescence, a suitable laboratory method for the microscopic demonstration of antigens and antibodies in biological materials, useable, for example, to provide evidence for the pathogenesis of disease in histological or cytological preparations and for tumour cell differentiation. For this reason immunofluorescence has a decisive role as the method of choice for the diagnosis of auto-immune diseases. This primer on immunofluorescence techniques, which first appeared in 1979, is a richly illustrated handbook suitable for everyday practical work in the laboratory, useable as both an introduction to the subject as well as an atlas. In hardly any other area of medicine are there so many new findings to report. The second edition of this book is concerned not only with the detection methods which now form an essential and established part of diagnostic techniques, but also with the most recent research results such as the discovery of antibodies against Auerbach's plexus and against podocytes...

Preface

Foreword to the English edition

vii

Foreword to the 2nd German edition

Abbreviations

Introduction to the basic principles and techniques of immunofluorescence

(4)

Fluorescence, primary and secondary flurescence

(1)

Principles of immunoflurescence

(2)

Nomenclature used in immunoflurescence studies

(1)

Short description of the most important procedures for direct immunoflurescence

(1)

Production of antibodies and labelling with flurochromes

(14)

Structure and function of antibodies (immunoglobulins)

(1)

Production of antisera

(2)

Isolation of immunoglobulins from serum

(1)

Labelling of antisera with flurochromes

(4)

Fluorescent dyes (fluorochromes)

(1)

Requirements for flurochromes

(1)

Flurescein iso-thiocyanate (FITC)

(1)

Tetramethyl-rhodamine-iso-thiocyanate (TRITC)

(1)

Further flurochromes

(1)

Coupling with FITC

(1)

Coupling with TRITC

(1)

Purification of conjugates

(3)

Gel-filtration with Sephadex G25 and G50

(2)

Gel-filtration with Biogel P6

(1)

Fractionation on DEAE-Sephadex A50

(1)

Adsorption and immunological purification

(1)

Concentration of dilute protein solutions

(3)

Characterisation and quality control of antisera and conjugates

(16)

Identification data for conjugates

(1)

Methods for characterising antisera

(10)

Testing for free dye

(1)

Determination of the fluorochrome-protein ratio (F/P quotient)

(1)

Determination of the F/P ratio for FITC-conjugates

(3)

Determination of the molar fluorochrome-protein ratio for TRITC-labelled conjugates

(1)

Determination of the F/P ratio for conjugates labelled with other dyes

(1)

Cellulose acetate thin-layer electrophoresis

(1)

Testing the activity of antisera using double radial immunodiffusion in agar gels

(1)

Determination of the antibody concentration by reversed radial immunodiffusion

(1)

Determination of the antibody protein content using standard double gel diffusion

(1)

Specificity control by coupling antigens to artificial substrates, the socalled DASS method

(1)

Specificity control using biological systems

(1)

Testing using bone marrow cells

(1)

Special assays for membrane immunoflurescence

(1)

Suitability testing in special assay systems with determination of the optimal working dilution for the conjugate

(6)

Actual optical immunofluorescence techniques

(12)

Storage of sera

(1)

Preparation and production of sectioned specimens

(7)

Sectioning technique

(1)

Cryotome-sectioning technique (cryomicrotomy)

(1)

Paraffin embedding

(1)

Freeze-drying

(1)

Immunohistological staining

(1)

Carrying out the direct method

(1)

Carrying out the simple indirect method without fixation at room temperature

(1)

Carrying out the ``double-indirect'' method for the detection of complement-binding antibodies

(1)

Special methods

(2)

Background- and post- or counter-staining

(1)

Embedding

(1)

Preparation and production of specimens for membrane immunofluorescence

(1)

Membrane immunofluorescence for lymphocytes

(1)

Isolation of lymphocytes

(1)

Incubation

(1)

Embedding and preparation of permanent (long term) specimens

(1)

Membrane immunoflurescence for liver cells

(1)

Preparation of hepatocytes

(1)

Incubation

(1)

Undesirable flurescence and specificity controls during immunofluorescence investigations

(3)

Controls during indirect immunoflurescence for the detection of autoantibodies

(1)

Controls in direct immunofluorescence

(1)

Controls in the detection of complement-fixing antibodies

(1)

Instrumental technique and the documentation of findings

(10)

Microscope

(4)

Additional equipment

(6)

Setting up for photomicrography

(2)

Confocal laser-scanning flurescence microscopy

(4)

General advice

(2)

Sera, antibodies, conjugates and antigenic substrates

(1)

Analysis of results

(2)

Application of immunoflurescence

(138)

Overview of the field of application

(1)

Detection of immunoglobulins, immune complexes, auto-, allo-, xeno-and neo-antigens in biopsy material

(13)

Kidney biopsy

(6)

Liver biopsy

(5)

Skin biopsy

(2)

Detection of humoral antibodies

(115)

Antibodies against cell membrane antigens

(1)

Antibodies against liver cell membranes

(1)

Antibodies against thyroid cell membranes

(1)

Antibodies against membranes of pancreatic islet cells

(1)

Antibodies against lymphocyte membranes

(1)

Antibodies against tumour cell membrane antigens

(1)

Antibodies against components of the cytoplasm

(1)

Antibodies against thyroid antigens

(3)

Antibodies against the cells of the stomach

(1)

Antibodies against the parietal cells of the stomach

(1)

Antibodies against the gastrin-producing cells of the stomach

(1)

Antibodies against the chief cells of the stomach

(1)

Antibodies against pancreas

(1)

Antibodies against the pancreatic islet cells (Langerhans)

(1)

Antibodies against the pancreatic acinus cells

(1)

Antibodies against steroid-producing cells in the adrenal, testis, ovary and placenta

(1)

Antibodies against cells of the parathyroid

(2)

Antibodies against cells of the hypophysis

(1)

Antibodies against cells of the hypothalamus

(1)

Antibodies against the efferent ducts of the salivary gland excretory ducts

(1)

Antibodies against kidney antigens

(1)

Antibodies against the glomerular podocytes

(1)

Antibodies against the cells of Henle's loop

(1)

Antibodies against collecting tubules

(3)

Antibodies against the brush-border of the proximal tubules in rat kidney

(1)

Antibodies against various proximal and distal tubules

(1)

Antibodies against cells of the gut

(1)

Antibodies against the endometrium

(4)

Antibodies against nervous tissue

(1)

Antibodies against peripheral nerves

(3)

Antibodies against Auerbach's plexus

100

(1)

Antibodies against neurone cell nuclei

100

(1)

Antibodies against Purkinje cells

100

(1)

Antibodies against the retina

101

(1)

Antibodies against granulocytes and monocytes

101

(4)

Antibodies against thrombocytes

105

(1)

Antibodies against the cytoplasm of liver cells

106

(2)

Antibodies against the endoplasmic reticulum (so-called liver and kidney microsomes LKM)

108

(4)

Antibodies against mitochondria

112

(6)

Antibodies against ribosomes

118

(1)

Antibodies against cell nuclei

118

(6)

Antibodies showing nuclear membrane fluorescence

124

(1)

Antibodies showing homogeneous nuclear fluorescence

125

(2)

Antibodies showing speckled nuclear fluorescence

127

(3)

Antibodies showing punctate nuclear fluorescence

130

(3)

Antibodies showing nucleolar nuclear fluorescence

133

(3)

Antibodies showing mitosis-associated fluorescence

136

(5)

Antibodies against cytoplasmic antigens in cultured cells

141

(1)

Antibodies showing granular cytoplasmic flurescence

141

(8)

Antibodies showing filamentous cytoplasmic fluorescence

149

(1)

Antibodies against special components

149

(1)

Antibodies against spermatozoa

149

(1)

Antibodies against epidermis

150

(2)

Antibodies against the surface of the stratum corneum

152

(1)

Antibodies against the stratum corneum

152

(1)

Antibodies against the cell surface (inter-cellular substance) especially of the stratum spinosum (``Pemphigus'' -antibodies)

153

(2)

Antibodies against the basal cells of the epidermis

155

(1)

Antibodies against the basement membrane between the epidermis and corium of squamous epithelium (``Pemphigoid''-antibodies)

156

(3)

Antibodies against perinuclear antigens (perinuclear factors)

159

(1)

Antibodies against smooth muscle

160

(7)

Antibodies against skeletal muscle

167

(4)

Antibodies against cardiac muscle

171

(2)

Antibodies against vascular endothelium

173

(4)

Antibodies against connective tissue

177

(4)

Antibodies against renal glomerular basement membranes

181

(1)

Antibodies against renal tubule basement membranes

182

(2)

Antibodies against bile ducts, bile duct epithelium and bile canaliculi

184

(3)

Detection of immunoglobulin receptors on lymphocytes

187

(3)

Immunofluorescence using artificial spherical substrates

190

(3)

Detection of antibodies against D-penicillamine

191

(2)

Detection of complement activation by C-reactive protein (CRP)

193

(4)

Bibliography

197

(24)

Index

221

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Immunofluorescence in Clinical Immunology

Summary

Table of Contents

Supplemental Materials

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