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.

Secondary article
Article Contents
. Introduction . 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

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, doctors use laboratory data to help them to arrive at a denitive diagnosis. Note that in this article 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. Laboratory data greatly enhance the resolving power of diagnosis. A few relevant questions and a thorough examination may tell the physician that a patient is suering from infection, and it is probably bacterial rather than viral. Laboratory tests can identify the bacterial pathogen, can determine to which antibiotics the pathogen is sensitive, and can provide detailed information about the immune status of the patient. For example, has the pathogen been encountered before or is there some underlying reason for the failure of the patient to recover from the infection 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 can assist the pathologist include identication 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 specicity of antibodies, and the fact that they can be used in a variety of test formats, to provide qualitative and quantitative information.

Antibody as Diagnostic Probe

The antibody molecule has a binding site which will recognize a particular molecular shape. The immune system of mammals, birds and reptiles is capable of making an enormous diversity of antibody-binding sites. While this diverse antibody 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. Antibodies may be used in diagnostic tests requiring identication, 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.

Assay formats
An antibody reacts specically with a particular molecular structure, referred to as an epitope. That epitope is usually 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 specic probe for epitope, molecule or cell. 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 information required (identication, localization or quantitative assay) dictates the assay type required. Table 1 summarizes assay formats suitable for dierent 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 and the techniques that might be used to arrive at an answer.
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Monoclonal Antibodies: Diagnostic Uses

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

Cell

Tissue staining and microscopy

Table 2 A few typical diagnostic questions and the techniques that might be used to give an answer Question Does patient X express the HLA-B27 allele, which is associated with the inammatory back disorder ankylosing spondylitis? How many CD4 cells, the target cells for the human immunodeciency virus (HIV), does patient Y have per mililitre of blood? Is Ms D pregnant? Information required Identication of cells bearing a particular marker Enumeration of cells bearing a particular marker Identication of a particular molecule (hormone) in urine Quantitation of molecule (antibody) Suggested technique Flow cytometry

Flow cytometry

Particle agglutination

Laboratory worker P has just been exposed to Enzyme-linked patient serum. What level of antibody against immunoassay hepatitis B does she have, as a result of prior (protective) immunization? Does Patient Y, immunosuppressed to control Antigens coded for by the virus may be detected Tissue staining and rejection of a transplanted kidney, have on blood cells, or on tissue culture cells after (uorescence) cytomegalovirus infection? incubation with infected material. Identication microscopy of molecule (viral antigen) on cells An enlarged, rm and nontender lymph node is Identication and localization of particular cell Tissue staining and removed from the neck to determine if it is types and features microscopy malignant. Histological examination reveals inltration by small round neoplastic cells. What is the nature of these inltrating cells, and what is their lineage (do they have a lymphoid or an epithelial origin)?

Applications in Infectious Diseases

Medical significance
Infection remains a challenge to the pathologist. The multiplicity of related infectious agents, coupled with the need for appropriate and often immediate treatment, provide a strong stimulus for the development of laboratory diagnostic tests. Important as it is to arrive at a denite diagnosis in the interests of the patient, diagnosis
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of certain infectious diseases is even more important in the interests of public health. To illustrate with a few recent and recurring examples, identication of a new strain of inuenza in humans, derived from chickens, sounds warnings of a possible pandemic and leads to the destruction of infected or at-risk birds; diagnosis of a cluster of cases of haemolytic uraemic syndrome necessitates rapid identication 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

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Monoclonal Antibodies: Diagnostic Uses

to endemic areas, calls for urgent trapping of mosquitoes to conrm the source of this mosquito-borne virus and mosquito eradication measures if it is detected.

Analysis of the patients antibody response

As an alternative to antigen detection, the antibody response of the patient may provide valuable information. This approach is particularly valuable in viral infection, where antigen detection may be particularly dicult. 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 encountered 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, indicative of a current infection. Figure 1 shows the format of an enzyme immunoassay designed to titrate IgM and IgG against the dengue virus. The decision whether to base diagnosis on identication of the antigen or on analysis of the patients antibody (serology) 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.

Diagnostic options
The diagnosis of infection traditionally requires that the organism be identied in, or cultured from, infected material. This is often not possible with adequate sensitivity or specicity, and must be supplemented with highly sensitive and specic techniques. Antibody-based methods and molecular techniques such as the polymerase chain reaction (PCR) provide the necessary sensitivity and specicity. Antigens characteristic of particular infectious agents can be detected using antibodies in a variety of assay formats, including immunohistochemistry on tissue samples, enzyme-linked or uorescence-based immunoassays of tissue extracts or body uids, and a variety of technically simple tests, such as particle agglutination, which can be carried out in the eld. Assays vary in sensitivity, and crossreactivity between related organisms is a recurring problem. 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 antigens 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. It is thus important to know the natural history of the infectious organism and use antibodies against antigens which will provide the right level of specicity.

Identification of viruses
Viruses are detected usually in enzyme immunoassay format or by immunouorescence using antibody against viral 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 example, cytomegalovirus (CMV) produces a long-lasting infection, with latent virus inhabiting cells of
Anti-human IgM Anti-human IgG

Dengue 14 antigens Mix reconstituted dengue antigen with horseradish peroxidaselabelled monoclonal antibody tracer. Incubate 60 min at room temperature Add patients serum containing IgM & IgG. Incubate 60 min at 37C

60 min at 37C

Figure 1 Schematic representation of an enzyme-linked immunoassay to detect and titrate IgG and IgM antibodies against dengue virus. Courtesy Peter Devine, Jody Mitchell and Andrea Cuzzubo, PanBio Pty Ltd.

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Monoclonal Antibodies: Diagnostic Uses

the immune system. The virus can be grown in human broblasts in the laboratory, but it may take several weeks before the cells show cytopathic changes. However, CMV early antigens may be detected in the cells by immunouorescence within hours, or may be detected directly in patient blood cells (Drew, 1988).

Other pathogens
Apart from the viruses and bacteria, pathogens of medical signicance include unicellular and multicellular parasites, fungi, mycoplasma and chlamydia. Parasites are generally dicult to grow in culture but they tend to be larger than bacteria and were originally identied by microscopy. Antibody-based methods have greatly enhanced dierential diagnosis, permitting identication of individual species with certainty. Antibodies are used with microscopy or ow cytometry to detect parasites such as Giardia, which can be transmitted in contaminated drinking water, and the insect-borne malaria parasite, Plasmodium. Fungal infections are important in immune compromised patients, including acquired immune deciency syndrome (AIDS), and can be detected with a range of antibody-based assays including microscopic immunouorescence and enzyme immunoassay directed against specic fungal antigens.

Bacterial infection
A major advantage for direct isolation of bacteria from patient 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 identication is advantageous to allow early treatment with appropriate antibiotics. Monoclonal antibodies are used to conrm identication of bacterial pathogens, and in situations where identication by other means is complex and timeconsuming. An example is in the diagnosis of Neisseria gonorrhoea and its dierentiation from other Neisseria species, which would otherwise require complex biochemical testing. Monoclonal antibodies are also used in rapid agglutination 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 uorochrome-labelled antibody coupled with either uorescence microscopy or ow cytometry.

Applications in Disorders of the Immune System

Introduction
The immune system has evolved to defend us from infectious disease, and it does that eectively. However, immune responses are frequently the cause of disease, when they target self tissues (autoimmunity), when they are ineective against infection (immune deciency, frequently caused by a genetic defect in a component of the immune system), when they are overzealous (hypersensitivity disorders, including allergies), or when they perform their task well, but are inconvenient where medicine has tinkered with nature (transplant rejection, for example). The immune system is a complex network of positive and negative interactions between several types of cells and involving a multitude of proteins, hormones and their receptors working together with cell surface or soluble ligandreceptor pairs. Defects, qualitative or quantitative, in any of these components, or in the downstream signalling pathways evoked by their interactions, can lead to malfunction of the immune system. Diagnostic immunopathology requires the analysis of the mediators of immunity (antibody, complement components, eector cells). Detailed analysis of the interacting components of the immune system can shed light on mechanisms of immunopathology, and can frequently guide the selection of specic treatment options.

Antibody-based assays and molecular biological assays

Assays based on molecular biological techniques, particularly the PCR, are highly sensitive and specic. Where detection of the pathogen is appropriate, molecular techniques can often replace antibody-based assays, although they cannot at present compete with the speed and simplicity of methods based on particle agglutination. Furthermore, serological assays, which detect antibody made by the patient, yield information of a dierent nature, and are unlikely to be replaced by molecular methods. Monoclonal antibodies can often complement molecular techniques, for example by providing a preliminary enrichment of pathogen prior to PCR. This approach is particularly valuable when the pathogen has to be concentrated from a large sample in the presence of potentially inhibitory or confounding impurities (e.g. patient stools).
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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

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Monoclonal Antibodies: Diagnostic Uses

Dierentiation Antigens (HLDA) have named 168 CD molecules, most of them cell surface molecules (Kishimoto et al., 1997). The majority of these molecules had not been identied before the availability of monoclonal antibodies. Once identied with monoclonal antibodies, the genes coding these molecules could be cloned, and the control of their expression analysed. Cells express molecules involved in their function on their surface. While some cell surface molecules are expressed by all cells, others are expressed in a restricted way which 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 methods such as PCR, the use of antibodies generally is much preferred, because it demonstrates that the molecule is actually expressed, and because the combination of monoclonal antibodies and ow cytometry allows cell-bycell analysis. Multiparameter analysis allows the simultaneous analysis of several cell markers. Once an antibody is available as a marker for a particular 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 distinguish 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. 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 ow cytometry. It is also the example most relevant to studies of the immune system, because the cellular composition of blood and lymphoid tissue provides a window allowing the analysis and monitoring of the immune system. Monoclonal antibodies against cell surface markers are most easily and powerfully used as uorescent conjugates, by ow cytometry for cells in suspension or uorescence

microscopy for studies of solid tissues. Fluorescence is capable of very high sensitivity, matching that of radioisotopic methods. The wavelength change characteristic of uorescence, and the ability to measure the uorescent emission at 908 to the incident light, allows very favourable signal/noise ratios, both in microscopy and in ow cytometry. Flow cytometry In ow cytometry, cells in suspension are forced to ow in single le 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 scatter) 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 dierentiation of cell populations such as platelets, red cells, lymphocytes, granulocytes and monocytes in blood. Cell types which cannot be distinguished on the basis of their scatter properties can be distinguished using monoclonal antibodies conjugated to uorochromes. By using a number of antibodies labelled with dierent uorochromes with distinct uorescent spectra several markers can be examined simultaneously. This provides a high level of analytical power for example cells which are T cells (CD3) and also express a marker for the T-helper cell subset (CD4) can be identied using two-colour analysis; use of cytoplasmic staining with a third uorochrome allows these cells to be divided into those that do and do not express interferon g. Analysis of cell function Evaluation of function of the cellular components of the immune system is an important part of diagnostic immunopathology. Monoclonal antibodies can be applied to the study of cell function in a number of ways. Antibodies can be used to purify, remove or destroy subpopulations of cells and thereby determine their

Table 3 The most widely used cell surface markers in diagnostic ow cytometry Marker CD3 CD4 CD8 CD19 CD14 or CD68 CD45 CD45R0 CD45RA CD5 Cells identied T cells T cell subset: helper/inducer T cell subset: suppressor/killer B cells Monocytes All leucocytes Antigen-experienced (memory) T cells Antigen-inexperienced (naive) T cells T cells, B-cell subset, chronic lymphocytic leukaemia References Kung et al. (1979) Kung et al. (1979) Reinherz et al. (1980) Nadler et al. (1983)

Young et al. (1997) Young et al. (1997) Kipps (1989)

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Monoclonal Antibodies: Diagnostic Uses

function. Antibodies against cell surface molecules frequently initiate responses including activation, proliferation, or apoptosis. These agonistic antibodies may be mimics of the physiological ligands, and provide an in vitro model for studies of cellular activation and its regulation. Alternatively, antibodies may be inhibitory, suppressing the function normally mediated by the natural ligand. Cell activation or dierentiation is often accompanied by changes in the expression of cell surface or cytoplasmic molecules, including cytokines, their receptors, and receptorligand pairs involved in intercellular interactions. Antibodies 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, monoclonal antibodies have also contributed to the renement 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 frequently important to know the class of autoantibody involved, and this is best done using monoclonal anti-immunoglobulins. Monoclonal antibodies are also useful reagents in assays of components of the immune system, such as immunoglobulin subclasses and complement proteins, in assessing immune deciency. In the study of allergy monoclonal antibodies can be useful directly, for example in assaying IgE or cytokines, or indirectly, in the preparation of puried allergen for test development.

essential part of the diagnostic evaluation of leukaemia and lymphoma (Jennings and Foon, 1997). This analysis is carried out for the leukaemias almost invariably by ow cytometry with CD markers. For the related solid tumours, 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 lymphocytic 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 bleeding 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 antibody, which traverses the placenta and causes haemolytic disease of the newborn.

60

Mean channel fluorescence = 91

180

Mean channel fluorescence = 352

M1 0 (a) 100 160 101 102 103 104 0 (b) 100 100 101 102 103

M1 104

Mean channel fluorescence = 859

Mean channel fluorescence = 195

Applications in Haematology and Blood Transfusion

Antibodies against the leucocyte markers described in the previous section are powerful reagents for dierential diagnosis and monitoring in the haematological malignancies, which include the leukaemias and lymphomas, as well as multiple myeloma. Each case represents the progeny of a single malignant leucocyte. Because leucocytes are a heterogeneous 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 stage of dierentiation (stem cell, early progenitor, mature cell). The cell type and stage of dierentiation seem to predict to some degree the behaviour of the malignant clone and its response to therapy. Phenotypic analysis has become an
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M1 0 (c) 100 101 102 103 104 0 (d) 100 101 102 103 M1 104

Figure 2 Typical application of monoclonal antibody in differential diagnosis in leukaemia. Patterns of fluorescence intensity following FMC-7 staining in three different disease states and in a healthy individual. (a) Bcell chronic lymphocytic leukaemia (B-CLL). FMC-7 expression weakly positive. Clinical features: high lymphocyte count, lymphoadenopathy and nonaggressive clinical course. (b) Prolymphocytic leukaemia (PLL). FMC-7 expression strongly positive. Clinical features: enlarged spleen, high lymphocyte count, no lymphadenopathy, aggressive clinical course. (c) Hairy cell leukaemia (HCL). FMC-7 expression strongly positive. Clinical features: enlarged spleen, marrow fibrosis, low white cell count, lymphadenopathy. (d) Healthy individual. FMC-7 expression moderately positive on a normal B-cell population. Courtesy Dean Moss and Sean Meehan, AMRAD Biotech Pty Ltd.

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Monoclonal Antibodies: Diagnostic Uses

Figure 3 Detection of rhesus D-positive fetal cells in maternal blood. Courtesy Dean Moss and Sean Meehan, AMRAD Biotech Pty Ltd.

Table 4 Examples of analytes measured using monoclonal antibody-based immunoassay in the chemical pathology laboratory Antigen Parathyroid hormone, parathyroid hormone-related protein HCG (human chorionic gonadotrophin) a-glycoprotein, carcinoembryonic antigen (CEA) Prolactin, luteinizing hormone Digoxin, cyclosporin A C-reactive protein b2-microglobulin Immunoreactive trypsinogen Haemoglobin A1c Asialo transferrin Diagnostic application Bone disease, bone metastases (Guise, 1997) Oncology (von Kleist, 1986) Reproductive medicine Drug dose monitoring Inammation Renal disease Cystic brosis Diabetes Alcohol abuse

Applications in Chemical Pathology

Chemical pathology is the term used to describe the laboratory measurement of a broad range of substances, ranging from blood glucose to mutations in enzymes. The chemical pathology laboratory uses a range of measurement techniques, chosen on the basis of their specicity, sensitivity, accuracy, precision, speed and cost-eectiveness. Immunoassays score well enough on these criteria to be selected for many assays. Immunoassays are used in a variety of formats. Radioimmunoassays score well on most of the criteria listed above, but are increasingly unpopular because of the hazards to laboratory workers associated with the use of radioisotopes. 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 uorescence 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. The range of analytes is enormous; limited only by the availability of antibody. This essentially means that immunoassays are generally applied to proteins and other large molecules, which are immunogenic small analytes, 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.
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Monoclonal Antibodies: Diagnostic Uses

Applications in Tissue Pathology

The pathologist derives a great deal of information of diagnostic value by examining thin sections of tissue in the microscope. Tissue pathology is particularly relevant to the early diagnosis of cancer or premalignant states, and to the assessment of immunologically mediated disorders including inammation and transplant rejection. The microscopic examination of tissue has traditionally depended on the use of dyes which stained distinct types of cells dierently. A logical extension of this is to use antibody to stain specic cell or tissue markers immunohistology, or immunohistochemistry.

The tissue can be either xed and embedded in paran or polymeric material for sectioning, or cut from frozen tissue (cryostat material). Fixed/embedded material generally shows superior morphology, but many molecules are modied by the xative and embedding material and no longer recognized by antibody. Immunohistology was well established with polyclonal antisera before the availability of monoclonal antibodies. Most monoclonal antibodies are ineective on xed/ embedded tissue, and pathologists are reluctant to sacrice the excellent morphology obtainable only with xed/ embedded tissue. This has led to a greater eort to make monoclonal reagents that will work on xed tissue, or to

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-specic enolase, insulin, gastrin Human chorionic gonadotropin, human placental lactogen Thyroid-stimulating hormone, placental lactogen, growth hormone Diagnostic application Subtyping lymphomas Distinguishing mesothelioma from metastatic adenocarcinoma in pleura Typing of anaplastic metastases Hormonal receptor status (prognostic marker) in breast cancer Neuroendocrine dierentiation 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.

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Monoclonal Antibodies: Diagnostic Uses

treat xed/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 uorescence. Pathologists generally prefer enzymatic methods because they allow counterstaining and visualization of staining in the context of clear morphology. Immunouorescence generally yields greater sensitivity and contrast, but does not allow visualization of surrounding unstained material. Table 5 lists some examples of antibodies used in important diagnostic immunohistochemical applications, and Figure 4 shows, as an example the detection of megakaryocytes (stained brown) in a bone marrow preparation, using a monoclonal antibody against the platelet-specic antigen glycoprotein IIb/IIIa.

Other Applications
Monoclonal antibodies nd uses wherever specicity and reproducibility are of paramount importance. In situations where polyclonal antisera showed adequate specicity users did not nd 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 elds such as forensic pathology and veterinary pathology.

Kipps TJ (1989) The CD5 B cell. Advances in Immunology 47: 117185. Kishimoto T, Goyert S, Kikutani H et al. (1997) CD antigens 1996. Blood 89(10): 3502. von Kleist S (1986) The clinical value of the tumor markers CA 19/9 and carcinoembryonic antigen (CEA) in colorectal carcinomas: a critical comparison. International Journal of Biological Markers 1(1): 38. Kung P, Goldstein G, Reinherz EL and Schlossman SF (1979) Monoclonal antibodies dening distinctive human T cell surface antigens. Science 206(4416): 347349. Nadler LM, Anderson KC, Marti G et al. (1983) B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes. Journal of Immunology 131(1): 244250. Reinherz EL, Kung PC, Goldstein G and Schlossman SF (1980) A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously dened by a heteroantiserum termed TH2. Journal of Immunology 124(3): 13011307. Young JL, Ramage JM, Gaston JS and Beverley PC (1997) In vitro responses of human CD45R0-brightRA- and CD45R0-RA bright T cell subsets and their relationship to memory and naive T cells. European Journal of Immunology 27(9): 23832390.

Further Reading
Eisenbarth GS and Jackson RA (1982) Application of monoclonal antibody techniques to endocrinology. Endocrinology Reviews 3(1): 2639. Herrman JE (1995) Immunoassays for the diagnosis of infectious diseases. 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 Immunopathology. Cambridge: Cambridge University Press.

References
Drew WL (1988) Diagnosis of cytomegalovirus infection. Review of Infectious Diseases suppl. 3: S468S476. Guise TA (1997) Parathyroid hormone-related protein and bone metastases. Cancer 80(8 suppl.): 15721580. Jennings CD and Foon KA (1997) Recent advances in ow cytometry: Application to the diagnosis of hematologic malignancy. Blood 90(8): 28632892.

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