Monoclonal Antibodies:
Diagnostic Uses
Heddy Zola, Child Health Research Institute, Adelaide, Australia
Peter Roberts Thomson, Flinders Medical Centre, Adelaide, Australia
Diagnosis of disease depends frequently on the identification, quantitation or localization
of particular molecules, cells or organisms in tissue or body fluids. Antibodies, because of
their exquisite specificity, are particularly useful reagents in this context, and monoclonal
antibodies are often superior to polyclonal mixtures of antibodies. Monoclonal antibodies
are used widely in the diagnostic laboratory.
Introduction
The diagnosis of disease has always relied on the skill,
knowledge and intuition of the doctor. Increasingly, as
the mechanisms of physiological processes and the ways
in which they can go wrong have been understood, doc-
tors use laboratory data to help them to arrive at a de-
finitive diagnosis. Note that in this paper the term
‘diagnosis’ is used in its broad sense to include all tests
that help to identify or understand a disease process,
even if they are not registered as diagnostic assays by
regulatory authorities. It is also used to include moni-
toring of disease severity and response to treatment, after
the original diagnosis.
Laboratory data greatly enhance the resolving power of
diagnosis. A few relevant questions and a thorough exam-
ination may tell the physician that a patient is suffering
from infection, and it is probably bacterial rather than vi-
ral. Laboratory tests can identify the bacterial pathogen,
can determine to which antibiotics the pathogen is sensi-
tive, and can provide detailed information about the im-
mune status of the patient. For example, has the pathogen
been encountered before or is there some underlying rea-
son for the failure of the patient to recover from the in-
fection naturally?
Laboratory tests can add enormous analytical power to
the diagnostic skill of the physician; however, they do not
replace that skill. Asking the right question, making the
link between disparate symptoms, experience and insight
remain essential qualities for the diagnostician.
Laboratory tests which assist the pathologist include
identification of pathogens, measurement of the levels of
normal or abnormal blood and tissue components, and
detailed microscopic examination of tissue architecture
and composition. Antibodies can be powerful tools in all of
these tests. This is a consequence of the exquisite specificity
of antibodies, and the fact that they can be used in a variety
of test formats, to provide qualitative and quantitative in-
formation.
ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net
Advanced article
. Introduction
Article Contents
. Antibody as Diagnostic Probe
. Applications in Infectious Diseases
. Applications in Disorders of the Immune System
. Applications in Haematology and Blood Transfusion
. Applications in Chemical Pathology
. Applications in Tissue Pathology
. Other Applications
doi: 10.1038/npg.els.0004019
Antibody as Diagnostic Probe
The antibody molecule has a binding site which recognizes
a particular molecular shape. The immune system of mam-
mals, birds and reptiles is capable of making an enormous
diversity of antibody-binding sites. While this diverse an-
tibody repertoire has presumably evolved to protect us
from infection by diverse and rapidly evolving pathogens,
we can subvert it to prepare, under controlled conditions,
antibody against essentially any molecular structure we
choose. See also: Antibodies
Antibodies may be used in diagnostic tests requiring
identification, quantitation and localization of molecules
and cells, whether foreign or derived from the host. They
may be used in a variety of assay formats, and are used in
the diagnostic disciplines of microbiology, immunology,
haematology and blood transfusion, chemical pathology,
tissue pathology, forensic pathology and veterinary
pathology. See also: Monoclonal antibodies
Assay formats
An antibody reacts specifically with a particular molecular
structure, referred to as an epitope. That epitope is usually
a part of a larger molecule, which may be free in solution or
may be part of a larger structure, a cell or a tissue. Thus,
antibody can be used as a specific probe for epitope, mol-
ecule or cell. See also: Epitopes
Depending on the question being asked, antibody is used
to identify, localize or quantitate the target molecule or
cell. The target (molecule or cell) and the type of informa-
tion required (identification, localization or quantitative
assay) dictate the assay type required. Table 1 summarizes
assay formats suitable for different purposes, showing
some of the major techniques available to answer questions
of identity, quantity and localization of molecules and
cells. Table 2 lists a few examples of diagnostic questions
1
 Monoclonal Antibodies: Diagnostic Uses

Table 1 Assay formats suitable for answering different questions about molecular and cellular targets
Nature of question
Target Identify Quantify Localize
Molecule Western blot Radioimmunoassay Tissue staining and microscopy
Immunoprecipitation Enzyme-linked immunoassay
Particle agglutination
Cell Microscopy Flow cytometry Tissue staining and microscopy
Flow cytometry Video image analysis

and the techniques that might be used to arrive at an an-
swer. See also: Enzyme-linked immunosorbent assay; Flow
cytometers; Fluorescence microscopy
Applications in Infectious Diseases
Medical significance
Infection remains a challenge to the pathologist. The mul-
tiplicity of related infectious agents, coupled with the need
for appropriate and often immediate treatment, provide a
strong stimulus for the development of laboratory diag-
nostic tests. Important as it is to arrive at a definite diag-
nosis in the interests of the patient, diagnosis of certain
infectious diseases is even more important in the interests
of public health. To illustrate with a few recent and recur-
ring examples, identification of a new strain of influenza in
humans, derived from chickens, sounds warnings of a pos-
sible pandemic and leads to the destruction of infected or
at-risk birds; diagnosis of a cluster of cases of haemolytic
uraemic syndrome necessitates rapid identification of the
contaminated food source; diagnosis of dengue fever in an
area in which it is not normally found, in patients who have
not recently travelled to endemic areas, calls for urgent
trapping of mosquitoes to confirm the source of this mos-
quito-borne virus and mosquito eradication measures if it
is detected. See also: Influenza epidemics
Diagnostic options
The diagnosis of infection traditionally requires that the
organism be identified in, or cultured from, infected ma-
terial. This is often not possible with adequate sensitivity or
specificity, and must be supplemented with highly sensitive
and specific techniques. Antibody-based methods and mo-
lecular techniques such as the polymerase chain reaction
(PCR) provide the necessary sensitivity and specificity.
See also: Polymerase chain reaction (PCR)
Antigens characteristic of particular infectious agents
can be detected using antibodies in a variety of assay for-
mats, including immunohistochemistry on tissue samples,
enzyme-linked or fluorescence-based immunoassays of tis-
sue extracts or body fluids, and a variety of technically
simple tests, such as particle agglutination, which can be
carried out in the field. Assays vary in sensitivity, and cross-
reactivity between related organisms is a recurring prob-
lem. Certain components of microorganisms, for example,
bacterial lipopolysaccharide, are shared by many species.
Conversely, some antigens are subject to rapid variation in
expression, and tests based on antibodies against these an-
tigens may fail to identify a pathogen. Subtyping related
organisms, for example, herpes simplex types I and II, may
be an important part of the diagnosis. See also: Herpes-
viruses (human); Immunoassay
It is thus important to know the natural history of the
infectious organism and use antibodies against antigens
that will provide the right level of specificity.
Analysis of the patient’s antibody response
As an alternative to antigen detection, the antibody re-
sponse of the patient may provide valuable information.
This approach is particularly valuable in viral infection,
where antigen detection may be particularly difficult. The
antibody concentration and class (immunoglobulin M
(IgM), characteristic of an early immune response, or IgA
and IgG, the classes characteristic of the later stages of a
response or a memory response to a previously encoun-
tered antigen) are particularly informative. Detection of
antibody against, for example, the dengue virus, is not by
itself informative unless we know whether it is IgM, indic-
ative of a current infection. Figure 1 shows the format of an
enzyme immunoassay designed to titrate IgM and IgG
against the dengue virus. See also: Antibody classes;
Dengue fever viruses
The decision whether to base diagnosis on identification
of the antigen or on analysis of the patient’s antibody (sero-
logy) depends on a number of factors, including primarily
the availability of well-validated assay kits. In practice,
serology is used very widely, because it is rapid, technically
straightforward, and capable of application on serum
samples, wherever the tissue site of infection.

2
 Monoclonal Antibodies: Diagnostic Uses

Table 2 A few typical diagnostic questions and the techniques that might be used to give an answer
Question Information required Suggested technique
Does patient X express the HLA-B27 Identification of cells bearing a particular Flow cytometry
allele, which is associated with the in- marker
flammatory back disorder ankylosing
spondylitis?
How many CD4 cells, the target cells for Enumeration of cells bearing a particular Flow cytometry
the human immunodeficiency virus marker
(HIV), does patient Y have per millilitre
of blood?
Is Ms D pregnant? Identification of a particular molecule Particle agglutination
(hormone) in urine
Laboratory worker P has just been ex- Quantitation of molecule (antibody) Enzyme-linked immunoassay
posed to patient serum. What level of
antibody against hepatitis B does the
worker have, as a result of prior (pro-
tective) immunization?
Does patient Y, immunosuppressed to Antigens coded for by the virus may be Tissue staining and (fluorescence) mi-
control rejection of a transplanted kid- detected on blood cells, or on tissue cul- croscopy
ney, have cytomegalovirus (CMV) in- ture cells after incubation with infected
fection? material. Identification of molecule (viral
antigen) on cells
An enlarged, firm and nontender lymph Identification and localization of partic- Tissue staining and microscopy
node is removed from the neck to deter- ular cell types and features
mine if it is malignant. Histological ex-
amination reveals infiltration by small
round neoplastic cells. What is the nature
of these infiltrating cells, and what is their
lineage (do they have a lymphoid or an
epithelial origin)?

Identification of viruses
Viruses are detected usually in enzyme immunoassay for-
mat or by immunofluorescence using antibody against vi-
ral antigens, often after infecting cells in culture using tissue
isolates thought to contain the virus. The most appropriate
test format depends on the biology of infection. For ex-
ample, CMV produces a long-lasting infection, with latent
virus inhabiting cells of the immune system. The virus can
be grown in human fibroblasts in the laboratory, but it may
take several weeks before the cells show cytopathic chang-
es. However, early CMV antigens may be detected in the
cells by immunofluorescence within hours, or may be de-
tected directly in patient blood cells (Drew, 1988). See also:
Cytomegalovirus infections in humans
Bacterial infection
A major advantage for direct isolation of bacteria from pa-
tient material is the analysis of antibiotic sensitivity of the
particular strain. However, this is not always feasible, since
some species do not grow rapidly enough or do not grow at
all in the laboratory. Rapid identification is advantageous to
allow early treatment with appropriate antibiotics.
Monoclonal antibodies are used to confirm identifica-
tion of bacterial pathogens, and in situations where iden-
tification by other means is complex and time-consuming.
An example is in the diagnosis of Neisseria gonorrhoea and
its differentiation from other Neisseria species, which
would otherwise require complex biochemical testing.
Monoclonal antibodies are also used in rapid aggluti-
nation tests, and in the detection of bacterial products such
as toxins.
Assay formats include antibody-coated latex beads for
rapid agglutination tests, enzyme-linked immunoassays
and detection of bacteria in tissue or culture using fluoro-
chrome-labelled antibody coupled with either fluorescence
microscopy or flow cytometry. See also: Agglutination
techniques for detecting antigen–antibody reactions; Flow
cytometers

3
 Monoclonal Antibodies: Diagnostic Uses
Figure 1 Schematic representation of an enzyme-linked immunoassay to detect and titrate IgG and IgM antibodies against dengue virus. (Courtesy of
Peter Devine, Jody Mitchell and Andrea Cuzzubo, PanBio Pty Ltd.)
Antibody-based assays and molecular
biological assays
Assays based on molecular biological techniques, partic-
ularly the PCR, are highly sensitive and specific. Where
detection of the pathogen is appropriate, molecular tech-
niques can often replace antibody-based assays, although
they cannot at present compete with the speed and sim-
plicity of methods based on particle agglutination. Fur-
thermore, serological assays, which detect antibody made
by the patient, yield information of a different nature, and
are unlikely to be replaced by molecular methods.
Monoclonal antibodies can often complement molecu-
lar techniques, for example, by providing a preliminary
enrichment of pathogen prior to PCR. This approach is
particularly valuable when the pathogen has to be concen-
trated from a large sample in the presence of potentially
inhibitory or confounding impurities (e.g. patient stools).
Other pathogens
Apart from viruses and bacteria, pathogens of medical
significance include unicellular and multicellular parasites,
fungi, mycoplasma and chlamydia. Parasites are generally
difficult to grow in culture but they tend to be larger than
bacteria and were originally identified by microscopy. An-
tibody-based methods have greatly enhanced differential
diagnosis, permitting identification of individual species
with certainty. Antibodies are used with microscopy or
flow cytometry to detect parasites such as Giardia, which
can be transmitted in contaminated drinking water, and
the insect-borne malaria parasite, Plasmodium. Fungal in-
fections are important in immune-compromised patients,
including acquired immune deficiency syndrome (AIDS),
4
and can be detected with a range of antibody-based assays
including microscopic immunofluorescence and enzyme
immunoassay directed against specific fungal antigens.
See also: Acquired immune deficiency syndrome (AIDS);
Malaria; Protozoan pathogens: identification
Applications in Disorders of the Immune
System
Introduction
The immune system has evolved to defend us from infec-
tious disease, and it does that effectively. However, im-
mune responses are frequently the cause of disease, when
they target self-tissues (autoimmunity), when they are in-
effective against infection (immune deficiency, frequently
caused by a genetic defect in a component of the immune
system), when they are overzealous (hypersensitivity dis-
orders, including allergies), or when they perform their
task well, but are inconvenient where medicine has tink-
ered with nature (transplant rejection, for example).
See also: Autoimmune disease: pathogenesis; Graft rejec-
tion: mechanisms; Hypersensitivity: immunological; Im-
munodeficiency
The immune system is a complex network of positive and
negative interactions between several types of cells and
involves a multitude of proteins, hormones and their recep-
tors working together with cell surface or soluble ligand–
receptor pairs. Defects, qualitative or quantitative, in any of
these components, or in the downstream signalling path-
ways evoked by their interactions, can lead to malfunction
of the immune system. See also: Immunoregulation
 Monoclonal Antibodies: Diagnostic Uses

Diagnostic immunopathology requires the analysis of
the mediators of immunity (antibody, complement com-
ponents, effector cells). Detailed analysis of the interacting
components of the immune system can shed light on mech-
anisms of immunopathology, and can frequently guide the
selection of specific treatment options.
Leucocyte markers
Monoclonal antibodies have made possible a detailed
analysis of the functional molecules on the cell surface. To
date, the International Workshops on Human Leucocyte
Differentiation Antigens (HLDA) have named 247 ‘CD’
markers, corresponding to 320 molecules, most of them
cell surface molecules (Mason et al., 2002). The majority of
these molecules had not been identified before the avail-
ability of monoclonal antibodies. Many of them were first
identified with monoclonal antibodies, the genes coding
these molecules were then cloned, and the control of their
expression analysed.
Cells express molecules involved in their function on
their surface. While some cell surface molecules are ex-
pressed by all cells, others are expressed in a restricted way
that allows them to be used as ‘markers’ of particular cell
types. Thus, all leucocytes and only leucocytes express
CD45, all T cells and only T cells express CD3. Table 3
provides a list of some of the more useful cell markers used
in analysis of the components of the immune system.
While these markers can be detected by molecular meth-
ods such as PCR, the use of antibodies generally is much
preferred, because it demonstrates that the molecule is ac-
tually expressed, and because the combination of mono-
clonal antibodies and flow cytometry allows cell-by-cell
analysis. Multiparameter analysis allows the simultaneous
analysis of several cell markers.
Once an antibody is available as a marker for a partic-
ular cell type, it can be used to identify the cells in tissue, to
count cells in tissue or in a suspension, to separate out cells
with the help of physical separation procedures which dis-
tinguish cells coated with antibody from uncoated cells, or
to destroy the cells using a cytotoxic agent linked to the
antibody. Antibody against some surface molecules can be
used as probes for cellular function. See also: Cell
separation techniques used in immunology
The use of cell-membrane markers to study leucocyte
composition in blood and tissue serves as an example of the
analytical power of monoclonal antibodies, particularly in
combination with flow cytometry. It is also the example
most relevant to studies of the immune system, because the
cellular composition of blood and lymphoid tissue pro-
vides a ‘window’, allowing the analysis and monitoring of
the immune system.
Monoclonal antibodies against cell-surface markers are
most easily and powerfully used as fluorescent conjugates,
by flow cytometry for cells in suspension or fluorescence
microscopy for studies of solid tissues. Fluorescence is ca-
pable of very high sensitivity, matching that of radioiso-
topic methods. The wavelength change characteristic of
fluorescence, and the ability to measure the fluorescent
emission at 908 to the incident light, allows very favourable
signal to noise ratios, both in microscopy and in flow
cytometry.
Flow cytometry
In flow cytometry, cells in suspension are forced to flow in a
single file through a laser beam. Each cell scatters light as it
passes through the beam; the intensity of the scattered light
in a forward direction (low-angle scatter or forward scat-
ter) depends on the size of the cell, while the intensity of the
scatter signal at 908 (side scatter) depends on a number of
physical properties including complexity of the cell surface
and interior. Together, side scatter and forward scatter
allow differentiation of cell populations such as platelets,
red cells, lymphocytes, granulocytes and monocytes in
blood.
Cell types that cannot be distinguished on the basis of
their scatter properties can be distinguished using mono-
clonal antibodies conjugated to fluorochromes. By using a
number of antibodies labelled with different fluoro-
chromes with distinct fluorescent spectra, several markers

Table 3 The most widely used cell surface markers in diagnostic flow cytometry
Marker Cells identified References
CD3 T cells Kung et al. (1979)
CD4 T-cell subset: helper/inducer Kung et al. (1979)
CD8 T-cell subset: suppressor/killer Reinherz et al. (1980)
CD19 B cells Nadler et al. (1983)
CD14 or CD68 Monocytes
CD45 All leucocytes
CD45RO Antigen-experienced (‘memory’) T cells Young et al. (1997)
CD45RA Antigen-inexperienced (‘naive’) T cells Young et al. (1997)
CD5 T cells, B-cell subset, chronic lymphocy- Kipps (1989)
tic leukaemia

5
 Monoclonal Antibodies: Diagnostic Uses

can be examined simultaneously. This provides a high level Applications in Haematology and Blood
of analytical power – for example, cells that are T cells
(CD3) and also express a marker for the T-helper cell sub-
Transfusion
set (CD4) can be identified using two-colour analysis; use
Antibodies against the leucocyte markers described in the
of cytoplasmic staining with a third fluorochrome allows
previous section are powerful reagents for differential di-
these cells to be divided into those that do and those that do
agnosis and monitoring in the haematological malignan-
not express interferon g. Instruments available widely in
cies, which include the leukaemias and lymphomas, as well
diagnostic laboratories analyse 3-5 colours, while research
as multiple myeloma. Each case represents the progeny of a
instruments are available that can analyse up to 12 colours.
single malignant leucocyte. Because leucocytes are a het-
See also: Flow cytometers
erogeneous population, their malignancies are equally
variable. A particular case may derive from a malignant
lymphoid or myeloid cell, from a particular subpopulation
(for example, T or B lymphocyte) and from a particular
Analysis of cell function stage of differentiation (stem cell, early progenitor and
Evaluation of the function of the cellular components of mature cell). See also: Haematopoiesis; Leukaemias and
the immune system is an important part of diagnostic lymphomas
immunopathology. Monoclonal antibodies can be applied The cell type and stage of differentiation seem to predict
to the study of cell function in a number of ways. Anti- to some degree the behaviour of the malignant clone and its
bodies can be used to purify, remove or destroy sub- response to therapy. Phenotypic analysis has become an
populations of cells and thereby determine their function. essential part of the diagnostic evaluation of leukaemia and
Antibodies against cell-surface molecules frequently initi- lymphoma (Jennings and Foon, 1997). This analysis is
ate responses including activation, proliferation, or carried out for the leukaemias almost invariably by flow
apoptosis. These ‘agonistic antibodies’ may be mimics of cytometry with CD markers. For the related solid tumours,
the physiological ligands, and provide an in vitro model for
studies of cellular activation and its regulation. Alterna-
tively, antibodies may be inhibitory, suppressing the func-
tion normally mediated by the natural ligand.
Cell activation or differentiation is often accompanied
by changes in the expression of cell surface or cytoplasmic
molecules, including cytokines, their receptors and recep-
tor–ligand pairs involved in intercellular interactions. An-
tibodies to these molecules can be used to monitor and
understand functional responses.

Other applications in diagnostic

immunopathology
While the study of leucocytes has been transformed by
the availability of a large panel of monoclonal antibodies
against a variety of cell markers and products, mono-
clonal antibodies have also contributed to the refinement
of many other diagnostic procedures in immunology.
Autoantibodies are detected usually by analysis of the
interaction of patient serum with target tissue, either by
microscopy or by enzyme-linked immunoassay. It is fre- Figure 2 Typical application of monoclonal antibody in differential
quently important to know the class of autoantibody diagnosis in leukaemia. Patterns of fluorescence intensity following FMC-7
staining in three different disease states and in a healthy individual. (a) B-
involved, and this is best done using monoclonal anti-
cell chronic lymphocytic leukaemia (B-CLL). FMC-7 expression weakly
immunoglobulins. Monoclonal antibodies are also useful positive. Clinical features: high lymphocyte count, lymphoadenopathy and
reagents in assays of components of the immune system, nonaggressive clinical course. (b) Prolymphocytic leukaemia (PLL). FMC-7
such as immunoglobulin subclasses and complement expression strongly positive. Clinical features: enlarged spleen, high
proteins, in assessing immune deficiency. In the study of lymphocyte count, no lymphadenopathy and aggressive clinical course. (c)
Hairy cell leukaemia (HCL). FMC-7 expression strongly positive. Clinical
allergy, monoclonal antibodies can be useful directly, for
features: enlarged spleen, marrow fibrosis, low white cell count and
example, in assaying IgE or cytokines, or indirectly, in lymphadenopathy. (d) Healthy individual. FMC-7 expression moderately
the preparation of purified allergen for test development. positive on a normal B-cell population. (Courtesy of Dean Moss and Sean
See also: Allergy Meehan, AMRAD Biotech Pty Ltd.)

6

Figure 3 Detection of rhesus D-positive fetal cells in maternal blood. (Courtesy of Dean Moss and Sean Meehan, AMRAD Biotech Pty Ltd.)
the lymphomas, the equivalent analysis is carried out either
in the same way using cell suspensions, or on tissue sections
by microscopy.
Figure 2 shows the reactivity of a monoclonal antibody,
FMC-7, which reacts with normal and malignant B cells
but is helpful in distinguishing between chronic lymph-
ocytic leukaemia (generally weak to negative) from the
more aggressive prolymphocytic and hairy cell leukaemias
(strongly positive).
Blood transfusion laboratories make extensive use of
monoclonal antibodies to type the blood of donors and
recipients, to ensure compatibility. Monoclonal antibody
against the rhesus (D) antigen is useful in detecting bleed-
ing of fetal cells into the maternal circulation (Figure 3).
Exposure of a rhesus (D)-negative mother to rhesus (D)-
positive infant cells can result in the production of anti-
body, which traverses the placenta and causes haemolytic
disease of the newborn. See also: Blood groups and
transfusion science; Rhesus haemolytic disease of the
newborn
Applications in Chemical Pathology
Chemical pathology is the term used to describe the lab-
oratory measurement of a broad range of substances,
ranging from blood glucose to mutated enzymes. The
chemical pathology laboratory uses a range of measure-
ment techniques, chosen on the basis of their specificity,
sensitivity, accuracy, precision, speed and cost-effective-
ness. Immunoassays score well enough on these criteria to
be selected for many assays. See also: Immunoassay
Monoclonal Antibodies: Diagnostic Uses
Immunoassays are used in a variety of formats. Radio-
immunoassays score well on most of the criteria listed
above, but are increasingly unpopular because of the haz-
ards to laboratory workers associated with the use of ra-
dioisotopes. Enzyme immunoassays are generally inferior
to radioimmunoassay in precision, and span a narrow
concentration range, but are widely used because of their
convenience and ease of automation. Assays based on flu-
orescence as an endpoint are increasingly favoured because
they can achieve the precision of radioimmunoassay and
span the same wide concentration range, and are capable of
automation. By carrying out the immunoassay on beads
that can be analysed in a cytometer, multiplex assays can
measure a number of different analytes simultaneously on
a small sample of serum (Vignali, 2000).
The range of analytes is enormous, limited only by the
availability of antibody. This essentially means that
immunoassays are generally applied to proteins and oth-
er large molecules, which are immunogenic – small anal-
ytes, and analytes which are found universally in all
species, for example glucose, are not readily analysed by
immunoassay. Widely used immunoassays include those
for hormones, enzymes, tumour antigens and some drugs.
Table 4 provides a few examples. See also: Tumour
immunology; Tumours: targeting of monoclonal antibo-
dies for imaging and potential for therapy
Applications in Tissue Pathology
The pathologist derives a great deal of information of di-
agnostic value by examining thin sections of tissue in the
7
 Monoclonal Antibodies: Diagnostic Uses
Table 4 Examples of analytes measured using monoclonal antibody-based immunoassay in the chemical pathology laboratory
Antigen
Parathyroid hormone, parathyroid hormone-related protein
Human chorionic gonadotrophin a-glycoprotein, carcinoem-
bryonic antigen
Prolactin, luteinizing hormone
Digoxin, cyclosporin A
C-reactive protein
b2-microglobulin
Immunoreactive trypsinogen
Haemoglobin A1c
Asialo transferrin
microscope. Tissue pathology is particularly relevant to the
early diagnosis of cancer or premalignant states, and to the
assessment of immunologically mediated disorders includ-
ing inflammation and transplant rejection. See also:
Cancer; Inflammation: acute; Inflammation: chronic;
Graft rejection: mechanisms
The microscopic examination of tissue has traditionally
depended on the use of dyes which stain distinct types of
cells differently. A logical extension of this is to use anti-
body to stain specific cell or tissue markers – immunohis-
tology or immunohistochemistry.
The tissue can be either fixed and embedded in paraffin
or polymeric material for sectioning or cut from frozen
tissue (cryostat material). Fixed/embedded material gen-
erally shows superior morphology, but many molecules are
modified by the fixative and embedding material and no
longer recognized by antibody. See also: Immunohisto-
chemical detection of tissue and cellular antigens
Immunohistology was well established with polyclonal
antisera before the availability of monoclonal antibodies.
Many monoclonal antibodies are ineffective on fixed/em-
Table 5 Examples of important diagnostic markers used in tissue pathology
Markers
CD3, CD20, CD15, CD30
Cytokeratin; mesothelial antigen
Markers for melanoma (e.g. S100, HMB45), carcinoma
(cytokeratins), lymphoma (CD markers)
Oestrogen and progesterone
Chromogranin, synaptophysin, neuron-specific enolase, in-
sulin, gastrin
Human chorionic gonadotropin, human placental lactogen
Thyroid-stimulating hormone, placental lactogen, growth
hormone
8
Diagnostic application
Bone disease, bone metastases (Guise, 1997)
Oncology (von Kleist, 1986)
Reproductive medicine
Drug-dose monitoring
Inflammation
Renal disease
Cystic fibrosis
Diabetes
Alcohol abuse
bedded tissue, and pathologists are reluctant to sacrifice
the excellent morphology obtainable only with fixed/em-
bedded tissue. This has led to a greater effort to make
monoclonal reagents that will work on fixed tissue, or to
treat fixed/embedded material in a way that allows the use
of the large panel of available monoclonal antibodies.
The binding of antibody can be visualized either by
enzymatic methods or by fluorescence. Pathologists gen-
erally prefer enzymatic methods because they allow coun-
terstaining and visualization of staining in the context of
clear morphology. Immunofluorescence generally yields
greater sensitivity, contrast and more precise quantitation,
but does not allow visualization of surrounding unstained
material. See also: Antigen–antibody binding
Table 5 lists some examples of antibodies used in impor-
tant diagnostic immunohistochemical applications, and
Figure 4 shows, as an example, the detection of me-
gakaryocytes (stained brown) in a bone marrow prepara-
tion, using a monoclonal antibody against the platelet-
specific antigen glycoprotein IIb/IIIa. See also: Bone
marrow
Diagnostic application
Subtyping lymphomas
Distinguishing mesothelioma from metastatic adenocarcino-
ma in pleura
Typing of anaplastic metastases
Hormonal receptor status (prognostic marker) in breast cancer
Neuroendocrine differentiation
Germ cell tumours
Typing of pituitary adenomas

Figure 4 Detection of megakaryocytes (brown staining) in bone marrow
preparation, using monoclonal antibody against platelet-specific
glycoprotein.
Other Applications
Monoclonal antibodies find uses wherever specificity and
reproducibility are of paramount importance. In situations
where polyclonal antisera showed adequate specificity, us-
ers did not find any immediate reasons to change, but it did
not take long for the antiserum producers to appreciate
that monoclonal antibodies, once made and characterized,
improved reproducibility and reduced the cost of quality
control. Monoclonal antibodies are used more and more
widely in fields such as forensic pathology and veterinary
pathology. See also: Bacteriophage display of combinator-
ial antibody libraries
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astases. Cancer 80(Suppl. 8): 1572–1580.
Jennings CD and Foon KA (1997) Recent advances in flow cytometry:
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Monoclonal Antibodies: Diagnostic Uses
Kipps TJ (1989) The CD5 B cell. Advances in Immunology 47: 117–185.
von Kleist S (1986) The clinical value of the tumor markers CA 19/9 and
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Further Reading
Eisenbarth GS and Jackson RA (1982) Application of monoclonal an-
tibody techniques to endocrinology. Endocrinology Reviews 3(1): 26–
39.
Herrman JE (1995) Immunoassays for the diagnosis of infectious dis-
eases. In: Murray, PR; Baron, EJ; Pfaller, MA; Tenover, FC and
Yolken, RH (eds) Manual of Clinical Microbiology, 6th edn, pp. 110–
122. Washington, DC: American Society for Microbiology.
Isenberg HD (ed.) (1992) Clinical Microbiology Procedures Handbook,
vol. 2, sect. 9. Washington, DC: American Society for Microbiology.
Murray PR, Baron EJ, Pfaller MA, Tenover FC and Yolken RH (1995)
Manual of Clinical Microbiology. Washington, DC: ASM Press.
Rose NR, Conway de Macario E, Folds JD, Lane HC and Nakamura
RM (1997) Manual of Clinical Laboratory Immunology. Washington
DC: ASM Press.
White DO and Fenner FJ (1994) Medical Virology, 4th edn. San Diego,
CA: Academic Press.
Zola H, Roberts Thomson P and McEvoy R (1995) Diagnostic Immuno-
pathology. Cambridge: Cambridge University Press.