Autoantibodies: Methods and Protocols

Gunnar Houen
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Methods in
Molecular Biology 1901

Gunnar Houen Editor

Auto-
antibodies
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Autoantibodies

Methods and Protocols

Edited by

Gunnar Houen
Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense,
Denmark
Editor
Gunnar Houen
Department of Autoimmunology
Statens Serum Institut
Copenhagen, Denmark
Department of Biochemistry and Molecular Biology
University of Southern Denmark
Odense, Denmark

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8948-5 ISBN 978-1-4939-8949-2 (eBook)
https://doi.org/10.1007/978-1-4939-8949-2
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Preface

Autoimmune diseases are a heterogeneous group of unknown etiology. Very broadly, they
have traditionally been divided in systemic rheumatic connective tissue diseases and organ-
specific diseases. This distinction is somewhat arbitrary as the diseases often overlap with
each other.
Generally, it is known that autoimmune diseases are caused by a combination of genetic
predisposing factors in combination with environmental factors.
Due to the importance of autoimmune diseases, methods for their discovery, character-
ization, use in diagnostics, and possible pathophysiological roles are extremely important.
This volume of MiMB covers methods for determination of rheumatic connective tissue
diseases and organ-specific diseases. It is intended to be helpful for all persons working with
research and development of autoimmune laboratory diagnostics and for clinicians using
autoantibody tests in daily work with patients.
I thank all contributors and editorial staff, who have been helpful during the work. Also,
I thank all my students and collaborators through the years.
A special thanks to my wife Eva for support in difficult times.
Finally, I am grateful to the series editor, John Walker, for support and for giving me the
opportunity to edit this volume of MiMB.

Copenhagen, Denmark Gunnar Houen

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Autoantibodies as Diagnostic Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Gunnar Houen
2 Nonspecific Binding in Immunoassays for Autoantibodies . . . . . . . . . . . . . . . . . . . 13
Gunnar Houen
3 Detection of Autoantibodies by Indirect Immunofluorescence
Cytochemistry on Hep-2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Alessandra Dellavance and Luis Eduardo Coelho Andrade
4 Detection of Anti-neutrophil Cytoplasmic Antibodies (ANCA)
by Indirect Immunofluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
J. J. B. C. van Beers, J. Vanderlocht, C. Roozendaal,
and J. Damoiseaux
5 Determination of Subset-Restricted Anti-neutrophil Cytoplasmic
Antibodies (ANCA) by Immunofluorescence Cytochemistry . . . . . . . . . . . . . . . . . 63
Firoozeh Amirbeagi, Amanda Welin, Pontus Thulin,
and Johan Bylund
6 Determination of Anti-aquaporin 5 Autoantibodies
by Immunofluorescence Cytochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Jehan Alam, Sumin Jeon, and Youngnim Choi
7 Determination of cN1A Autoantibodies by Cell-Based
Immunofluorescence Cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Satoshi Yamashita and Nozomu Tawara
8 Determination of Agonistically Acting Autoantibodies to the
Adrenergic Beta-1 Receptor by Cellular Bioassay. . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Hanna Davideit, Annekathrin Haberland, Sabine Bartel,
Sarah Schulze-Rothe, Johannes Müller, and Katrin Wenzel
9 Determination of Autoantibodies to Salivary Gland Antigens. . . . . . . . . . . . . . . . . 103
Li Cui, Xinyuan Zhao, and Shen Hu
10 Measurement of Autoantibodies to Gastric H+,K+-ATPase
(ATP4A/B) Using a Luciferase Immunoprecipitation
System (LIPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Edith Lahner, Ilaria Marzinotto, Cristina Brigatti,
Howard Davidson, Janet Wenzlau, Lorenzo Piemonti,
Bruno Annibale, and Vito Lampasona
11 Detection of DNA Autoantibodies by Electrophoretic Mobility
Shift Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Jacqueline Keyhani and Ezzatollah Keyhani

vii
viii Contents

12 Antigen-Specific Detection of Autoantibodies Against Myeloperoxidase

(MPO) and Proteinase 3 (PR3). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
J. Vanderlocht, J. J. B.C. van Beers, P. C. Limburg,
J. Damoiseaux, and C. Roozendaal
13 Analysis of C3 Nephritic Factors by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Lillemor Skattum
14 Analysis of Anti-C1q Autoantibodies by Western Blot . . . . . . . . . . . . . . . . . . . . . . . 183
Anci Verlemyr, Lennart Truedsson, and Lillemor Skattum
15 Anti-factor H Autoantibodies Assay by ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Marie Sénant and Marie-Agnes Dragon-Durey
16 Determination of Autoantibodies to Transglutaminase
by Electrochemiluminescence (ECL) Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Zhiyuan Zhao, Yong Gu, Dongmei Miao, Eric Hoffmeyer,
Yu Liu, and Liping Yu
17 Determination of CRP Autoantibodies by SPR Immunoassay . . . . . . . . . . . . . . . . 205
Qiu-Yu Li and Hai-Yun Li
18 Histone Protein Epitope Mapping for Autoantibody Recognition
in Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Feliciana Real-Fernández, Federico Pratesi, Paola Migliorini,
Paolo Rovero
19 Detection of SSA and SSB Antibodies Associated with Primary
Sjögren’s Syndrome Using Enzyme-Linked Immunosorbent
Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Nicole Hartwig Trier
20 Assessment of Peptidylarginine Deiminase Activity by ELISA
Using Human Fibrinogen as Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Dres Damgaard and Claus H. Nielsen
21 Use of a Citrullinated Peptide Panel for Detection of Anti-Citrullinated
Protein Antibodies by Enzyme-Linked Immunosorbent Assay. . . . . . . . . . . . . . . . 243
Nicole Hartwig Trier
22 Determination of Autoantibodies by Line Immunoblotting . . . . . . . . . . . . . . . . . . 255
Louise Sternbæk, Tina Friis, and Gunnar Houen
23 Determination of Rheumatoid Factors by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Nicole Hartwig Trier and Gunnar Houen
24 Detection of Autoantibodies to Complement Components
by Surface Plasmon Resonance-Based Technology . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Remi Noe, Sophie Chauvet, Shambhuprasad K. Togarsimalemath,
Maria Chiara Marinozzi, Maria Radanova, Vasil V. Vasilev,
Veronique Fremeaux-Bacchi, Marie-Agnes Dragon-Durey,
and Lubka T. Roumenina

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Contributors

JEHAN ALAM  School of Dentistry, Dental Research Institute, Seoul National University,
Seoul, Republic of Korea; Mary and Dick Holland Regenerative Medicine Program,
University of Nebraska Medical Center, Omaha, NE, USA
FIROOZEH AMIRBEAGI  Clinical Immunology and Transfusion Medicine, Sahlgrenska
University Hospital, Gothenburg, Sweden
LUIS EDUARDO COELHO ANDRADE  Rheumatology Division, Escola Paulista de Medicina,
Universidade Federal de São Paulo, São Paulo, Brazil; Immunology Division, Fleury
Medicine and Health Laboratories, São Paulo, Brazil
BRUNO ANNIBALE  Medical-Surgical Department of Clinical Sciences and Translational
Medicine, Sant’Andrea Hospital, Sapienza University of Rome, Rome, Italy
SABINE BARTEL  Berlin Cures GmbH, Berlin, Germany
CRISTINA BRIGATTI  Beta Cell Biology Unit, Diabetes Research Institute, IRCCS San
Raffaele Scientific Institute, Milan, Italy
JOHAN BYLUND  Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska
Academy at University of Gothenburg, Gothenburg, Sweden
SOPHIE CHAUVET  INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Paris,
France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France; Université Paris
Descartes, Sorbonne Paris Cité, Paris, France; Assistance Publique–Hôpitaux de Paris,
Service de néphrologie, Hôpital Européen Georges Pompidou, Paris, France
YOUNGNIM CHOI  School of Dentistry, Dental Research Institute, Seoul National University,
Seoul, Republic of Korea
LI CUI  UCLA School of Dentistry, Los Angeles, CA, USA; UCLA Jonsson Comprehensive
Cancer Center, Los Angeles, CA, USA
DRES DAMGAARD  Institute for Inflammation Research, Center for Rheumatology and Spine
Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; Section
for Periodontology, Microbiology and Community Dentistry, Department of Odontology,
Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
J. DAMOISEAUX  Central Diagnostic Laboratory, Maastricht University Medical Centre,
Maastricht, The Netherlands
HANNA DAVIDEIT  Berlin Cures GmbH, Berlin, Germany
HOWARD DAVIDSON  Barbara Davis Center for Diabetes, University of Colorado Anschutz
Medical Campus, Aurora, CO, USA
ALESSANDRA DELLAVANCE  Research and Development Division, Fleury Medicine and Health
Laboratories, São Paulo, Brazil
MARIE-AGNES DRAGON-DUREY  INSERM, UMR_S 1138, Centre de Recherche des
Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France;
Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Assistance
Publique–Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital Européen Georges
Pompidou, Paris, France

ix
x Contributors

VERONIQUE FREMEAUX-BACCHI  INSERM, UMR_S 1138, Centre de Recherche des

Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France;
Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Assistance
Publique–Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital Européen Georges
Pompidou, Paris, France
TINA FRIIS  Department of Autoimmunology, Statens Serum Institut, Copenhagen,
Denmark
YONG GU  Barbara Davis Center for Childhood Diabetes, University of Colorado, Aurora,
CO, USA; Department of Endocrinology, First Affiliated Hospital of Nanjing Medical
University, Nanjing, Jiangsu, China
ANNEKATHRIN HABERLAND  Berlin Cures GmbH, Berlin, Germany
ERIC HOFFMEYER  Barbara Davis Center for Childhood Diabetes, University of Colorado,
Aurora, CO, USA
GUNNAR HOUEN  Department of Autoimmunology, Statens Serum Institut, Copenhagen,
Denmark; Department of Biochemistry and Molecular Biology, University of Southern
Denmark, Odense, Denmark
SHEN HU  UCLA School of Dentistry, Los Angeles, CA, USA; UCLA Jonsson Comprehensive
Cancer Center, Los Angeles, CA, USA
SUMIN JEON  School of Dentistry, Dental Research Institute, Seoul National University,
Seoul, Republic of Korea
EZZATOLLAH KEYHANI  Laboratory for Life Sciences, Tehran, Iran
JACQUELINE KEYHANI  Laboratory for Life Sciences, Tehran, Iran
EDITH LAHNER  Medical-Surgical Department of Clinical Sciences and Translational
Medicine, Sant’Andrea Hospital, Sapienza University of Rome, Rome, Italy
VITO LAMPASONA  Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific
Institute, Milan, Italy
HAI-YUN LI  MOE Key Laboratory of Environment and Genes Related to Diseases, School of
Basic Medical Sciences, Xi’an Jiaotong University, Xi’an, Shanxi, People’s Republic of
China
QIU-YU LI  Department of Respiratory and Critical Care Medicine, Peking University
Third Hospital, Beijing, People’s Republic of China
P. C. LIMBURG  Department of Laboratory Medicine, University Medical Center Groningen,
University of Groningen, Groningen, The Netherlands
YU LIU  Department of Endocrinology, The Second Hospital of Jilin University, Jilin, China
MARIA CHIARA MARINOZZI  INSERM, UMR_S 1138, Centre de Recherche des Cordeliers,
Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France; Université Paris
Descartes, Sorbonne Paris Cité, Paris, France
ILARIA MARZINOTTO  Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific
Institute, Milan, Italy
DONGMEI MIAO  Barbara Davis Center for Childhood Diabetes, University of Colorado,
Aurora, CO, USA
PAOLA MIGLIORINI  Clinical Immunology and Allergy Unit, Department of Clinical and
Experimental Medicine, University of Pisa, Pisa, Italy
JOHANNES MÜLLER  Berlin Cures GmbH, Berlin, Germany
CLAUS H. NIELSEN  Institute for Inflammation Research, Center for Rheumatology and
Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark;
Section for Periodontology, Microbiology and Community Dentistry, Department of
Odontology, Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark
 Contributors xi

REMI NOE  INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France;
Sorbonne Universités, UPMC Univ Paris 06, Paris, France; Université Paris Descartes,
Sorbonne Paris Cité, Paris, France
LORENZO PIEMONTI  Beta Cell Biology Unit, Diabetes Research Institute, IRCCS San
Raffaele Scientific Institute, Milan, Italy
FEDERICO PRATESI  Clinical Immunology and Allergy Unit, Department of Clinical and
Experimental Medicine, University of Pisa, Pisa, Italy
MARIA RADANOVA  Department of Biochemistry, Molecular Medicine and Nutrigenomics,
Medical University of Varna, Varna, Bulgaria
FELICIANA REAL-FERNÁNDEZ  Laboratory of Peptide and Protein Chemistry and Biology,
Division of Pharmaceutical Sciences and Nutraceutics, Department of Neurofarba,
University of Florence, Sesto Fiorentino, Italy
C. ROOZENDAAL  Department of Laboratory Medicine, University Medical Centre
Groningen, University of Groningen, Groningen, The Netherlands
LUBKA T. ROUMENINA  INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Paris,
France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France; Université Paris
Descartes, Sorbonne Paris Cité, Paris, France
PAOLO ROVERO  Laboratory of Peptide and Protein Chemistry and Biology, Division of
Pharmaceutical Sciences and Nutraceutics, Department of Neurofarba, University of
Florence, Sesto Fiorentino, Italy
SARAH SCHULZE-ROTHE  Berlin Cures GmbH, Berlin, Germany
MARIE SÉNANT  Laboratoire d’Immunologie, Hôpital Européen Georges Pompidou, Paris,
France; Faculté de Médecine, Université Paris Descartes, Paris, France
LILLEMOR SKATTUM  Department of Laboratory Medicine, Section of Microbiology,
Immunology and Glycobiology, Lund University, Lund, Sweden; Clinical Immunology and
Transfusion Medicine, Region Skåne, Sweden
LOUISE STERNBÆK  Phase Holographic Imaging, Lund, Sweden
NOZOMU TAWARA  Department of Neurology, Graduate School of Medical Sciences,
Kumamoto University, Kumamoto, Japan
PONTUS THULIN  Clinical Immunology and Transfusion Medicine, Sahlgrenska University
Hospital, Gothenburg, Sweden
SHAMBHUPRASAD K. TOGARSIMALEMATH  INSERM, UMR_S 1138, Centre de Recherche des
Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France;
Université Paris Descartes, Sorbonne Paris Cité, Paris, France
NICOLE HARTWIG TRIER  Department of Autoimmunology and Biomarkers, Statens Serum
Institut, Copenhagen, Denmark
LENNART TRUEDSSON  Department of Laboratory Medicine, Section of Microbiology,
Immunology and Glycobiology, Lund University, Lund, Sweden
J. J. B. C. VAN BEERS  Central Diagnostic Laboratory, Maastricht University Medical
Centre, Maastricht, The Netherlands
J. VANDERLOCHT  Central Diagnostic Laboratory, Maastricht University Medical Centre,
Maastricht, The Netherlands
VASIL V. VASILEV  Nephrology Clinic, University Hospital ‘Tsaritsa Yoanna-ISUL’, Medical
University, Sofia, Bulgaria
ANCI VERLEMYR  Clinical Immunology and Transfusion Medicine, Region Skåne, Sweden
AMANDA WELIN  Rheumatology and Inflammation Research, Institute of Medicine,
Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
KATRIN WENZEL  Berlin Cures GmbH, Berlin, Germany
xii Contributors

JANET WENZLAU  Barbara Davis Center for Diabetes, University of Colorado Anschutz
Medical Campus, Aurora, CO, USA
SATOSHI YAMASHITA  Department of Neurology, Graduate School of Medical Sciences,
Kumamoto University, Kumamoto, Japan
LIPING YU  Barbara Davis Center for Childhood Diabetes, University of Colorado, Aurora,
CO, USA
XINYUAN ZHAO  Department of Endodontics, Stomatological Hospital of Southern Medical
University and Guangdong Provincial Stomatological Hospital, Guangzhou, China
ZHIYUAN ZHAO  Barbara Davis Center for Childhood Diabetes, University of Colorado,
Aurora, CO, USA; Department of Endocrinology, The Second Hospital of Jilin University,
Jilin, China
 Chapter 1

Autoantibodies as Diagnostic Tools

Gunnar Houen

Abstract
Autoimmune diseases are very diverse and include many common diseases of unknown etiology.
Diagnosis can be challenging but can be facilitated by the identification of characteristic autoantibodies
(AuAbs), which are present in varying frequencies. Identification of such AuAbs requires a range of different
techniques, depending on the autoantigens in question. Each individual AuAb assay is characterized by
analytical sensitivity and specificity, which in turn determines clinical sensitivity and specificity in relation to
diseases. Clinical sensitivities and specificities vary much, but many AuAb analyses can be of significant help
in establishing correct diagnoses. It remains unsettled whether AuAbs are generally pathogenic, but it is
generally agreed that autoimmune diseases are caused by a combination of genetic and environmental
factors, and that early and correct diagnosis facilitates treatment.

Key words Autoantibodies, Autoantigens, Autoimmune diseases, Diagnostics, Sensitivity, Specificity,

ROC curve

1 Introduction

Autoimmune diseases (ADs) are a diverse collection including

many common diseases like rheumatoid arthritis (RA), diabetes
type 1, multiple sclerosis, and many more (Table 1) [20, 21]. Tradi-
tionally, autoimmune diseases have been divided in systemic auto-
immune diseases (SADs), also denoted rheumatic or connective
tissue diseases (CTDs) and organ-specific autoimmune diseases
(OSADs). However, this distinction is quite artificial, as many of
the diseases overlap in several respects and often occur together.
Consequently, many (or all) of these diseases may be regarded as
syndromes, and some are actually named as such (e.g., Sjögren
syndrome (SS), Goodpasture syndrome, anti-phospholipid syn-
drome (APS, Hughes’ syndrome), and paraneoplastic syndromes
(Table 1).
The etiology of ADs appears to be complex and several theories
exist, including loss of self-tolerance, infections, bystander activa-
tion, molecular mimicry, self-antigen (posttranslational) modifica-
tion, epigenetic changes, and more [20–26]. These theories are not

Gunnar Houen (ed.), Autoantibodies: Methods and Protocols, Methods in Molecular Biology, vol. 1901,
https://doi.org/10.1007/978-1-4939-8949-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

1
2 Gunnar Houen

Table 1
Examples of autoimmune diseases and associated autoantibodies (see Note 11)

Chapters,
Disease AuAbs Assays [references]
Anti-phospholipid syndrome PL/β2GPI Abs ELISA, TLCIB [1–5]
(APS)
Autoimmune dilated Aβ1R Abs Bioassay 8
cardiomyopathy
Autoimmune encephalitis NMDAR Abs ICC [6]
Celiac disease TGase Abs ECLIA 16, [7]
Diabetes type 1 Insulin Abs, GAD65 IHC, RIA, ECL [8–10]
Abs
Goodpasture syndrome Collagen IV Abs ELISA, ECL [11, 12]
Granulomatosis with ANCA, PR3 Abs ICC, ELISA 4, 5, and 12
polyangiitis (GPA)
Inclusion body myositis ANA, cN1A ICC, LIA 3, 7, and 22
Multiple sclerosis MBP Abs, MOG abs ICC [13–16]
Paraneoplastic syndromes Multiple AuAbs ICC, IHC, ELISA, LIA, 22, [6, 17, 18]
(PNS) RIA
Pernicious anemia/ H+, K+-ATPase Abs IP, IHC, ELISA, RIA 10, [19]
autoimmune gastritis
Rheumatoid arthritis (RA) RFs, CdAbs ELISA 18, 20, 21, and 23
Sjögreen syndrome (SS) ANA, SSA/B Abs, ICC, ELISA, LIA, Ag array 3, 6, 19, and 22
Aqp5 Abs
Systemic lupus ANA, DNA Abs, C’ ICC, ELISA, LIA, EMSA, 3, 11, 13, 14, 15, 19,
erythematosus (SLE) Abs SPR, WIB and 22
Scleroderma ANA ICC, ELISA, LIA 3 and 22
Abbreviations: Ab antibody, Aβ1R adrenergic beta-1 receptor, ACA anti-cell antibodies, Ag antigen, ANA anti-nuclear
antibodies, ANCA anti-neutrophil granulocyte cytoplasm antibodies, Aqp aquaporin, AuAb autoantibody, C0 comple-
ment components, C3NF C3 nephritic factor, CdAbs citrulline-dependent antibodies, CRP C-reactive protein, ECL
enhanced chemiluminescence, ECLIA electrochemiluminescence immunoassay, ELISA enzyme-linked immunosorbent
assay, EMSA electrophoretic mobility shift assay, fH factor H, GAD glutamate decarboxylase, ICC immunocytochemis-
try, IHC immunohistochemistry, IP immunoprecipitation, LIA line immunoblotting, MBP myelin basic protein, MOG
major oligodendrocyte glycoprotein, NMDAR N-methyl-D-aspartate receptor, PL phospholipid, PR3 proteinase 3, RF
rheumatoid factor, RIA radioimmunoassay, SPR surface plasmon resonance, SSA/B Sjögreen Syndrome antigen A and B,
TGase transglutaminase, TLCIB thin layer chromatography immunoblotting, TPO thyroid peroxidase, TSHR thyroid-
stimulating hormone receptor, WIB western immunoblotting

mutually exclusive and several mechanisms are most likely opera-

tional in the development of ADs. However, it is generally accepted
that ADs are caused by a combination of genetic, epigenetic, and
environmental factors [21, 23–33]. The relative contribution of
genetic and environmental factors varies from disease to disease
 Autoantibodies as Diagnostic Tools 3

and is also reflected in very different sex (female–male) ratios, with

about 20 ADs having a strong female preponderance and about
20 ADs having a male dominance [20, 21, 30–33].
Characteristics of ADs also vary much according to the disease,
and stringent criteria for ADs are difficult to define. However, some
traits are common to several diseases, including fatigue, inflamma-
tion, and the occurrence of AuAbs and/or autoreactive T cells with
varying frequencies, a trait which has given rise to the collective
name of the diseases [20, 21, 34].
Most AuAbs are characteristic of certain diseases (Table 1) and
the frequency of different AuAbs among patient groups and healthy
persons ranges from negligible to essentially 100% [1–33]. Surpris-
ingly, some AuAbs may actually be more frequent in healthy per-
sons (e.g., dense fine speckles 70 (DSF 70) AuAbs)
[35, 36]. AuAbs may be present many years before clinical symp-
toms appear and intriguingly, they may persist to varying degrees
after treatment [37–41].
Whether AuAbs are generally pathogenic or not remains an
open question, and their relative contribution to the various ADs
may vary, depending on the disease, from causal (e.g., antibodies to
cell surface receptors) to merely being a consequence of the disease
[20–22, 26, 27, 37, 38, 40, 41].
However, the diagnostic and prognostic values of AuAb mea-
surements are firmly established, which makes AuAb assays highly
valuable clinical tools. For some diseases, measurement of several
AuAbs and different isotypes may be desirable (e.g., rheumatoid
factor (RF) IgM/IgA, citrulline-dependent Abs (CdAbs) IgG in
RA), while for others, it may be sufficient to measure only a single
AuAb and a single isotype (Table 1) [1–41].

2 Materials

1. Materials (see Chapters 2–24).

(a) Common immunological laboratory materials (microtiter
plates, microscope slides, etc.)
(b) Autoantigens (AuAgs) (see Note 1).
(c) Cell lines/AuAg arrays.
(d) Isolated cells (e.g., neutrophils).
(e) Tissue slides/tissue arrays.
(f) Labelled secondary antibodies (i.e., anti-IgM/IgA/IgG
conjugates).
2. Genetic tests (e.g., MHC typing) (see Note 2).
3. Apparatus (see Chapters 2–24).
(a) Common laboratory equipment (pipettes, etc.)
(b) Incubators.
4 Gunnar Houen

(c) Fluorescence microscope.

(d) Microplate reader(s) (absorbance, chemiluminescence).
(e) Scanning densitometry reader(s).

3 Methods

Whether working in a laboratory or in a clinical environment, it is

important to understand basic concepts of AuAb assay design and
performance, including their strengths and limitations.
1. AuAb assays are carried out according to established protocols
and guidelines (see Chapters 3–24) (see Note 3). When setting
up a new assay, follow simple guidelines, focusing on appropri-
ate controls and the final use of the assay (see Chapters 2–24
and [42–45]).
2. For each assay, the analytical (functional) sensitivity is estab-
lished by determining the smallest concentration measurable
with reasonable confidence and by characterizing the change in
signal by increasing concentrations [42]. Also the upper mea-
surable concentration should be determined to define a mea-
suring range for a given dilution of samples (Fig. 1).

( A - D)
Y= +D
4 parameter logistic fit é æ X öB ù
ê1 + ç ÷ ú
êë è C ø úû

Upper detection limit -

Signal

Cut off -
Lover detection limit -
X

Concentration
Fig. 1 Typical dose–response curve (four-parameter logistic fit). The analytical
cutoff value (determined as the mean of 100 healthy control samples +2–3
STDs) is indicated together with lower and upper measuring limits
 Autoantibodies as Diagnostic Tools 5

3. An appropriate dose–response curve fitting equation is estab-

lished, when applicable (e.g., a polynomial or four-parameter
logistic fit (Fig. 1) [46]).
4. The analytical/clinical cutoff value is determined by measuring
a minimum number of healthy samples in the assay (e.g., 100)
and calculating the mean + 2 or 3 standard deviations (STDs).
If relevant, a grey zone may be defined (e.g., the interval +/
1 STD around the cutoff value; or another appropriate assay-
specific interval). For assays with discrete scales (e.g., negative
(0), grey zone (/+ or ½), weakly positive (+ or 1), medium
positive (++ or 2), and strongly positive (+++ or 3)), essentially
the same procedure is followed, except for using discrete inter-
vals instead of decimal numbers.
5. Clinical sensitivity and specificity values are determined by
measuring a minimum numbers of samples from relevant dis-
ease categories (e.g., 100 from each) (see Note 4) using the
established cutoff value. The clinical sensitivities and specifici-
ties are calculated as shown in Fig. 2 (see Note 5) [46, 47].
6. A receiver-operating-characteristic (ROC) curve may be con-
structed by varying the cutoff value from zero to maximum
reading, recording the number of positives for each cutoff
value, and plotting sensitivity as a function of 1-specificity
(Fig. 3, Note 6) [42, 48, 49].
7. Positive predictive value (PPV, true positive predictive rate) and
negative predictive value (NPV, true negative predictive rate)
can be calculated using the equations in Fig. 4 (see Note 7)
[42, 46, 47].
8. Diagnostic odds ratios combine clinical sensitivity and specific-
ity into a single number and may be calculated as shown in
Fig. 5 (not considering grey zones) (see Note 7) [42, 47].

Sick Healthy

PS
Positive PS PH Sensitivity: ———— 100 %
PS + NS

NH
Negative NS NH Specificity: ———— 100 %
PH + NH

Fig. 2 Assay clinical sensitivity (true positive rate) and specificity (true negative
rate). PS, test positive sick person; NS, test negative sick person; PH, test
positive healthy person; NS, test negative healthy person. For a perfect test,
both are 100%
6 Gunnar Houen

Fig. 3 Receiver operating characteristic (ROC) curve (a). A hypothetical, perfect

test has an area under the curve (AUC) of 1 (100% sensitivity, 100% specificity),
whereas a test unable to differentiate between sick and healthy persons has an
AUC of 0.5. In practice, good test have AUCs >0.7 and are able to separate sick
from healthy persons with the chosen cutoff value, which determines specificity
and sensitivity (b). TP (true positive) ¼ PS (positive sick), FN (false negative) ¼
NS (negative sick), FP (false positive) ¼ PH (positive healthy), TN (true
negative) ¼ NH (negative healthy) in Fig. 2. Figure by Sharpr (CC BY-SA 3.0,
https://commons.wikimedia.org/w/index.php?curid¼44059691)

Sick Healthy

PS
Positive PS PH PPV: ———— 100 %
PS + PH

NH
Negative NS NH NPV: ———— 100 %
NS + NH

Fig. 4 Assay positive predictive value (PPV) and negative predictive value (NPV).
PS, test positive sick person; NS, test negative sick person; PH, test positive
healthy person; NS, test negative healthy person. For a perfect test, both are
100%
 Autoantibodies as Diagnostic Tools 7

Sick Healthy

Positive PS PH
Diagnostic PS/NS
————
odds ratio: PH/NH
Negative NS NH

Fig. 5 Assay diagnostic odds ratio (DOR) (disease-specific). PS, test positive sick
person; NS, test negative sick person; PH, test positive healthy person; NS, test
negative healthy person

9. Prevalence of an AD is the absolute number of cases per popu-

lation unit a given time (e.g., cases per 100,000) (see Note 8)
[20, 21, 30–33].
10. Incidence of an AD is the number of new cases per population
unit per time unit (e.g., new cases per 100,000 per year) (see
Notes 8–10) [20, 21, 30–33].

4 Conclusion

AuAbs are highly valuable diagnostic tools, provided that assays are
used with caution and are corrected for nonspecific binding. AuAbs
may be present long before diagnosis and may disappear or persist
after treatment, depending on the disease and the treatment.
Each assay is characterized by dose–response curve, an analyti-
cal sensitivity and a cutoff value, which in turn determines clinical
specificity and sensitivity (true positive and true negative rate),
which describe the clinical usefulness.
It remains unsettled whether AuAbs are generally pathogenic,
that is, are they a cause or a consequence of disease? This question is
reflected in the many theories about ADs, ranging from defective
self-tolerance (e.g., resulting in AuAbs against cell surface receptors
(e.g., NMDAR AuAbs) to (chronic) infections, resulting in cell
death and tissue inflammation with concomitant AuAb production
against released self-antigens (e.g., ANA). However, it is generally
agreed that early and correct diagnosis facilitates treatment and that
increased understanding of the role of AuAbs in ADs may facilitate
development of curative treatments and eventually prevention of
these diseases.
8 Gunnar Houen

5 Notes

1. Peptides, proteins (native or recombinant), phospholipids, gly-

colipids, etc. (commercial or in house-produced).
2. Genetic tests complement AuAb assays in many diseases (e.g.,
MHC II with “shared epitope” (SE) motives in RA, and MHC
I types DQ2, DQ8 in celiac diseases, etc.) [50, 51].
3. Be aware on nonspecific binding and correct for this whenever
possible (Chapter 2).
4. Be sure to measure (approximately) the same number of sam-
ples and healthy controls and distribute these equally between
plates/setups (do not measure samples from each category/
disease sequentially in different plates/setups.
5. A general problem is the absence of a universal “golden stan-
dard” to compare the results of new assays with. In the absence
of this, the results are compared with “state of the art” clinical
diagnostics (not including results of the assay in question).
6. The area under the curve (AUC) is a measure of assay (clinical)
performance and should be as close to 1 as possible).
7. Sensitivity/specificity, positive/negative predictive values and
diagnostic odds ratios are different ways of presenting the same
results and are related by the equation below (Eq. 1,
Fig. 6) [47].
8. Prevalence should not be confused with incidence (and vice
versa) (see Note 9).
9. The relation between prevalence and incidence is not straight-
forward (i.e., prevalence ¼ incidence x time), even if the inci-
dence remains relatively constant, and calculations must take
into account deaths from the disease and all other causes).
10. Incidence rate is the incidence of an AD relative to person-time
(e.g., new cases per 100,000 per 1000 person years).
11. For more comprehensive lists and for resources of ADs and
AuAbs see [20, 21, 52] and Table 2.

(sensitivity) (specificity) (PPV) (NPV)

DOR = =
(1-sensitivity) (1-specificity) (1-PPV) (1-NPV)

Fig. 6 Equation 1 [47]


Table 2
Autoimmune disease resources
Resource
Autoimmune disease database
Epitope database
Immunoassay methods
Medline Plus Autoimmune diseases
NIH Autoimmune diseases
PubMed
World Health Organisation International
Classification of Diseases (WHO ICD10)
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 Chapter 2

Nonspecific Binding in Immunoassays for Autoantibodies

Gunnar Houen

Abstract
Immunoassays are invaluable for detection and quantification of numerous analytes, including autoanti-
bodies. However, human sera often yield high nonspecific binding in such assays, and this may result in false
positive or sometimes false negative results. The causes of nonspecific binding are numerous and it
correlates with inflammatory parameters. Since the results of autoantibody testing are used for diagnosis
and treatment of autoimmune diseases, it is mandatory to be aware of all possible causes of nonspecific
binding for each individual assay and to correct for it whenever possible. General guidelines for this are
described in this chapter.

Key words Autoantibodies, Immunoassay, Immunocytochemistry, Immunohistochemistry, False

positive, False negative, Nonspecific binding, Positive predictive value, Negative predictive value,
Sensitivity, Specificity

1 Introduction

Immunoassays can be defined as any assay involving antibodies and

they are invaluable for the measurement of multiple analytes,
including antigens (Ags) and (auto)antibodies (Au)Abs [1–4]. Cur-
rent immunoassays routinely used for measurement of AuAbs
include bioassays, immunocytochemistry (ICC), immunohisto-
chemistry (IHC), electrophoretic mobility shift assay (EMSA),
enzyme-linked immunosorbent assay (ELISA), line blotting, west-
ern blotting, nephelometry/turbidimetry, and lateral flow assays
(Table 1). Some immunoassay formats with certain drawbacks
(e.g., radioactive materials or cumbersome procedures) may still
be used routinely or occasionally (e.g., radioimmunoassay (RIA),
crossed immunoelectrophoresis (CIE)), while other assay formats
mainly have educational or historical value (e.g., hemagglutination,
immunodiffusion (Ouchterlony), or countercurrent electrophore-
sis) [1–6, 13].
In many immunoassays, nonspecific binding (NSB) is a signifi-
cant but often overlooked problem [4, 6, 12, 14–19]. NSB can be

Gunnar Houen (ed.), Autoantibodies: Methods and Protocols, Methods in Molecular Biology, vol. 1901,
https://doi.org/10.1007/978-1-4939-8949-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

13
14 Gunnar Houen

Table 1
Examples of immunoassays for AuAb determinationa

Chapters,
Method/principle Example(s) [references]
Agglutination assays RFs 23, [5, 6]
Antigen arrays Salivary gland AuAbs 9
Bioassay Aβ1R Abs, TSHR AuAbs 8, [7]
Electrochemiluminescence TGase Abs 16
Electrophoretic mobility shift assay DNA Abs 11
Enzyme-linked immunosorbent PR3/MPO Abs, SSA/SSB Abs, fH Abs, 12, 13, 15, 19–21,
assay (ELISA) C3NF, CdAbs, RFs and 23
Immunocytochemistry (ICC) ACA, ANA, ANCA, Aqp5 Abs, cN1A Abs 3–7
Immunohistochemistry (IHC) Insulin Abs, H+, K+-ATPase Abs 10, [6, 8]
Immunoprecipitation H+, K+-ATPase Abs 10
Lateral flow assays PR3 Abs, MPO Abs [9]
Line immunoblotting assay (LIA) ACA, ANA, cytoplasmic Ag Abs 22
Nephelometry/turbidimetry RFs [5, 6]
Radioimmunoassay (RIA) TPO Abs, GAD 65 Abs [6, 10, 11]
Surface plasmon resonance (SPR) CRP Abs, C’ Abs 17 and 24
Western immunoblotting (WIB) C1q Abs, ANA 14, [12]
a
Abbreviations: Ab antibody, Aβ1R adrenergic beta-1 receptor, ACA anti-cell antibodies, Ag antigen, ANA anti-nuclear
antibodies, ANCA anti-neutrophil granulocyte cytoplasm antibodies, Aqp aquaporin, AuAb autoantibody, C0 comple-
ment components, C3NF C3 nephritic factor, CdAbs citrulline-dependent antibodies, CRP C-reactive protein, fH
factor H, GAD glutamate decarboxylase, MPO myeloperoxidase, PR3 proteinase 3, RF rheumatoid factor, SSA/B
Sjögreen Syndrome antigen A and B, TGase transglutaminase, TPO thyroid peroxidase, TSHR thyroid-stimulating
hormone receptor

defined as any reaction not related to the analyte of interest, and

may cause false positive results or may overshadow weakly positive
results, causing false negative results. The problem is especially
important, when it is vital to distinguish between infection and
autoimmunity, since NSB appears to correlate with inflammatory
parameters [14].
NSB may involve ionic interactions, hydrogen bonds and
hydrophobic interactions or a combination of these and depending
on the interactions involved, they may be suppressed by salt, pH
changes, hydrogen bond breakers, organic solvent, detergents,
temperature changes, proteins, carbohydrates, or other agents.
However, NSB in immunoassays may actually also occur as a result
of specific (unwanted) reactions, in which case specific precautions
are required to avoid, neutralize, or compensate for this (e.g.,
endogenous biotin detection in ICC and IHC with detection
 Nonspecific Binding in Immunoassays for Autoantibodies 15

involving biotin-labelled reagents and labelled avidin or

streptavidin [20].
The well-known causes of NSB are as follows:
1. Adsorption of Abs to solid surfaces. This problem is mainly
seen with IgG, is most problematic with more hydrophobic
surfaces, that is, high-binding capacity ELISA plates and seems
to correlate with inflammatory markers [14].
2. Bridging of capture Abs with serum IgG by rheumatoid factors
(RFs) or heterophilic Abs (HAbs) (e.g., capture assay for PR3
AuAbs) [15, 18].
3. Adsorption of immune complexes and/or complement com-
ponents [14, 21].
4. Antibodies to blocking agents (e.g., bovine serum albumin
(BSA), casein, gelatin, etc.) [12, 22].
5. Cross-reaction of tissue Abs with conjugates or cross-reaction
of tissue Ags with serum Abs (e.g., carbohydrate Ags) [6, 23].
The examples mentioned are the most common causes of NSB,
but the list of potential causes of NSB is longer and unfortunately,
no general solutions to avoidance/minimization of NSB and false
positive/negative results exist, besides the use of proper controls.
Solutions to minimize NSB must be tailored to each specific assay,
and therefore only general guidelines are described here.

2 Materials

1. Solid phase immunoassays (arrays, ELISAs, ECL, LIAs, WIB,

IP) (this volume, Chapters 9–23).
2. Cell/tissue-based assays (ICC, IHC) (this volume, Chapters
3–7).
3. Bioassays (this volume, Chapter 8).

3 Methods

3.1 General All assays must include proper general controls: Positive control
(preferably both a high positive control and a low positive control),
negative control, blank control (buffer).
All results must be carefully evaluated in order to exclude false
positive or false negative results.

3.2 Solid-Phase ELISA, RIA, LIA, immunoblotting.

Immunoassays It is recommended to include controls with noncoated wells/
omission of Ag for all samples, including standard and general
controls. The value of the noncoated wells is subtracted from the
16 Gunnar Houen

coated wells. Omission of sample (serum) can help to identify

and compensate for unwanted interactions between conjugate
and Ag(s).
If NSB is too high, the blocking/incubation/washing buffer
may have to be optimized. Common incubation buffers are based
on phosphate or Tris and may include 1% BSA or HSA and/or
0.05–0.1% nonionic detergent (e.g., Tween 20). The blocking
protein may be exchanged with casein (1%) or 1% animal serum
(preferably the same species as the conjugate is from). The deter-
gent concentration may be increased to 1%. The salt (i.e., NaCl)
concentration may also be increased from physiological concentra-
tion (0.15 M) to for example 0.3 M in order to suppress unwanted
ionic interactions. These changes may be made individually or
combined. An efficient and simple blocking/incubation/washing
buffer is the TTN buffer (50 mM Tris, pH 7.5, 1% Tween
20, 0.3 M NaCl). This buffer is designed to have a high detergent
concentration for minimization of hydrophobic interactions and a
relatively high salt concentration to minimize unwanted ionic inter-
actions [12, 14–16]. This buffer may be supplemented with 1%
animal serum (e.g., rabbit serum or goat serum for conjugates
produced in rabbits or goats respectively) [16].

3.3 Cell- and Tissue- ICC/IHC/bioassays.

Based Assays Traditionally, ICC and IHC assays for AuAbs have often been
carried out in simple buffers without additives (e.g., PBS or Tris
buffers). This however, has necessitated testing of samples in higher
dilutions to avoid too many low positive samples. With this modifi-
cation, traditional ANA and ANCA assays yield useful results (i.e.,
good clinical sensitivity and specificity).
Despite the good results in ICC and IHC with simple buffers, it
may be advisable to include blocking agents (BSA/HSA, detergent,
animal sera) in concentrations from 0.1 to 1% in blocking, washing,
and incubation buffers. If this is warranted, Tween 20 or Triton
X-100 may be used.
Moreover, as for the solid-phase immunoassays, appropriate
controls are mandatory, that is, omission of sample, negative sam-
ple, positive sample.
In some cases, for example, when developing/validating an
assay, absorption controls with the Ag in question may be per-
formed (e.g., with recombinant antigen). In this case, the sample
is preincubated overnight in incubation buffer with an excess of
(recombinant) Ag (100–1000 fold excess, corresponding to
approximately 0.1 mg/mL) or higher if possible. This should
remove (absorb) the specific staining.
 Nonspecific Binding in Immunoassays for Autoantibodies
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M (2008) New trends in immunoassays. Adv
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man BL (1985) Measurement of rheumatoid
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ogy, assay methods, and clinical applications.
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9. Offermann N, Conrad K, Fritzler MJ, Fooke
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and MPO (pANCA). J Immunol Methods
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12. Jørgensen CS, Hansen KB, Jacobsen S,
Halberg P, Ullman D, Mikkelsen TL, Weile B,
Madsen MH, Heegaard NHH, Wiik A, Houen
G (2005) Absence of high affinity calreticulin
autoantibodies in patients with systemic rheu-
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Lab Invest 65:403–412
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 Chapter 3

Detection of Autoantibodies by Indirect

Immunofluorescence Cytochemistry on Hep-2 Cells
Alessandra Dellavance and Luis Eduardo Coelho Andrade

Abstract
Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular
antigens for more than 50 years. Originally using rodent tissue as substrate, the method was optimized by
using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA
platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the
samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the auto-
antibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence
(pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate
autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the Interna-
tional Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of
the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized
its nomenclature. The former designation “antinuclear antibody test” has been progressively replaced by
the term “anti-cell antibody test,” due to the recognition that the HEp-2/IFA method in fact allows the
detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.
The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell
culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-
conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading
mounting medium. The several steps of the procedure must be carefully performed in order to avoid the
formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum
standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every
new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the
most relevant AC patterns. Special attention should be dedicated to the training of personnel for the
analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all
relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.

Key words Indirect immunofluorescence, HEp-2 cells, Antinuclear antibody, Autoantibodies, Auto-
immunity testing, Assay standardization, Methods in autoimmunity testing

1 Introduction

In 1941, Coons, Creech, and Jones established the immunofluo-

rescence technique to identify pneumococcal antigens in tissues
[1, 2]. Since then this technique has undergone multiple

Gunnar Houen (ed.), Autoantibodies: Methods and Protocols, Methods in Molecular Biology, vol. 1901,
https://doi.org/10.1007/978-1-4939-8949-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

19
20 Alessandra Dellavance and Luis Eduardo Coelho Andrade

methodological advances and has become a well-established

method for a variety of immunological applications. The indirect
immunofluorescence assay (IFA) is a laboratory technique in which
specific antibodies present in biological fluids (primary antibody)
are immobilized by reacting with their cognate antigens on appro-
priate substrates and then detected by secondary antibodies labeled
with fluorochromes. Substrates for the capture and immobilization
of the primary antibodies are prepared using a variety of protocols
and biological matrices: cultured cells and tissue cryo-sections are
frequently used. Cryo-sections of animal tissues, often kidney or
liver, were the substrate of choice for decades for the detection of
autoantibodies to cellular antigens by IFA. Monolayer cell culture
substrates offer a greater spectrum of information and allow for
optimal visualization of subcellular structures. In addition, they
yield more reproducible and more easily standardized results than
tissue cryo-sections. Therefore, IFA on HEp-2 cells (HEp-2/IFA)
has become the gold standard laboratory method for screening for
autoantibodies in systemic rheumatic diseases (SARD) [3]. The
HEp-2 cell line is an immortal cell line derived from a human larynx
carcinoma (HEp-2—American Type Culture Collection ATCC®
CCL-23™). ATCC reported this cell line to be positive for the
presence of human papilloma viral DNA sequences and chromosome
markers from HeLa cells, a cell line derived from a human uterine
cervical cancer (data present in the HEp2 ATCC® CCL23 ™
Product Sheet available from the ATCC) [4].
HEp-2/IFA allows detection of a large number of autoantibo-
dies relevant to a variety of clinical conditions [5]. The test offers
three important pieces of information: (1) the presence or absence
of autoantibodies in the sample, (2) the antibody titer, and (3) the
immunofluorescence pattern. The titer represents a semiquantita-
tive assessment of the autoantibody concentration in serum. This is
clinically valuable information, since autoantibodies occur in a
certain fraction of the general population, and most of these non-
autoimmune individuals present low titers, while patients with
systemic autoimmune diseases frequently present moderate-to-
high titers [6–9]. The HEp-2/IFA pattern also adds valuable infor-
mation because it helps to discriminate nonautoimmune indivi-
duals from autoimmune patients [9], as further discussed ahead.
In addition, the HEp-2/IFA pattern often indicates the autoanti-
body specificities most likely to be present in a given sample, thus
guiding the next step in serologic analysis [5, 10–12]. Previously
known as the antinuclear antibody (ANA) test, this test has recently
been renamed as the anti-cell antibody test due to the realization
that HEp-2/IFA also detects autoantibodies against antigens in the
cytoplasm and mitotic apparatus. The nomenclature for 30 anticell
patterns (AC patterns) to be reported in the HEp-2/IFA test was
recently defined and harmonized by the International Consensus
on ANA Patterns initiative (ICAP) [13]. These patterns are
described in Table 1.
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 CHAPTER VII.

THE CATASTROPHE.

James Beresford was not brave. He was very kind and tender and good; but
he had not courage to meet the darker emergencies of life. He felt as he
rushed downstairs from his wife’s presence that he had but postponed the
evil day, and that many another dreadful argument on this subject, which
was not within the range of arguing, lay before him. What could he say to
her? He felt the abstract justice of her plea. A hopeless, miserable, lingering,
loathsome disease, which wore out even love itself, and made death a
longed-for relief instead of a calamity. What could he say when she appealed
to him to release her from that anguish of waiting, and hasten the deliverance
which only could come in one way? He could not say that it would be
wicked or a sin; all that he could say was, that he had not the courage to do it
—had not the strength to put her away from him. Was it true, he asked
himself, that he would rather watch out her lingering agonies than deprive
himself of the sight of her, or consent to part with her a day sooner than he
must? Was it himself he was thinking of alone, not her? Could he see her
anguish and not dare to set her free? He knew that, in the case of another
man, he would have counselled the harder self-sacrifice. But he, how could
he do it? He rushed out of the house, through the afternoon sunshine, away
to the first space he could find near, and struck across the open park, where
there was no one to disturb him, avoiding all the pleasant walks and paths
where people were. The open space and the silence subdued his excitement;
and yet what could really bring him peace? He had no peace to look for—
nothing but a renewed and ever-new painful struggle with her and with
himself. Yes, even with himself. If she suffered greatly, he asked, with a
shudder, how could he stand by and look on, knowing that he could deliver
her? And would not she renew her prayers and cries to him for deliverance?
God help him! It was not as if he had made an end of that mad prayer once
and for ever by refusing it. It would come back—he knew it would come
back—hour by hour and day by day.
Oh, how people talk (he thought) of such mysteries when the trouble is
not theirs! He himself had argued the question often, in her hearing, even
with her support. He had made it as clear as day to himself and to others. He
had asked what but cowardice—miserable cowardice—would keep a man
from fulfilling this last dread, yet tender service? Only love would dare it—
but love supreme, what will that not do, to save, to succour, to help, to
deliver? Love was not love which would shrink and think of self. So he had
often said with indignant, impassioned expansion of the heart—and she had
listened and echoed what he said. All this returned to him as he rushed
across the dewy grass, wet with spring rains, and untrodden by any other
foot, with London vague in mists and muffled noises all round. Brave words
—brave words! he remembered them, and his heart grew sick with self-pity.
How did he know it was coming to him? How could he think that this case
which was so plain, so clear, should one day be his own? God and all good
spirits have pity upon him! He would have bidden you to do it, praised you
with tears of sympathy for that tremendous proof of love; but himself? He
shrank, shrank, contracted within himself; retreated, crouching and slinking,
from the house. What a poor cur he was, not worthy the name of man! but he
could not do it; it was beyond the measure of his powers.
When he turned to go home the afternoon light was waning. Small heart
had he to go home. If he could have escaped anywhere he would have been
tempted to do so; and yet he was on the rack till he returned to her. Oh, that
Heaven would give her that sweet patience, that angelical calm in suffering,
which some women have! Was it only religious women who had that calm?
He asked himself this question with a piteous helplessness; for neither he nor
she had been religious in the ordinary sense of the word. They had been
good so far as they knew how—enjoying themselves, yet without
unkindness, nay, with true friendliness, charity, brotherly-heartedness to their
neighbours; but as for God, they had known little and thought less of that
supreme vague Existence whom they accepted as a belief, without knowing
Him as a person, or desiring to know. And now, perhaps, had their theory of
life been different they might have been better prepared for this emergency.
Was it so? He could not tell. Perhaps philosophy was enough with some
strong natures, perhaps it was temperament. Who can tell how human
creatures are moved; who touches the spring, and what the spring is, which
makes one rebellious and another submissive, sweet as an angel? He had
loved the movement, the variety, the indocility, the very caprice, of his wife,
in all of which she was so much herself. Submission, resignedness, were not
in that changeful, vivacious, wilful nature; but, oh, if only now the meekness
of the more passive woman could somehow get transfused into her veins, the
heavenly patience, the self courage that can meet anguish with a smile!
There was Cherry, his faded old maiden sister—had it been she, it was in her
to have drawn her cloak over the gnawing vulture, and borne her tortures
without a sign of flinching. But even the very idea of this comparison hurt
him while it flashed through his mind. It was a slight to Annie to think that
any one could bear this horrible fate more nobly than she. Poor Annie! by
this time had she exhausted the first shock? Had she forgiven him? Was she
asking for him? He turned, bewildered by all his dreary thoughts, and
calmed a little by fatigue and silence, to go home once more.
It was getting dusk. As he passed the populous places of the park the hum
of voices and pleasant sounds came over him dreamily like a waft of warmer
air. He passed through that murmur of life and pleasure, and hurried along to
the more silent stony streets among which his Square lay. As he approached
he overtook Maxwell walking in the same direction, who looked at him with
some suspicion. The two men accosted each other at the same moment.
‘I wanted to see you. Come with me,’ said Beresford; and——’ What is
the matter? Why did you send for me?’ the doctor cried.
Then Maxwell explained that a hurried message had come for him more
than an hour before, while he was out, and that he was on his way to the
Square now.
‘Has there been any—change?’ he said. After this they sped along
hurriedly with little conversation. There seemed something strange already
about the house when they came in sight of it. The blinds were down in all
the upper windows, but; at the library appeared Cara’s little white face
looking eagerly out. She was looking out, but she did not see them, and an
organ-man stood in front of the house grinding out the notes of the
Trovatore’s song ‘Ah, che la morte,’ upon his terrible instrument. Cara’s
eyes and attention seemed absorbed in this. James Beresford opened the door
with his latch-key unobserved by any one, and went upstairs direct, followed
by the doctor, to his wife’s room.
How still it was! How dark! She was fond of light, and always had one of
those tall moon lamps, which were her favourites; there was no lamp in the
room, however, now, but only some twinkling candles, and through the side
window a glimmer of chill blue sky. Nurse rose as her master opened the
door. She gave a low cry at the sight of him. ‘Oh, don’t come here, sir, don’t
come here!’ she cried.
 ‘Is she angry, still angry?’ said poor Beresford, his countenance falling.
‘Oh, go away, sir; it was the doctor we wanted!’ said the woman.
Meantime Maxwell had pushed forward to the bedside, He gave a cry of
dismay and horror, surprise taking from him all self-control. ‘When did this
happen?’ he said.
James Beresford pressed forward too, pushing aside the woman who tried
to prevent him; and there he saw—what? Not his wife: a pale, lovely image,
still as she never was in her life, far away, passive, solemn, neither caring for
him nor any one; beyond all pain or fear of pain. ‘My God!’ he said. He did
not seem even to wonder. Suddenly it became quite clear to him that for
years he had known exactly how this would be.
Maxwell put the husband, who stood stupefied, out of his way; he called
the weeping nurse, who, now that there was nothing to conceal, gave free
outlet to her sorrow. ‘Oh, don’t ask me, sir, I can’t tell you!’ she said among
her sobs. ‘Miss Carry rung the bell and I came. And from that to this never a
word from her, no more than moans and hard breathing. I sent for you, sir,
and then for the nearest as I could get. He came, but there was nothing as
could be done. If she took it herself or if it was give her, how can I tell? Miss
Carry, poor child, she don’t know what’s happened; she’s watching in the
library for her papa. The medicine-box was on the table, sir, as you see. Oh,
I don’t hold with them medicine-boxes; they puts things into folk’s heads!
The other doctor said as it was laudanum; but if she took it, or if it was give
her——’
Mr. Maxwell stopped the woman by a touch on the arm. Poor Beresford
stood still there, supporting himself by the bed, gazing upon that which was
no more his wife. His countenance was like that of one who had himself
died; his mouth was open, the under lip dropped; the eyes strained and
tearless. He heard, yet he did not hear what they were saying. Later it came
back to his mind; at present he knew nothing of it. ‘God help him!’ said the
doctor, turning away to the other end of the room. And there he heard the
rest of the story. They left the two together who had been all in all to each
other. Had he given her the quietus, he who loved her most, or had she taken
it? This was what neither of them could tell. They stood whispering together
while the husband, propping himself by the bed, looked at her. At her? It
was not her. He stood and looked and wondered, with a dull aching in him.
No more—he could not go to her, call her by her name. A dreary, horrible
sense that this still figure was some one else, a something new and unknown
to him, another woman who was not his wife, came into his soul. He was
frozen by the sudden shock; his blood turned into ice, his heart to stone.
Annie! oh, heaven, no; not that; not the marble woman lying in her place!
He was himself stone, but she was sculptured marble, a figure to put on a
monument. Two hours of time—light, frivolous, flying hours—could not
change flesh and blood into that; could not put life so far, and make it so
impossible. He did not feel that he was bereaved, or a mourner, or that he
had lost what he most loved; he felt only a stone, looking at stone, with a
dull ache in him, and a dull consternation, nothing more. When Maxwell
came and took him by the arm he obeyed stupidly, and went with his friend,
not moving with any will of his own, but only because the other moved him;
making no ‘scene’ or terrible demonstrations of misery. Maxwell led him
downstairs holding him by the arm, as if he had been made of wood, and
took him to the library, and thrust him into a chair, still in the same passive
state. It was quite dark there, and Cara, roused from her partial trance of
watching at the window, stumbled down from her chair at the sight of them,
with a cry of alarm, yet relief, for the lamps outside had beguiled the child,
and kept her from perceiving how dark it had grown till she turned round.
No one had thought of bringing in the lamp, of lighting the candles, or any
of the common offices of life in that house where Death had so suddenly set
up his seat. The doctor rang the bell and ordered lights and wine. He began
to fear for James: his own mind was agitated with doubts, and a mingled
severity and sympathy. He felt that whatever had happened he must find it
out; but, whatever had happened, how could he do less than feel the
sentiment of a brother for his friend? He did not take much notice of the
child, but stooped and kissed her, being the friend of the house, and bade her
go to her nurse in a softened tender tone. But he scarcely remarked that Cara
did not go. Poor child, who had lost her mother! but his pity for her was of a
secondary kind. It was the man whom he had to think of—who had done it,
perhaps—who, perhaps, was his wife’s innocent murderer—yet whom,
nevertheless, this good man felt his heart yearn and melt over. When the
frightened servant came in, with red eyes, bringing the wine, Maxwell
poured out some for the chief sufferer, who sat motionless where he had
placed him, saying nothing. It was necessary to rouse him one way or other
from this stupefaction of pain.
‘Beresford,’ he said curtly, ‘listen to me; we must understand each other.
It is you who have done this? Be frank with me—be open. It is either you or
she herself. I have never met with such a case before; but I am not the man
to be hard upon you. Beresford! James! think, my dear fellow, think; we
were boys together; you can’t suppose I’ll he hard on you.’
‘She asked me—she begged of me,’ said Beresford, slowly. ‘Maxwell,
you are clever, you can do wonders.’
‘I can’t bring those back that have gone—there,’ said the doctor, a sudden
spasm coming in his throat. ‘Don’t speak of the impossible. Clever—God
knows! miserable bunglers, that is what we are, knowing nothing. James! I
won’t blame you; I would have done it myself in your place. Speak out; you
need not have any reserves from me.’
‘It isn’t that. Maxwell, look here; they’ve spirited my wife away, and put
that in her place.’
‘God! he’s going mad,’ said the doctor, feeling his own head buzz and
swim.
‘No,’ was the answer, with a sigh. ‘No, I almost wish I could. I tell you it
is not her. You saw it as well as I. That my wife? Maxwell——’
‘It is all that remains of her,’ said the doctor, sternly. ‘Mind what I say; I
must know; no more of this raving. Did you do it? Of course she asked you,
poor soul!’ (Here the doctor’s voice wavered as if a gust of wind had blown
it about.) ‘She never could endure the thought of pain; she asked you, it was
natural: and you gave her—opium?’
‘Nothing. I dared not,’ he said, with a shiver. ‘I had not the courage. I let
her plead; but I had not the courage. What? put her away from me,
willingly? how could I do it? Yes; if she had been in a paroxysm; if I had
seen her in agony; but she was calm, not suffering, and she asked me to do it
in cold blood?’
‘What then?’ The doctor spoke sternly, keeping the tone of authority to
which in his stupefied state poor Beresford appeared to respond. Cara from a
corner looked on with wide-open eyes, listening to everything.
‘Nothing more,’ he said, still sighing heavily. ‘It was more than I could
bear. I rushed away. I went out to calm myself—to try and think; and I met
you, Maxwell; and now——’
He lifted his hands with a shuddering gesture. ‘That is all—that is all! and
this desolate place is my—home; and that is—Annie! No, no! Maxwell,
some of your doctors—your cruel doctors—have taken her away to try their
experiments. Oh, say it is so, and I’ll thank you on my knees!’
 ‘Be quiet, Beresford! Try and be a man. Don’t you see what I have got to
do? If it was not you it was herself. I don’t blame her, poor soul, poor soul!
the thought of all she had to go through made her mad. Be silent, man, I tell
you! We must not have her branded with the name of suicide, James,’ cried
the doctor, fairly sobbing. ‘Poor girl, poor girl! it is not much wonder if she
was afraid; but we must not let them say ill of her now she is gone. I
remember her before you married her, a lovely creature; and there she is,
lying—but they must not speak ill of her. I’ll say it was—— Yes. if it’s a lie
I can’t help that—my conscience will bear it—there must not be talk, and an
inquest. Yes, that’s what I’ll say.’
‘An inquest!’ said the wretched husband, waking up from his stupor with
a great cry.
‘I’ll take it upon myself,’ said Maxwell, going to the writing-table. Then
he saw Cara leaning out of her chair towards them with great strained wide-
open eyes.
‘Cara! have you heard all we were saying?’
‘I don’t understand, I don’t understand!’ said the child with sudden sobs.
‘What have you done to my mamma?’
The door of the library opened softly, and they all started as if at the
approach of a new calamity.
‘If you please, sir,’ said John, addressing Maxwell with natural
recognition of the only source of authority, ‘I came to see if you wouldn’t
have some dinner—and master——’
With a moan, Beresford hid his face in his hands. Dinner must be,
whosoever lives or dies—if the world were breaking up—if hope and love
had failed for ever. John stood for a moment against the more powerful light
of the gas in the hall, for his answer, and then, not getting any, he had the
grace to steal quietly away.
But this wonderful intrusion of the outer ordinary life disturbed the
melancholy assembly. It roused Beresford to a sense of what had befallen
him. He got up and began to pace up and down the long room, and Cara’s
sobs broke the silence, and Maxwell at the table, with a spasm in his throat,
compiled the certificate of the death. In what medical form he put it I cannot
tell; but he strained his conscience and said something which would pass,
which nobody could contradict; was not that enough? ‘I hope I may never do
anything more wicked,’ he said, muttering to himself. The nurse came to call
the child, which was the first thing that had seemed natural to Cara in the
whole miserable day’s proceedings. She did not resist the command to go to
bed, as they had all resisted the invitation to dine. She got up quickly when
nurse called her, glad of something she was used to.
‘It’s the only place as we’re all fit for,’ said nurse, with a sigh of
weariness; ‘your poor papa, Miss Carry, as well as the rest.’ Then she turned
to the gentlemen with a touch of natural oratory. ‘What is the use of talking,’
she said; ‘I’m one as has loved her since first she drew breath. She was my
child, she was; and look you here, I’m glad—her old nurse is glad. I’ll not
cry nor make no moan for her,’ said nurse, the tears running down her
cheeks. ‘I’d have give her that dose myself if the darling had asked me; I
would, and never have trembled. I’d have done it and stood up bold and told
you I done it, and I don’t blame her. She’s seen what it was, and so have I.’
‘Nurse, you are a good woman,’ said the doctor, coming hastily forward
and grasping her hand. ‘Nurse, hold your tongue, and don’t say a word.
Don’t let those idiots talk downstairs. I’m ready to give them the reason of it
whoever asks. I did not know it would come on so quickly when I left to-
day; but I know what it is that has carried her off. It was to be expected, if
we hadn’t all been a parcel of fools.’
Nurse looked him anxiously in the face. ‘Then it wasn’t—it wasn’t?——
Ah!’ she added, drawing a long breath, ‘I think I understand.’
‘Now, hold your tongue,’ he cried, curtly, ‘and stop the others. You are a
sensible woman. My poor little Cara, good-night.’
‘Don’t speak to him,’ nurse whispered, drawing the child away. ‘Leave
your poor papa alone, darling. God help him, he can’t say nothing to you to-
night. Here’s Sarah coming to put you to bed, and glad I’d be to be there too:
it’s the only place as we’re fit for now.’
Sarah, who was waiting outside, had red eyes overflowing with tears. She
hugged the little girl and kissed her, bursting out into fits of subdued crying.
But Cara’s own sobs were stilled and over. Her head ached with bewildering
pain; her mind was full of confused bewildering thoughts.
 CHAPTER VIII.

CONSOLATION.

‘This is indeed an affliction, dear Miss Beresford. We came up directly we

heard of it; I would not let a moment pass. Oh, how little we know! We were
thinking of your poor niece as having returned from her foreign tour; as
being about to enter upon the brilliant society of the season. I don’t know
when I have received such a shock; and my poor Maria, her feelings were
almost beyond control; but she would not stay away.’
‘I thought she would come,’ said Miss Charity. ‘Maria always likes to get
news from the fountain-head, and to see how people are bearing their
troubles. Yes, my dear, I am bearing mine very well, as you see. Poor Annie!
she was only my niece by marriage after all. At my age one sees even one’s
own nieces, women with families, die without great trouble. It may sound
hard, but it’s true. When a woman is married, and has her own children
about her, you can’t but feel that she’s less to you. It’s dreadful for them; but,
so far as you are concerned, you lost her long ago.’
‘Oh, dear Miss Beresford, you like to pretend you are calm, to hide how
soft-hearted you are! But we know you better than that. I myself, though I
knew (comparatively) so little of poor Mrs. James——’
‘And I thought you did not like each other, so it is all the more kind of
you to cry. Cherry will cry too as much as you please, and be thankful for
your sympathy. Have you had a pleasant walk? I think the primroses are
thicker than ever this spring. We have been sending up basketsful. She was
fond of them——’ Here the old lady faltered for a moment. This was the
kind of allusion that melted her, not straightforward talk. She was in
profound black, a great deal more crape than the dressmaker thought at all
necessary, but Miss Charity had her own views on these subjects. ‘Put
double upon me, and take it off the child,’ she had said, to the wonder of the
tradespeople, who felt that the mourning for a niece by marriage was a very
different thing from that which was required for a mother. Mrs. Burchell
respected her greatly for her crape. She knew the value of it, and the
unthriftiness, and felt that this was indeed showing respect.
 ‘We heard it was very sudden at last,’ said the Rector, ‘that nobody had
the least idea—it was a very lingering disorder that she was supposed to
have? So we heard, at least. Do you happen to know how the doctors
accounted for its suddenness at last? There is something very dreadful to the
imagination in so sudden a death.’
‘I wish I could think I should have as quick an end,’ said Miss Charity;
‘but we Beresfords are strong, and die hard. We can’t shake off life like that.
We have to get rid of it by inches.’
‘My dear lady,’ said the Rector, ‘I don’t mean to say that I would put any
trust in death-bed repentances; but surely it is a privilege to have that time
left to us for solemn thought, for making sure that we are in the right way.’
‘I never think much when I am ill, my dear Rector; I can’t. I think why
the flies buzz so, and I think if I was Martha it would make me unhappy to
have such a red nose; and if you came to me, instead of listening to what you
said, I should be thinking all the time that your white tie was undone’ (here
the Rector furtively and nervously glanced down, and instinctively put up his
hand to feel if the remark was true) ‘or your coat rusty at the elbows. I say
these things at a hazard, not that I ever remarked them,’ she added, laughing.
‘You are tidiness itself.’
The Rector was put out by these chance possibilities of criticism, and
could not but feel that Miss Charity’s quick eyes must have seen him with
his white tie untidy, loosely unfastened, under his beard. He had grown a
beard, like so many clergymen, and it was not an improvement. Instead of
looking clean, as he once did, he looked black and coarse, a mixture of sea-
captain and divine. He kept putting up his hand stealthily all the time he
remained, and inviting his wife with nervous glances, to let him know if all
was right. Unfortunately he could not see it under the forest of black beard.
‘We heard,’ said his wife, coming to his relief, ‘that there was something
about an opiate—an over-dose, something of that sort—that poor Mrs. James
had taken it without measuring it, or—you know how everything is
exaggerated. I was quite afraid, and so glad to see the death in the paper
without any inquest or formalities of that kind, which must be so painful.
Was there really nothing in the story of the opiate? It is so strange how
things get about.’
‘I don’t think it at all strange, Maria. The servants call in a strange doctor,
in their fright, who does not know anything about her case or temperament.
He hears that she has to take some calming drops to relieve her pain, and of
course he jumps in his ignorance to the idea of an overdose. It is the
fashionable thing now-a-days. It is what they all say——’
‘And there was no truth in it?’
‘None whatever,’ said Miss Charity, who, safest of all advocates,
implicitly believed what she was saying, not knowing that any doubt had
ever existed on the subject. She sat facing them in her new mourning, so
freshly, crisply black. Miss Charity knew of no mystery even, and her calm
certainty had all the genuine force of truth.
The Rector and his wife looked at each other. ‘It shows that one should
not believe the tenth part of what one hears,’ he said. ‘I was told confidently
that poor Mrs. James Beresford held strange ideas about some things.’
‘That you may be quite sure of, Rector. I never knew any one yet worth
their salt who did not hold odd ideas about something——’
‘Not about fundamentals, my dear lady. I am not straitlaced; but there are
some matters—on some things, I am sure, none of us would like to give an
uncertain sound. Life, for example—human life, is too sacred to be trifled
with; but there is a set of speculatists, of false philosophers—I don’t know
what to call them—sceptics, infidels they generally are, and at the same time
radicals, republicans——’
‘Ah, politics? I dare say poor Annie was odd in politics. What did it
matter? they were not political people. If James had been in Parliament,
indeed, as I should like to have seen him—but unfortunately he was a man
of fine tastes; that is fatal. A man of fine tastes, who is fond of travelling,
and collecting, and rapt up in his wife, will never become a public man; and
I should like to have seen James in Parliament. Strange ideas! oh, yes, queer
to the last degree. If there is anything worse than republicanism (is there?) I
should think poor Annie went in for that.’
‘That is bad enough, but it is not exactly what I meant,’ said the Rector;
and then he rose up with an air of the deepest conventional respect. ‘My
dear, here is your kind friend, Miss Cherry,’ he said.
Mrs. Burchell sprang up at the intimation, and rushed forward with open
arms. She had put on a black merino dress instead of her usual silk, and a
black shawl, to mark her sense of the calamity—and swallowed up poor slim
Miss Cherry in the entanglements of that embrace, with solemn fervour.
Cherry had not much sense of humour, and she was too good to pass any
judgment upon the sudden warmth of affection thus exhibited; but it was a
little confusing and suffocating to find herself without any warning engulfed
in Mrs. Burchell’s old merino and the folds of her shawl.
‘Oh, my dear, dear Cherry, if I could but tell you how I feel for you! How
little did we think when we last met——’
‘You are very kind,’ said Miss Cherry, drawing herself forth somewhat
limp and crushed from this embrace. ‘I am sure you are very kind.’ Her lips
quivered and the tears came to her eyes; but she was not so overwhelmed as
her consoler, who had begun to sob. ‘It is my poor brother I think of,’ said
Miss Cherry. ‘It is little to us in comparison with, what it is to him. I think of
him most; more than of poor Annie, who is safe out of all trouble.’
‘We must hope so, at least,’ said the Rector, shaking his head; and his
wife stopped sobbing, and interchanged a glance with him, which was full of
meaning.
‘Poor Mrs. James! It was so sudden. I fear there was no time for
preparation—no time even for thought?’
‘Men soon get the better of these things,’ said Miss Charity, ‘and the
more they feel it at the time the more easily they are cured. Cherry there will
think of her longer than her husband will. I don’t mean to say your grief’s so
great, my dear, but it will last.’
‘Oh, aunt, you do James injustice! He thought of nothing but Annie. The
light of his eyes is gone, and the comfort of his house, and all he cares for in
life.’
Here poor Miss Cherry, moved by her own eloquence, began to cry,
picturing to herself this dismal future. Nothing at Sunninghill was changed:
the room was as full of primroses as the woods were; great baskets of them
mingled with blue violets filled every corner; the sunshine came in
unclouded; the whole place was bright. It struck the tender-hearted woman
with sudden compunction: ‘We are not touched,’ she said; ‘we have
everything just the same as ever, as bright; but my poor James, in that house
by himself; and the child! Oh, Aunt Charity, when I think of him, I feel as if
my heart would break.’
Miss Charity took up her work and began to knit furiously. ‘He will get
over it,’ she said, ‘in time. It will be dreadful work at first; but he will get
over it. He has plenty of friends, both men and women. Don’t upset me with
your talk; he will get over it—men always do.’
 ‘And let us hope it will lead him to think more seriously,’ said Mrs.
Burchell. ‘Oh, I am sure if you thought my dear husband could be of any use
—we all know he has not been what we may call serious, and oh, dear Miss
Beresford, would not this affliction be a cheap price to pay for it, if it
brought him to a better state of mind?’
‘His wife’s life? It would be a high price for any advantage that would
come to him, I think. Dry your eyes, Cherry, and go and put on your bonnet.
This is Mr. Maxwell’s day, and you had better go back to town with him.’
‘Was it Mr. Maxwell who attended poor Mrs. James? I hope he is
considered a clever man.’
‘How oddly you good people speak! Do you want to insinuate that he is
not a clever man? He takes charge of my health, you know, and he has kept
me going long enough. Eh! yes, I am irritable, I suppose; we are all put out.
You good quiet folks, with all your children about, nothing happening to you
——’
‘Indeed, Miss Beresford, you do us great injustice,’ said Mrs. Burchell,
stung, as was natural, by such an assertion, while the Rector slowly shook
his head. ‘We do not complain; but perhaps if we were to tell all, as some
people do. Nothing happening to us!—ah, how little you know!’
‘Well, well, let us say you have a great many troubles; you can feel then
for other people. Ah, here is Mr. Maxwell. Don’t talk of me now; don’t think
of me, my good man. I am as well—as well—a great deal better than a poor
useless woman of nearly threescore and ten has any right to be when the
young are taken. How is James?’
The doctor, who had come in by the open window with a familiarity
which made the Rector and his wife look at each other, sat down by the old
lady’s side and began to talk to her. Miss Cherry had gone to put on her
bonnet, and by-and-by Mr. and Mrs. Burchell rose to take their leave.
‘I am so glad to hear that, sad as it was, it was a natural death, and one
that you expected,’ said the Rector, taking Maxwell aside for a moment.
The doctor stared at him, with somewhat fiery eyes. ‘A natural death?
Mrs. Beresford’s? What did you expect it to be?’
‘Oh, my dear sir, I don’t mean anything! We had heard very different
accounts—so many things are said——’
‘You should put a stop to them, then,’ said the other, who was not without
temper; and he and Miss Charity paused in their sadder talk, as the visitors
disappeared, to interchange some remarks about them which were not
complimentary.
‘What they can mean by making up such wicked lies, and putting a slur
on her memory, poor child!’ said the old lady with a sudden gush of hot
tears.
The doctor said something very hotly about ‘meddlesome parsons,’ and
hastily plunged again into descriptions of poor James. The other was not a
subject on which he could linger. ‘I never saw a man so broken-hearted; they
were always together; he misses her morning, noon, and night. Cherry must
come to him; she must come at once,’ he said, forgetting how long it was
since he had spoken of Cherry before by her Christian name. But Miss
Charity noticed it with the keen spectator instinct of her age, and ruminated
in an undercurrent of thought even while she thought of ‘poor James,’
whether Maxwell’s faith in Cherry ‘meant anything,’ or if new combinations
of life might be involved in the sequences of that death scene.
The same thought was in the minds of the clerical pair as they went down
the hill. ‘Will that come to anything?’ they said to each other.
‘It is a nice little property,’ said the Rector, ‘and I suppose she will have
everything.’
‘But if I was Cherry,’ said Mrs. Burchell, ‘I should not like to be thrown
at his head in that very open way. Going with him to town! It is as good as
offering her to him.’
‘She is no longer young, my dear,’ said the Rector, ‘and people now-a-
days have not your delicacy.’
‘Oh, I have no patience with their nonsense!’ she cried; ‘and their
friendships, forsooth—as if men and women could ever be friends!’
And it is possible that in other circumstances Miss Cherry’s tranquil soul
might have owned a flutter at thought of the escort which she accepted so
quietly to-day; but she was absorbed with thoughts of her brother and of the
possible use she might be, which was sweet to her, notwithstanding her grief.
Miss Charity shook her head doubtfully. ‘It is not Cherry that will help him,’
she said, ‘but the child will be the better of a woman in the house.’
Really that was what Mr. Maxwell wanted, a woman in the house;
something to speak to, something to refer everything to; something to blame
even, if things were not all right. The funeral was over, and all that dismal
business which appals yet gives a temporary occupation and support to the
sorrowful. And now the blank of common life had recommenced.
‘Perhaps she will not help him much; but she will be there,’ said the
doctor. He was glad for himself that a soft-voiced, soft-eyed, pitying creature
should be there. There was help in the mere fact, whatever she might say or
do.
Cara had been living a strange life through these melancholy days. She
had not known, poor child, the full significance of that scene by her mother’s
bedside, of which she had been a witness. She did not fully understand even
now: but glimmers of horrible intelligence had come to her during that
interview in the library, and the things she had heard afterwards from the
servants had enlightened her still more. She heard the whispers that
circulated among them, terrified whispers, said half under their breath. That
she had done it herself—that she knew, poor dear, what she was doing—that
if anything had been known, there would have been an inquest, and things
would have come out. This was what Cara heard breathing about in half
whispers, and which filled her with strange panic, lest her secret should
escape her. She knew the secret, and she only. Nobody had questioned her,
but the child’s impulse to tell had bound her very soul for days after. She had
resisted it, though she had felt guilty and miserable to know something
which no one else knew; but she had kept her secret. ‘Don’t let us brand her
with the name of suicide.’ These words seemed to ring in her ears night and
day. She repeated them over and over to herself. ‘Don’t let us brand her with
the name of suicide.’
‘No, no,’ poor Cara said to herself, trembling; ‘no, no:’ though this
premature and horrible secret weighed down her heart like a visible burden.
Oh, if she could but have told it to nurse, or to Aunt Cherry! but she must
not, not even to papa. When her aunt arrived, it was mingled torture and
relief to the poor child. She clung round her with sobbing, longing so to tell;
but even to cling and to sob was consolatory, and Aunt Cherry wanted no
explanation of that unusual depth of childish distress. ‘Cara was not like
other children,’ she said to herself. She had feelings which were deeper and
more tender. She was ‘sensitive,’ she was ‘nervous.’ She was more loving
than the ordinary children, who cry one moment and forget the next. And
kind Cherry, though her own grief was of the milder, secondary kind, as was
natural, had always tears of sympathy to give for the grief of others. She
took the little girl almost entirely into her own care, and would talk to her for
hours together; about being ‘good,’ about subduing all her little irritabilities,
in order to please mamma, who was in heaven, and would be grieved in her
happiness to think that her child was not ‘good.’ Cara was greatly awed and
subdued by this talk. It hushed her, yet set her wondering; and those
conversations were sometimes very strange ones, which went on between
the two in their melancholy and silent hours.
‘Does everybody go to heaven who dies?’ said Cara, with awe-stricken
looks.
Miss Cherry trembled a little, having some fear of false doctrine before
her eyes. ‘Everybody, I hope, who loves God. There are bad people, Cara;
but we don’t know them, you and I.’
‘Who love God? but I never think of God, Aunt Cherry. At least, I do
now; I wonder. But if they did not do that, would they still go to heaven all
the same?’
‘God loves us, dear,’ said Cherry, with the tears in her soft eyes. ‘Fathers
and mothers love their children, whether their children love them or not.
That is all we know.’
‘Whatever they do? if they even laugh, and go wrong? Yes,’ said Cara,
very thoughtfully, ‘I suppose papa would not send me away, out into the
dark, if I did ever so wrong.’
‘I am sure he would not; but you must not think of such things, dear; they
are too difficult for you. When you are older, you will understand better,’
Cherry said, faltering, and with something in her heart which contradicted
her; for did not the child ‘understand’ better than she?
Then Cara started another difficulty, quite as appalling; facing it with
innocent confidence, yet wonder: ‘What sort of a place,’ she asked, softly,
looking up with her blue eyes full of serious faith and awe, ‘is heaven?’
‘Oh, my dear,’ said Miss Cherry, ‘you ask me what I would give all I
have in the world to know! There are so many whom I love there.’
‘But what do you think? Often when one doesn’t know, one has an idea. I
don’t know Italy or India; but I imagine something. Aunt Cherry, tell me
what you think.’
‘Oh, Cara, my darling, I don’t know what it is like! I know there is no
trouble or pain in it; and that God is not so far off as here. No, He is not far
off here; but we can’t see Him; and we are such poor dull creatures. And I
think, Cara, I think that our Lord must be always about there. That people
may go and stand on the roadside and see Him pass, and talk to Him, and be
satisfied about everything.’
‘How—be satisfied about everything?’
‘Oh, child! I should not want anything more. He sees both sides, my
darling, both here and there, and understands. I am sure they must be able to
speak to Him, and go to Him, whenever they will——’
This thought brought great tears, a suffusion of utter wistfulness yet
heart-content, to Cherry’s eyes. Little Cara did not know very well what was
meant by such words. She did not understand this conception of the great
Creator as a better taught child might have done. But she said to herself, all
secretly: ‘If there is One like that, whether it is in heaven or earth, I might
tell Him, and it would be no harm.’
While Miss Cherry dried her eyes, her heart lightened by that
overflowing. Perhaps, though they had not seen Him, He had passed that
way, and heard the babble—what was it more?—between the woman and the
child.
 CHAPTER IX.

THE HILL.

After this a long interval passed, which it is needless to describe in detail.

Five years is a long time in a life; how much it does! Makes ties and breaks
them, gives life and withdraws it, finds you happy and leaves you miserable,
builds you up or plucks you down; and at the same time how little it does!
Buffets you, caresses you, plays at shuttlecock with you; yet leaves you the
same man or woman, unchanged. Most of this time James Beresford had
spent in absences, now here, now there; not travels according to the old
happy sense, though in a real and matter-of-fact sense they were more travels
than those he had made so happily in the honeymooning days. But he did not
like to use the word. He called his long voyages absences, nothing more.
And they were of a very different kind from those expeditions of old. He
avoided the Continent as if pestilence had been there, and would not even
cross it to get the mail at Brindisi, but went all the way round from
Southampton when he went to the East. He went up the Nile, with a
scientific party, observing some phenomena or other. He went to America in
the same way. He was not a very good sailor, but he made up his mind to
that as the best way of fighting through those lonely years. Once he went as
far north as any but real Arctic explorers, with their souls in it, had ever
done. Once he tracked the possible path of Russia across the wildest border
wastes to the Indian frontier. He went everywhere languidly but persistently,
seldom roused, but never discouraged. A man may be very brave outside,
though he is not brave within; and weakness is linked to strength in ways
beyond our guessing. He went into such wilds once, that they gave him an
‘ovation’ at the Geographical Society’s meeting, not because of any
information he had brought them, or anything he had done, but because he
had been so far off, where so few people had ever been. And periodically he
came back to the Square; he would not leave that familiar house. His wife’s
drawing-room was kept just as she had liked it, though no one entered the
room: the cook and John the butler, who had married, having the charge of
everything. And when Mr. Beresford came back to England, he went home,
living downstairs generally, with one of his travelling companions to bear
him company. Maxwell and he had dropped apart. They were still by way of
being fast friends, and doubtless, had one wanted the other, would still have
proved so—last resource of friendship, in which the severed may still hope.
But, as nothing happened to either, their relations waxed cold and distant.
The doctor had never got clear of the suspicion which had risen in his mind
at Mrs. Beresford’s death. It is true that had James Beresford given the poor
lady that ‘strong sweet dose’ she once had asked for, Maxwell would have
forgiven his friend with all his heart. I do not know, in such a strange case,
what the doctor could have done; probably exactly as he did afterwards do,
invent a death-certificate which might be accepted as possible, though it was
not in accordance with the facts. But, anyhow, he would have taken up
warmly, and stood by his friend to his last gasp. This being the case, it is
impossible to tell on what principle it was that Maxwell half hated
Beresford, having a lurking suspicion that he had done it—a suspicion
contradicted by his own statement and by several of the facts. But this was
the case. The man who would have helped his wife boldly, heart-brokenly, to
escape from living agony, was one thing; but he who would give her a fatal
draught, or connive at her getting it, and then veil himself so that no one
should know, was different. So Mr. Maxwell thought. The inconsistency
might be absurd; but it was so. They positively dropped out of acquaintance.
The men who visited James Beresford when he was at home, were men with
tags to their names, mystic initials, F.G.S.’s, F.R.S.’s, F.S.A.’s, and others of
that class. And Maxwell, who was his oldest friend, dropped off. He said to
himself that if Beresford ever wanted him badly, he would find his
friendship surviving. But Beresford did not want Maxwell nor Maxwell
Beresford; and thus they were severed for a suspicion which would not have
severed them had it been a reality, or so at least Maxwell thought. The doctor
still went down once a week regularly to visit Miss Charity, and so kept up
his knowledge of the family; but ‘nothing came’ of the old fancy that had
been supposed to exist between him and Cherry. They all hardened down
unconsciously, these middle-aged folk, in their various ways. The doctor
became a little rougher, a little redder, a trifle more weather-beaten; and
Miss Cherry grew imperceptibly more faded, more slim, more prim. As for
Miss Charity, being now over seventy, she was younger than ever; her
unwrinkled cheeks smoother, her blue eyes as blue, her step almost more
alert, her garden more full of roses. ‘After seventy,’ she tersely said, ‘one
gets a new lease.’ And Mrs. Burchell, at the Rectory, was a little stouter, and
her husband a little more burly, and both of them more critical. Fifty is
perhaps a less amiable age than three-score and ten. I am not sure that it is
not the least amiable age of all; the one at which nature begins to resent the
fact of growing old. Of all the elder generation, James Beresford was the one
to whom it made least change, notwithstanding that he was the only one who
had ‘come through’ any considerable struggle. He was still speculative, still
fond of philosophical talk, still slow to carry out to logical conclusions any
of the somewhat daring theories which he loved to play with. He was as little
affected as ever by what he believed and what he did not believe.
As for Cara, however, these five years had made a great difference to her;
they had widened the skies over her head and the earth under her feet.
Whereas she had been but twelve, a child, groping and often in the dark,
now she was seventeen, and every new day that rose was a new wonder to
her. Darkness had fled away, and the firmament all around her quivered and
trembled with light; night but pretended to be, as in summer, when twilight
meets twilight, and makes the moment of so-called midnight and darkness
the merriest and sweetest of jests. Everything was bright around her feet, and
before her in that flowery path which led through tracts of sunshine. She was
no more afraid of life than the flowers are. Round about her the elders, who
were her guides, and ought to have been her examples, were not, she might
have perceived, had she paused to think, exuberantly happy. They had no
blessedness to boast of, nor any exemption from common ills; but it no more
occurred to Cara to think that she, she could ever be like her good Aunt
Cherry, or Mrs. Burchell, than that she could be turned into a blue bird, like
the prince in the fairy tale. The one transformation would have been less
wonderful than the other. She had lived chiefly at Sunninghill during her
father’s absence, and it was a favourite theory with the young Burchells, all
but two (there were ten of them), that she would progress in time to be the
Miss Cherry, and then the Miss Charity, of that maiden house. A fate was
upon it, they said. It was always to be in the hands of a Miss Beresford, an
old-maidish Charity, to be transmitted to another Charity after her. This was
one of the favourite jokes of the rectorial household, warmly maintained
except by two, i.e. Agnes, the eldest, a young woman full of aspirations; and
Roger, the second boy, who had aspirations too, or rather who had one
aspiration, of which Cara was the object. She would not die Charity
Beresford if he could help it; but this was a secret design of which nobody
knew. Cara’s presence, it may be supposed, had made a great deal of