MANUAL OF BIOLOGICAL MARKERS OF DISEASE

MANUAL OF BIOLOGICAL MARKERS OF DISEASE

Edited by

W.J.VAN VENROOIJ University of Nijmegen

The Netherlands

R.N. MAINI The Mathilda and Terence Kennedy Institute of Rheumatology London. UK

~. ,. KLUWER ACADEMIC PUBLISHERS DORDRECHT / BOSTON / LONDON

Library of Congress Catalog Card Number: 88-198312

Supplement 2 ISBN 0-7923-3808--1

Manual ISBN 0-7923-2219-3

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Contents

Section A: METHODS OF AUTOANTIBODY DETECTION

Introduction The Editors

1. International cooperative activities in standardization of antinuclear antibodies

E.M. Tan

2. Detection of antinuclear antibodies by immunofluorescence R.L. Humbel

1. Introduction 2. The immunofluorescence assay

2.1 The commercial substrate 2.2 The culturing of HEp-2 cells 2.3 Fixation of the cells 2.4 Preparation of the serum sample 2.5 The conjugate antibodies 2.6 The incubation procedure

3. Interpretation of the results 3.1 Nuclear fluorescence patterns 3.2 Nucleolar fluorescence patterns 3.3 Spindle apparatus fluorescence patterns 3.4 Cytoplasmic fluorescence patterns

4. Solutions and suppliers 4.1 Solutions for culturing the cells

3. Counterimmunoelectrophoresis and immunodiffusion for the detection of antibodies to soluble cellular antigens

C. Bunn and T. Kveder

1. Introduction 2. Antigen extract preparations

2.1 Extraction from acetone powders 2.2 Whole cell extract 2.3 Spleen extract for Ro 2.4 Liver extract for aminoacyl-tRNA synthetases 2.5 Nuclear extract 2.6 Ion-exchange chromatography

3. Procedures 3.1 Immunodiffusion 3.2 Counterimmunoelectrophoresis (CIE) 3.3 Staining with coomassie blue 3.4 Test planning and interpretation

4. Protein Blotting R. Verheijen, M. Salden and W.J. van Venrooij

1. Introduction 2. The antigens

2.1 Preparation of nuclear extracts 2.2 Preparation of cytoplasmic extracts

3. SDS polyacrylamide gel electrophoresis 3.1 Buffer selection 3.2 Power conditions

4. Protein blotting 4.1 Membrane selection 4.2 Blotting filter paper 4.3 Buffer selection 4.4 Semi-dry blotting 4.5 Tank blotting 4.6 Total protein staining 4.7 Blocking reagents 4.8 Serum samples 4.9 Immunoblot assay

5. Antigen appearance on the immunoblot 5.1 Nuclear antigens 5.2 Nucleolar antigens 5.3 Cytoplasmic antigens

5. Enzyme-linked immunosorbant assay in the rheumatological laboratory

P.J. Charles and R.N. Maini

1. Introduction 2. General notes

2.1 Solid phase 2.2 Conjugates 2.3 Substrates 2.4 Blocking agents 2.5 Antigens and antigen presentation

3. Assay methods 3.1 Solid phase ELISA 3.2 The chequerboard titration 3.3 Antigen capture (sandwich) ELISA

3.4 Dot immunobinding (dot blot) assay 4. Evaluation and improvement of the assay system

4.1 Evaluation 4.2 Improvement of the assay system

5. Validation of the assay system 6. Validation of a commercial assay system 7. Summary 8. Solutions and materials

8.1 Materials 8.2 Coating buffers 8.3 Dilution/washing buffer 8.4 Chromogenic substrate solution 8.5 Stop solutions

9. Laboratory notes on ELISA systems for the detection of anti-nuclear, anti-cytoplasmic and anti-phospholipid antibodies

9.1 Anti-nuclear antibodies 9.2 Anti-dsDNA 9.3 Anti-histone 9.4 Anti-Ro/SS-A 9.5 Anti-La 9.6 Anti-nRNP 9.7 Anti-Sm 9.8 Anti-ribosomal RNP (P protein) 9.9 Anti-Scl70 (Topoisomerase 1) 9.10 Anti-Ki 9.11 Anti-Jo-l 9.12 Anti-Cardiolipin 9.13 Anti-Centromere

6. Immunoprecipitation of labelled proteins E.M. Tan and C.L. Peebles

1. Introduction 2. Protein immunoprecipitation procedures

2.1 Preparation of 3sS-methionine radiolabeled extract 2.2 Preparation of the gels

3. Interpretation 3.1 Sm and UIRNP 3.2 SS-B/La and SS-A/Ro 3.3 PCNA, Jo-l, rRNP and Ku

4. Materials 4.1 Reagents and equipment

5. Solutions 5.1 Tissue culture media 5.2 Acrylamide solutions and electrophoresis buffers

5.3 Other buffers and solutions

7. Analysis of autoimmune sera by immunoprecipitation of cellular RNPs

O. Zieve, M. Fury and E.J.R. Jansen

1. Introduction 2. Preparation of cellular extracts

2.1 Radioactive labeling of RNAs 2.2 Cell fractionation

3. Immunoprecipitation 3.1 Binding of antibodies to agarose beads and

immunoprecipitation of RNP complexes 4. RNA gel electrophoresis

4.1 10OJo acrylamide/S.3 M urea gel 4.2 6-15% gradient acrylamide gels 4.3 Silver staining procedure

5. Reagents and suppliers

8. Measurement of antibodies to DNA R. Smeenk

1. Introduction 2. Procedures

2.1 ELISA 2.2 1FT on Crithidia luciliae 2.3 PEG assay 2.4 Farr assay

3. Reagents and solutions 3.1 ELISA 3.2 1FT on Crithidia luciliae 3.3 PEG assay 3.4 Farr assay

9. Methods to detect autoantibodies to neutrophilic granulocytes A. Wiik, N. Rasmussen and J. Wieslander

1. Introduction 2. Demonstration of neutrophil-reactive autoantibodies by lIF

2.1 Steps in the procedure 2.2 Solutions 2.3 Interpretation, artifacts, control cells

3. EIA procedures 3.1 Preparation of a-granules as described by Borregaard

et al. 3.2 EIA for quantification of ANCA using a-granules

3.3 EIA for quantification of Proteinase-3 (PR-3) ANCA 3.4 EIA for quantification of myeloperoxidase (MPO) ANCA

10. Detection of antiperinuciear factor and anti keratin antibodies R.M.A. Hoet

1. Introduction 2. Detection of the APF

2.1 Antigen substrate and their donors 2.2 Preparation of buccal mucosa cells for

immunofluorescence 2.3 Serum dilution and immunofluorescence procedure 2.4 Criteria for positivity and reproducibility of the test 2.5 Reagents and solutions

3. Detection of antikeratin antibodies (AKA) 3.1 Antigen substrate 3.2 Serum dilution and immunofluorescence procedure 3.3 Criteria for positivity of the AKA test 3.4 Reagents and solutions

11. Standards and reference preparations T.E. W. Fe/tkamp

1. Introduction 2. Standards 3. Reference preparations 4. How to use a standard 5. Why are standards often not used? 6. Examples of the use of standards 7. Characteristics of standards and reference preparations 8. How to obtain the standards?

Contents

Section B: Autoantigens

Introduction The Editors

1. Autoantigens in Rheumatoid Arthritis (RA)

1. RF (rheumatoId factor) R.A. Mageed 2. APF (antiperinuclear factor) J.-M. Berthelot. C. Vincent.

3. RA-33 (hnRNP-A2) 4. Collagen II 5. The autoantigens defined by

antikeratin antibodies (AKA)

G. Serre & P. Youinou G. Steiner D. G. Williams C. Vincent. J.-M. Berthelot. P. Youinou and G. Serre

2. Autoantigens in Systemic Lupus Erythematosus (SLE)

1. DNA R.J. T. Smeenk 2. Histones R. W Burlingame & R. L. Rubin 3. Ubiquitin S. Muller 4. Sm S.O. Hoch 5. Ribosomal RNP K.B. Elkon 6. Ku WH. Reeves. M. Satol!. J.

A.K. Ajmani 7. peNA Y. Muro & EM. Tan 8. Phospholipids EN. Harris

3. Autoantigens in SLE-overlap syndromes

1. The U I snRNP complex J. M. T. Klein Gunnell'iek & WJ. van Venrooij

4. Autoantigens in Sjogren's syndrome

I. Ro/SS-A 2. La/SS-B

EK.L. Chan & J.P. Buyon G.J. M. Pruijn

Wang &

5. Autoantigens in Scleroderma

1. DNA Topoisomerase 1 2. Centromere Proteins 3. Fibrillarin 4. PM/Sci

6. Autoantigens in Myositis

1. Transfer RNA synthetases

7. Autoantigens in Vasculitis

l. Proteinase 3 (c-ANCA) 2. Myeloperoxidase (MPO)

8. Autoantigens in other diseases

R. Verheijen R. Verheijen R. Verheijen F.A. Baut:: & M. Bliihtner

P. Plot:: & I.N. Targoff

J. Wieslander & A. Wiik J. Wieslander & A. Wiik

l. Mitochondrial autoantigens P.S.c. Leung, I. Mackay & M.E. Gershwin

2. P80 coilin M. Carmo-Fonseca, K. Bollmann, M. T. Carvalho & A.I. Lamond

Contents

Section C: Clinical Significance of Autoantibodies

Introduction The Editors

1. Autoantibodies in Rheumatoid Arthritis:

1.1 Rheumatoid factor 1.2 Antiperinuclear factor and

anti-keratin antibodies 1.3 Anti-RA33 antibodies 1.4 Anti-collagen antibodies

2. Autoantibodies in SLE

2.1 Autoantibodies to double-stranded DNA

2.2 Autoantibodies to histones, Sm and ubiquitins

2.3 Autoantibodies to Ku and related antigens

2.4 Autoantibodies to PCNA 2.5 Autoantibodies to

phospholipids

J.s. Smolen

A.J. G. Swaak & R.J. T. Smeenk

R. M. Bernstein

w.H. Reeves, M. Satoh & J J. Langdon Y. Takasaki & E.M. Tan V Morris & C. Mackworth- Young

3. Autoantibodies in SLE-overlap syndromes:

3.1 Autoantibodies to UlsnRNP

F.H.f. van den Hoogen & L.B.A. van de Putte

4. Autoantibodies in Sjogren's syndrome: Anti-Ro/SSA and A.G. T::ioufas & H.M. Moutsopoulos anti-La/SSB antibodies

5. Autoantibodies in Scleroderma:

5.1 Anti-centromere antibodies N.F. Rotlifleld 5.2 Anti-DNA

topoisomerase I antibodies 5.3 Anti-RNA polymerase antibodies 5.4 Anti-nucleolar and other autoantibodies

6. Autoantibodies in Myositis:

6.1 Transfer RNA synthetases I.N. Targoff & P.H. Plot::

7. Autoantibodies in Vasculitis:

7.1 Anti-neutrophil cytoplasmic C. G. M. Kallenberg antibody

8. Autoantibodies in other diseases:

8.1 Anti-mitochondrial antibody in primary biliary cirrhosis

8.2 Autoantibodies to the coiled body and other nuclear bodies

I.R.Mackay & M.E. Gershwin

L. E. C. Andrade