Antibody detection
Klaus Hermann, Markus Ollert and Johannes Ring
Introduction
The ACCURATE and reliable determination of biologi-
cally active endogenous compounds in plasma,
urine and other body fluids for scientific or diagnos-
tic purposes has been a challenge for many
decades. In the past, the assessment of such com-
pounds was difficult and tedious because specific
and practical analytical tools were not available. In
the early days, decisive and convincing conclusions
about the significance of a biologically active mole-
cule in disease or health could only be made after
purification and isolation of the ANALYTE and identifi-
cation of its chemical structure. A definite improve-
ment in the analysis of biologically active endoge-
nous compounds was the introduction of bioassays
based on an entire animal model or in vitro tissue
preparations.Although the bioassays possessed suffi-
cient sensitivity, there were problems with their lack
of SPECIFICITY. Other analytical procedures such as liq-
uid chromatography, electrophoresis or photometric
procedures have also been developed for in vitro
diagnosis. However, these approaches are either
tedious and time-consuming or require expensive
equipment and specially trained staff.A landmark in
diagnostics was the introduction of immunoassays
(IAs) which are inexpensive and easy to perform
with high reproducibility, SENSITIVITY and SPECIFICITY.
Basic principle of immunoassays
IAs are based on an antigen-ANTIBODY (Ab) reaction
utilizing the exceptional capability of the immune
system to produce specific Abs which can recognize
and discriminate between a practically infinite num-
ber of foreign compounds.The basic requirement for
B1
setting up an IA is the production of a specific Ab to
a given antigen or hapten by immunizing animals
such as mice, rabbits, goats, horses or others. High
molecular weight compounds like proteins are usu-
ally immunogenic and may serve as an antigen
which can be directly injected into the host animal
to produce Abs. In contrast, low molecular weight
compounds such as drugs, peptides or steroids are
haptens which do not induce immune responses.
They can be rendered immunogenic after coupling
to a carrier. The Abs obtained from the serum after
several booster injections with the immunogen are
HETEROGENEOUS polyclonal Abs (pAbs). A more
sophisticated approach was the generation of mono-
clonal Abs (mAbs), which was first introduced by
Köhler and Milstein in 1975 [1]. This technique
allows the production of HOMOGENEOUS Ab species in
nearly infinite quantities with high SPECIFICITY for a
certain antigen.The method involves the isolation of
spleen cells from an immunized animal containing
Ab-producing B CELLS.The B CELLS are then fused with
myeloma cells. After selection and screening of the
desired Ab-secreting cell line (hybridoma), the Ab
can be harvested in the supernatant. These hybrido-
mas can be grown in large volumes for the produc-
tion of huge quantities of the mAb.
Basic essentials for the characterization of the
Abs produced are high affinity, SPECIFICITY and sensi-
tivity. The affinity is a measure of the strength of the
binding interaction between the antigen and the Ab
and can be experimentally determined; according to
the law of mass action, it is expressed as equilibrium
constant of dissociation, Kd (mol/L), or of associa-
tion, Ka (L/mol). The lower the Kd the greater the
affinity.The SPECIFICITY depends on the recognition of
the ANALYTE by the Ab and the extent of crossreaction
with closely related or structurally similar ANALYTES.
The less the crossreaction, the higher the SPECIFICITY.
164 Antibody detection
FIGURE 1. STRUCTURE OF IMMUNOGLOBULINS
For further details, see text
This in turn is closely related to the ability of the Ab
to discriminate between negative and positive sam-
ples.The SENSITIVITY describes the detection limit and
is defined by the dose-response curve of the antigen
to the Ab. The lower the detection limit, the higher
the sensitivity.
Antibody structure
Abs, among other serum proteins such as albumins,
belong to the gammaglobulin or immunoglobulin
(Ig) fraction according to their electrophoretic
mobility.They are glycoproteins which are chemical-
ly very similar in structure and are constructed of
two identical light chains with an approximate
molecular weight (MW) of 25 kDa per chain and two
identical heavy chains with an approximate MW of
55 kDa per chain. Each of the two light chains is
covalently attached to one heavy chain via a single
disulfide bond.Likewise,the heavy chains are bound
to each other by multiple disulfide bridging. In addi-
tion, the three-dimensional Ab structure is stabilized
by noncovalent intra- and interchain-interactions.
Two locally distinct binding domains are prominent
on Ig molecules. One is the antigen binding site
(Fab) for the binding and recognition of antigens
and the other is the receptor binding site (Fc) for
binding to specific receptors on various cells
involved in immunological functions such as
mononuclear phagocytes, NATURAL KILLER CELLS, mast
cells or basophil LEUKOCYTES (Fig. 1). Although Igs
share an overall similarity, they can be divided into
different classes and subclasses according to their
size,charge,solubility and their behavior as antigens.
At present, the Ab molecules in humans can be clas-
sified into IgA, IgD, IgE, IgG and IgM. IgA and IgG can
be further subdivided into their subclasses IgA1 and
IgA2 and IgG1, IgG2, IgG3 and IgG4 (see Chapters A.3
and C.2).
Clinical relevance of antibody
detection
Initially, the identification and characterization of
specific Abs associated with pathophysiological con-
ditions were generally constrained to serve scientific
purposes. However, in many instances the measure-
ment of specific Abs evolved into clinically relevant
diagnostic markers in health and disease. Medical
conditions in which the determination of specific
Abs is routinely used are bacterial and viral infec-
tions, infestation with parasites, autoimmune dis-
eases and allergies.
Microbial infections
The determination of Abs in infectious diseases has
been known and used for a long time. Bacteria-spe-
cific Abs of the IgG and IgM class are routinely
detected in the serum of patients infected with differ-
ent bacteria such as Borrelia burgdorferi (lyme dis-
ease), Chlamydia trachomatis (sexually transmitted
infection of the urogenital tract), Legionella pneu-
mophila (legionnaire disease), Staphylococcus and
Streptococcus and Treponema pallidum (lues).
The assessment of IgG and IgM Abs is also a very
valuable parameter in the diagnosis of viral infec-
tions such as hepatitis, measles, epidemic parotitis

and rabies, and infections with the Epstein-Barr virus
(mononucleosis), herpesvirus (herpes simplex and
herpes zoster) and arborvirus (tick encephalitis).
Infestations with parasites leading to diseases such
as leishmaniasis, amoebiasis, malaria, toxoplasmosis,
schistosomiasis, echinococcosis, tichinellosis, filaria-
sis or others can induce the Ab formation of different
Ig classes. In the diagnosis of parasitosis, it is recom-
mended that the identity of the parasite be ascer-
tained first.In addition,serological methods are avail-
able to identify circulating antigens,antigen-Ab com-
plexes or circulating Abs.
Autoimmune diseases
Another area of pathophysiological abnormalities in
which the measurement of Abs has predictive and
diagnostic value is that of the numerous autoim-
mune diseases (ADs) in which the self-tolerance of
the immune system against certain structures of the
host is abrogated. Examples of an organ-specific AD
are Hashimoto’s thyroiditis with circulating Abs
against thyroglobulin, and myasthenia gravis with
auto-Abs against the acetylcholine receptor. Exam-
ples of non-organ-specific ADs are Sjögren’s syn-
drome, rheumatoid arthritis, scleroderma and sys-
temic lupus erythematosus affecting the skin, joints
and muscles with Abs against nuclear antigens such
as DNA, RNA or histones.
Allergy
The most important immunglobulins for the in vitro
diagnosis of allergic diseases, either immediate-type
HYPERSENSITIVITY reactions such as rhinitis,conjunctivi-
tis, allergic bronchial ASTHMA and ANAPHYLAXIS or LATE
PHASE reactions, e.g., allergic contact dermatitis, are
Abs of the IgE class [2, 3]. Clinically relevant are the
measurement of total IgE or allergen-specific IgE in
the patients’ serum for the determination of IgE-
mediated sensitization.Total serum IgE levels of >100
(kilo units) KU/l in adults are a good indicator for
atopy, a disease characterized by familial HYPERSENSI-
TIVITY to exogenous environmental agents associated
with high IgE Ab titers and altered reactivity against
Antibody detection methods 165
various pharmacological stimuli. However, high total
IgE Abs can also be induced by parasitic worm infes-
tations. Extremely high values, above 10,000 KU/l, are
indicative of IgE-producing myelomas or hyper-IgE
syndrome.The measurement of allergen-specific IgG
Abs as IgE-blocking Abs for monitoring the success
of immune therapy with insect venoms in patients
with hymenoptera venom allergy has been used ten-
tatively, but with discrepant results.
Antibody detection methods
The detection of Abs in the circulation or in tissue
has become a useful analytical tool for the in vitro
immunodiagnosis of numerous diseases. Several
immunological techniques are available for the rou-
tine identification of IgA,IgD,IgE,IgG or IgM Ab class-
es in the laboratory of clinical chemistry. The most
commonly used methods are discussed briefly.
Immunoprecipitation assay
Immunoprecipitation is a very simple and easy to
perform in vitro assay for the identification and semi-
quantification of soluble Abs. The addition of the
antigen to the Ab results in the formation of a three-
dimensional, insoluble network of precipitating
aggregates which can be detected by, e.g., a neph-
elometer. The assay is very similar to a volumetric
acid/base titration.The bulk of precipitate, formed at
equivalent concentrations of Ab and antigen, is a
measure of the concentration of the Ab. The assay
can also be used in reverse to measure the antigen
concentration by adding Abs.
A variation of the immunoprecipitation assay is
the hemagglutination test and the complement fixa-
tion test.The hemagglutination test allows the identi-
fication of Abs to red blood cell antigens or the
detection of Abs to antigens which are covalently or
noncovalently attached to the red cell surface. The
complement fixation test is a three-step assay in
which the Ab-containing serum is initially incubated
with a fixed amount of antigen to form an immune
complex. In the second step, COMPLEMENT is added,
firmly incorporated by the immune complexes and
166 Antibody detection
consumed to some extent. Finally, Ab-coated red
blood cells are added as indicator cells for titration
of the remaining quantity of COMPLEMENT; the less the
red blood cells are lysed the more the immune com-
plexes have been generated before.
Immunocytochemistry
Immunocytochemistry is a technique for the in situ
detection of an Ab in tissue slices.Frozen tissue or tis-
sue embedded in various embedding media is cut
into thin slices and then immobilized on a slide.After
fixation of the tissue with formaldehyde, glutaralde-
hyde,alcohol or acetone,the tissue is incubated with
a specific primary Ab directed against the Ab to be
detected. In the direct assay, the primary Ab is chem-
ically coupled to a fluorescent dye, which allows the
detection of the ANALYTE by fluorescence microscopy.
In the indirect assay, excess primary Ab is thoroughly
washed off, and the tissue is incubated with a sec-
ondary Ab to form a sandwich. The secondary Ab
can be fluoresceinated or coupled to an enzyme,
e.g., alkaline phosphatase (ALP) or peroxidase. This
allows the visualization of the ANALYTE by fluores-
cence microscopy or by light microscopy after addi-
tion of a colorless SUBSTRATE which is enzymatically
converted to a colored product. Only cells which
contain the ANALYTE will light up under the micro-
scope.
Immunoblotting
The immunoblot or dot blot technique is similar to
the immunoprecipitation assay. However, in immuno-
blotting the antigen-Ab reaction takes place on the
solid phase, whereas in the immunoprecipitation
assay the Ab reacts with the antigen in solution.The
assay utilizes the capability of nitrocellulose or poly-
vinylidene fluoride (PVDF) membranes to bind anti-
gens. Antigens are applied in small dots, and the
membranes are dried. The membranes are treated
with blocking buffer (containing e.g. ovalbumin, gel-
atin or milk proteins) to prevent unspecific adsorp-
tion. After blocking, the membranes are incubated
with the serum and dilutions of the serum, which
contain the Ab. The membranes are washed to
remove abundant Ab. Next, the membranes are incu-
bated with a secondary Ab raised against the Ab of
interest which is conjugated with an enzyme.The for-
mation of the antigen-Ab-secondary Ab complexes
can be visualized by adding a SUBSTRATE which will
be converted by the secondary Ab-bound enzyme to
yield a colored spot.The use of secondary Ab-bound
enzymes represents the basic ELISA (enzyme-linked
immunoadsorbent assay) principle. The intensity of
the spots is proportional to the amount of Ab present
in the serum samples (Fig.2,left panel).Further char-
acterization of the Ab can be achieved by separating
the antigens electrophoretically. The separated com-
ponents are transferred from the gel to a membrane,
a process which is called Western blotting.The mem-
branes are then treated like dot blots as outlined
above. This methodology combines the high resolv-
ing power of electrophoresis and the discriminating
power of an immunological reaction. Components
that are recognized by the Ab show up on the West-
ern blot as colored bands (Fig. 2, right panel).
Immunoadsorbent assays
Immunoadsorbent assay (IAA) techniques are wide-
ly used for the quantitative measurement of serum
IgE and IgG Abs. An antigen, e.g., an allergen extract
from chicken meat,grass or tree pollen,or house dust
mites, is attached to an inert matrix such as the wall
of a reaction vial or microtiter plate wells, or it is
chemically coupled to a paper disc.The serum of an
allergic patient is incubated in a first-step reaction
with the allergen-coated matrix. IgE molecules that
recognize the allergen are bound. After removal of
excess serum, a secondary Ab, in this case an anti-
human IgE Ab raised in mice,rabbits,goats or horses,
is added which forms an allergen-IgE-anti-IgE Ab
complex (second-step reaction). Excess of the sec-
ondary Ab is likewise removed by washing. The for-
mation of the allergen-IgE-anti-IgE-Ab-complex
depends on the amount of specific IgE present in the
serum sample. Since the secondary Ab, also termed
detecting Ab, carries a covalently coupled LABEL or
tag, the formation of the allergen-IgE-anti-IgE Ab
complex, a sandwichlike structure, can be moni-

FIGURE 2. DOT BLOTS AND IMMUNOBLOTTING
Specific Abs to an antigen, e.g., an allergen extract, can
be detected with the dot blot technique. Further charac-
terization of the Ab or the antigens present in the aller-
gen extract can be achieved by immunoblotting. For fur-
ther details, see text. The various lanes are different aller-
gen extracts.
tored.Utilizing a standard curve with increasing con-
centrations of the allergens or, as in most commer-
cial specific IgE assays, with various amounts of the
World Health Organization’s International Reference
Preparation for IgE (2nd IRP75/502), the signal
obtained with allergen-IgE-anti-IgE Ab complex in
the serum sample can be compared with the signal
of the standard curve, which permits the quantifica-
tion of the IgE Abs.The assay format of an IAA in gen-
eral is summarized in Figure 3.
The radio-allergo-sorbent test (RAST) is a radio-
immunoassay (RIA) version [4] in which the aller-
gens are chemically coupled to a paper disc and the
FIGURE 3. IMMUNO-ABSORBENT ASSAY FORMAT
Reproduced with permission from DPC Biermann, Germany. For further details, see text.
Antibody detection methods 167
secondary Ab is radioactively labeled with I-125
(Pharmacia Uppsala, Sweden). Similarly, the assay
can be performed as an enzyme immunoassay
(EIA) in which the secondary Ab is labeled with the
enzyme β-galactosidase which can react with a col-
orless SUBSTRATE to form a colored reaction product
(EAST, Sanofi Diagnostics Pasteur, Chaska, MN, USA).
In addition to the RAST or EAST, a RAST or EAST
inhibition assay can be performed to confirm and
validate the results [5]. The serum samples are first
incubated in vitro with increasing concentrations of
the allergens prior to the RAST or EAST.The binding
inhibition of the Ab can be illustrated by a dose-
response curve which inversely correlates with the
concentration of allergens added; low allergen con-
centrations still give a high signal in the RAST or
EAST,whereas the signal vanishes with high concen-
trations. The concentration of the allergen at 50%
inhibition (IC50) can be calculated from the dose-
response curve. A low IC50 is a good indicator for a
high affinity and SPECIFICITY of the Ab to the allergens
(Fig. 4).
A further development of the RAST or EAST was
the introduction of a three-dimensional cellulose
sponge instead of the paper disc. This approach is
practiced by the ImmunoCAP system (Pharmacia
Uppsala, Sweden). The ImmunoCAP assay is per-
formed in microtiter plates and allows partial to
complete automation of the assay. The advantages
are higher binding capacity of the cellulose sponges
(CAP) and the use of the photometric detection of
the allergen-IgE-anti-IgE Ab complex with a micro-
168 Antibody detection
FIGURE 4. RAST INHIBITION ASSAY
Reproduced with permission from DPC Biermann, Germany. For further details, see text.
plate reader. Full automation of the CAP system for
IgE detection was achieved with the most recent
developments (UniCAP250 and 1000).Variations and
modifications of this basic assay format have been
made with respect to the characteristics of the aller-
gens, e.g., using allergens in liquid phase and replac-
ing the β-galactosidase-labeled secondary Ab with
an Ab that is chemically linked to ALP. This unique
concept is realized in several assay formats. The
AlaSTAT liquid allergen technology [Diagnostic
Product Corporation (DPC), Los Angeles, CA, USA]
[6] is based on a four-step reaction. In the first incu-
bation, the biotinylated allergens react with patients’
serum IgE. In the second incubation, avidin is added
which forms a complex between the biotinylated
allergens and IgE.In the third incubation,an enzyme-
labeled secondary anti-IgE Ab is added to form a
sandwich between the biotin of the reaction vessel,
avidin,the biotinylated allergen and the allergen-spe-
cific IgE Abs. Finally, the formation of the triple-sand-
wich complex can be photometrically monitored by
the enzymatic conversion of the SUBSTRATE to a col-
ored product (Fig. 5A).
A different assay technology is realized in the
Access Allergy Diagnostic System (Sanofi Diagnos-
tics Pasteur). The assay is also a liquid phase assay
using paramagnetic polystyrene beads coated with
an antibiotin Ab as a capturing Ab.In a first-step reac-
tion, the biotinylated allergens and the allergen-spe-
cific IgE form a triple-sandwich complex between
the polystyrene beads, the allergen and the IgE. The
second reaction is the incubation with an anti-
human IgE Ab (secondary Ab) labeled with ALP,
which results in the formation of a quadruple sand-
wich (paramagnetic polystyrene beads,allergens,IgE
and anti-IgE). Excess ALP-labeled secondary Ab is
also removed in the magnetic field. Finally, dioxe-
tane-phosphate is added as a SUBSTRATE which is con-
verted by ALP to a chemiluminescent product which
can be quantified in a luminometer (Fig. 5B).
In the absence of a recognized reference method
for in vitro sIgE measurement, the Pharmacia’s sec-
ond-generation ImmunoCAP® technology has
become a quasi-standard due to its widespread use,
analytical reliability and the generally adequate cor-
respondence of its results – on a positive/negative
basis – with the results of skin testing [7]. From their
inception,however,certain limitations of second-gen-
eration test systems for sIgE have been apparent.
They are labor-intensive and require specially
trained personnel, making them both costly to the
laboratory and potentially more prone to human
 Antibody detection methods 169

FIGURE 5. ADVANCED IMMUNOASSAY TECHNIQUES

(A) AlaSTAT liquid allergen technology (reproduced with permission from DPC Biermann, Germany). (B) Access Aller-
gy Diagnostics System (reproduced with permission from Sanofi Diagnostics Pasteur, USA). For further details, see text.

error than a fully automated system with barcoding “Third generation” systems for the in vitro measure-
as used in most other fields of clinical chemistry; ment of sIgE, addressing the major limitations associ-
moreover, turnaround time (≥ 3 hours) is still unde- ated with second-generation assays, have finally
sirably slow from the allergy specialist’s point of view. been developed [8, 9]. These are based on proven
170 Antibody detection
technology, have considerably shorter turn-around
times, and utilize chemiluminescence.The first fully-
automated, random-access assay to become avail-
able is DPC’s IMMULITE® 2000 Allergy (IML) [9, 10],
which represents the implementation of routine sIgE
testing on a family of systems already well estab-
lished in clinical chemistry for performing
immunoassays and immunometric assays, in both
hospital and reference laboratory settings. IML has a
working range of 0.10 to 100 kU/L and uses DPC’s liq-
uid allergen chemistry together with the Immulite
bead and wash technology and an enzyme-
enhanced chemiluminescent detection system [10].
Summary
Antibody detection is crucial for the differential
diagnosis of many different pathological conditions.
Determination of specific antibodies to bacterial
and viral pathogens as well as to parasites enables
the correct therapeutic measures to be taken. Anti-
bodies to organ-specific and systemic autoimmune
diseases are predictive for prognosis and detection
of IgE class antibodies is essential for the diagnosis
of immediate HYPERSENSITIVITY reactions.
Methods for detection of antibodies include
immunoprecipitation assay, in which Ag-Ab complex
aggregates are detected, often by hemagglutination;
immunocytochemistry, for in situ Ab detection in tis-
sue slices; immunoblotting (dot blot technique)
whereby Ag-Ab aggregates are trapped on mem-
branes and then detected with a secondary Ab to
yield spots; and immunosorbent assays, which are
similar to immunoblotting but,by using a tagged sec-
ondary antibody, allow the primary antibody to be
quantified. A variety of immunosorbent kits are avail-
able which permit rapid,specific,accurate and sensi-
tive detection particularly of IgE antibodies.
Selected readings
Tijssen P (1985) Practice and theory of enzyme immunoas-
says. Elsevier,Amsterdam
Abbas AK,Lichtman AH,Pober JS (eds) (1994) Cellular and
molecular immunology.W.B. Saunders, Philadelphia
Crowther JR (ed) (1995) ELISA. Humana Press,Totowa
Thomas L (ed) (1988) Labor und Diagnose. Medizinische
Verlagsgesellschaft, Marburg
Roitt IM, Brostoff J, Male DK (1986) Immunology. Mosby, St.
Louis
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