Immunoglobulin Titers and Immunoglobulin Subtypes

Chapter 12

Thinking is more interesting then knowing, but less interesting than looking. Johann Wolfgang von Goethe

IMMUNOGLOBULINS Immunoglobulins (antibodies) are a group of heterogeneous proteins that exhibit the unique property of being able to bind to proteins or polysaccharides that stimulated the production of the antibody. Antibodies are one of two important antigen recognition components of the immune system. Antibodies recognize antigen directly in a three-dimensional conformation while the other antigen recognition system, the T lymphocytes, recognizes antigen in conjunction with MHC molecules. Antibodies are expressed on the membrane of developing and mature B cells where they function as B-celt receptors for antigens. The interaction of antigen with membrane bound immunoglobulins initiates activation of the B cell and, with proper stimulation, there is synthesis and secretion of the specific antibody into the circulation. Immunoglobulins specifically recognize a particular antigenic determinant on a larger molecule. The site recognized by a specific antibody is called an 'epitope'. Antigen binding is the primary function of antibodies. Physiologic benefits provided by antibodies include helping to rid the body of disease-causing bacteria, protection from viral infection and neutralizing bacterial toxins. The significant biological effects of antibody are mediated Measuring Immunity, edited by Michael -£ Lotze and Angus W. Thomson ISBN 0-12-455900-X, London

by a variety of secondary 'effector functions' including activation of the complement cascade or attachment of antibody-covered bacterial antigens to phagocytic cells through the specific receptors on the phagocytic cell for the portion of the antibody molecule that does not directly bind to the antigen. In addition to their unique and central role in host protection, antibody contributes to the pathogenesis of a variety of reactions that are called hypersensitivity reactions. Hypersensitivity implies that the individual has been exposed to the antigen in the past and when exposed for a second time, there is a rapid onset of an immune reaction. Allergies are an example of a hypersensitivity reaction. There are three other types of hypersensitivity reaction in addition to the allergic type, which is called Type 1. Type 2 hypersensitivity reactions occur when antibodies specifically recognize and bind to receptors on cell membranes. If the receptor is involved with altering cell function there may be increased (for example, Graves' disease) or decreased (for example, myasthenia gravis) cell activity. Finally, antibodies binding to circulating cells could induce ceil removal or lysis and cause severe clinical conditions like anemia and thrombocytopenia. Type 3 hypersensitivity reactions occur when circulating antibody binds to the specific antigen and form immune complexes. The phagocytic cells of the spleen, liver and lung normally remove immune complexes from the circulation. If there are more complexes formed than can be removed by mononuclear phagocytes, the complexes Copyright © 2005, Elsevier. All rights reserved.

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

will become deposited in tissue. In tissue the complexes will activate the complement system. This will induce an inflammatory response and damage to the tissue that is caused by enzymes released from the inflammatory cells and anoxia (the lack of oxygen) caused by occlusion of small blood vessels. Type 4 hypersensitivity is mediated by T lymphocytes and does not involve antibody. All immunoglobulins consist of two identical low molecular weight polypeptide light chains (23 kD) and two identical polypeptide heavy chains (50-70 kD). The chains are held together by inter-chain disulfide bonds and by non-covalent interactions. While all immunoglobulin molecules share the same basic structural characteristics, the amino terminal end displays remarkable variability that accounts for the capability of antibodies of different specificities to bind to an almost infinite number of antigens. Based on the variability in the amino acid sequences, both the heavy and light chain can be divided into variable (V) and constant (C) regions. The antigen binding site involves the variable region. Structural differences outside their antigen binding sites, i.e. in the constant region, correlate with the different effector function mediated by antibodies, such as complement activation or binding to specific receptors expressed on different cell types. For example, mast cells have a receptor for the constant region of the IgE class of antibody and polymorphonuclear leukocytes have a receptor for the constant region of IgG. Based on the total number of different variable regions or antigen binding sites, it can be estimated that an individual can produce antibodies with more than 10 million different specificities. Analysis of immunoglobulin fragments produced by proteolytic digestion has been useful in elucidating structural and functional relationships of immunoglobulins. Digestion with papain breaks the immunoglobulin molecule into two identical fragments that contain the whole light chain and the VH and C H I domains of the heavy chain. They are called Fab fragments because they contain the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule is divalent. Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc fragment because it was easily crystallized. The effector functions of immunoglobulins are mediated by the Fc part of the molecule, while other domains in this fragment intercede in different functions. In contrast to the papain-mediated digestion, treatment of immunoglobulins with pepsin results in cleavage of the heavy chain in a fragment that contains both antigen-binding sites. This fragment was called F(ab')2 because it was divalent. The F(ab')2 strongly binds antigen but it does not mediate the effector functions of antibodies, since it misses the Fc part of the whole molecule.

IMMUNOGLOBULIN CLASSES The immunoglobulin classes are IgG, IgA, IgM, IgD and IgE. The constant region is the same for all immunoglobulins within a given class, indeed it is the constant region that provides a means of differentiating one class of immunoglobulin from another. Thus, all immunoglobulin molecules that are of the IgG class have identical aspects to their constant regions that allow them to be identified as IgG. Similarly IgA, IgM, IgD and IgE have identical characteristics to their constant regions. Not every part of the variable region is as variable as every other part of the variable region. The variable end of the H and L chains begins at the amino terminal end of the chain. The first 110 amino acids are involved in the variable portion of the H and L chains. The amino acids at positions 30-34, 48-56 and 94-99 are much more variable than the amino acids at the other positions. The highly variable regions are termed the hypervariable regions and the amino acids between the hypervariable regions are termed the framework regions. The intrachain disulfide bonds bring the hypervariable regions together to form the actual site that combines with the antigen. Another aspect of the immunoglobulin molecule is termed the hinge region. The hinge region is located between the first (CHI) and second (CH2) constant regions of the H chain. This region allows the two Fab portions to have mobility so that the two portions that combine with antigen can move. The primary shape of the immunoglobulin molecule is that of a 'Y' with flexibility of the two arms given by the hinge region. The heavy chain is assigned a specific designation for each isotype. Isotype

Heavy chain

IgG IgA IgM IgD

Gamma Alpha Mu Delta Epsilon

The molecular weight of the light chain is approximately 22 OOOdaltons. There are only two classes of L chains and they are termed kappa and lambda. The two L chains which comprise a single basic immunoglobulin unit are both kappa or both lambda. Thus each immunoglobulin class (IgG, IgA, IgM, IgD, IgE) is composed of kappa containing molecules and lambda containing molecules. The difference between kappa and lambda is due to different amino acid sequences. IgG Immunoglobulin G (IgG) is a major class of immunoglobulins found in the blood comprising 75 per cent of total serum immunoglobulins (Table 12.1). IgG is also the major immunoglobulin in the extravascular spaces and the only

Immunoglobulin Titers and Immunoglobulin Subtypes

type of immunoglobulin that binds to receptors on placental trophoblasts and crosses the placenta. Transferred maternal IgG provides immunity for the fetus and newborn. The IgG is capable of carrying out all of the functions of immunoglobulin molecules. In general, IgG is strong at activating complement and is the best antibody for phagocytosis of opsonized microorganisms through the Fey receptors on phagocytes. IgG also plays an important role in neutralizing bacterial toxins in the blood and tissues. Antibodies of the IgG class express their predominant activity during a secondary immune response. Thus, the appearance of specific IgG antibodies normally reflects the 'maturation' of the immune response, which is switched on upon repeated contact with a specific antigen. The subclasses of IgG are produced depending on the cytokine present (especially IL-4 and IL-2) and each subclass has its own specific activity (Table 12.2) (Schur, 1987). For instance, lgG2 does not cross the placenta, lgG4 does not fix complement and lgG2 and lgG4 do not bind to Fc receptors with a high affinity (Meulenbroek and Zeijiemaker, 1996). lgG2 levels are also slowly increased during childhood and reach adult levels only by 6-8 years of age. lgG2 antibodies are usually induced by some carbohydrate antigens, such as pneumococcal polysaccharide, whereas protein antigens induce predominantly IgGI and/or lgG3 responses. However, Haemophilus influenzae type b polyribose phosphate (H. influenzae vaccine) and Neisseria meningitidis capsular polysaccharide induce IgGI and lgG2 antibodies.

IgA Immunoglobulin A (IgA) can be detected in the circulation in low levels and in the monomeric form. IgA is the

second most common immunoglobulin in human serum after IgG (see Table 12.1). IgA is widespread, does not fix complement and is most active at mucosal surfaces such as bronchioles, nasal mucosa, prostate, vagina and intestine, where it presents as a dimeric protein (Fagarasan and Honjo, 2003). IgA is also common in saliva, tears and breast milk, especially colostrums (Van de Perre, 2003). Secretory IgA is found in association with another protein, the so-called secretory piece or T piece. Unlike the IgA that is produced by plasma cells, the secretory piece is expressed in epithelial cells and added to the IgA molecules as they pass into the secretions. The secretory piece mediates IgA transport across the mucosa and protects IgA from degradation. Secretory IgA can neutralize toxins, bind viruses, agglutinate bacteria, prevent bacteria from binding to mucosal epithelial cells and bind to various food antigens, thus preventing their entry into the general circulation. The role of serum IgA is less unclear IgA subclasses differ in distribution and function. IgAI is present mainly in the serum (it accounts for 85 per cent of serum IgA) and predominates in secretion in the upper intestine and in the various mucosal glands. lgA2 predominates in secretion in the large intestine and in the female genital tract. IgM Immunoglobulin M (IgM) is the third most common serum immunoglobulin accounting for 5-10 per cent of total levels. It is also a component of secretory immunoglobulins at the mucosal surfaces and in breast milk. Secreted IgM normally exists as a pentamer but it can be also detected as a monomer As a consequence of its pentameric structure.

Table 12.1 Estimated ranges for normal levels of serum immunoglobulins*

3 months 1 year 5 years 10 years Adult

IgG (mg/dl)

lgA(mg/dl)

IgM (mg/dl)

lgE(IU/ml)

140-650 250-1100 515-1450 770-1650 560-1770

1-45 10-100 25-200 40-360 40-390

20-120 25-160 40-200 40-250 60-360

<15.0 <60.0 <90.0 <200.0 < 100.0

* Normal ranges of serum immunoglobulins vary between laboratories, since the estimation of immunoglobulin concentration in serum specimens strongly depends on analytical methods, calibration curves, specificity and type of antiglobulin antibodies, provided standards.

Table 12.2 Properties of immunoglobulin G subclasses*

Serum concentration (adult ranges) (mg/dl) Proportion of total Ig (%) Serum half-life (days) Complement activation Response to protein antigens Response to polysaccharide antigens Placental cross-transfer

igGi

lgG2

IgGa

lgG4

420-1100

150-600

20-140

1-180

40-80 21

15-50 21

1.5-10 7

0.5-15 21

++

+ +/-

-\- + + ++

++

-\-

+/+

-h +

+ +

-\-

* Normal ranges of serum immunoglobulins vary between laboratories, since the estimation of immunoglobulin concentration in serum specimens strongly depends on analytical methods, calibration curves, specificity and type of antiglobulin antibodies, provided standards.

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

IgM is a powerful agglutinating and precipitating antibody, a strong complement fixing immunoglobulin and thus is efficient in leading to the lysis of microorganisms. It is also involved in neutralization of toxins (Stahl and Sibrowski, 2003). Biological significance of this immunoglobulin is based on the facts that IgM is the first immunoglobulin to be made by the fetus and the first antibody to be produced by virgin B cells after the primary antigen stimulation. IgM is the first antibody to be produced in response to infection since it does not require 'class switch' to another antibody class. However, it is only synthesized as long as antigen remains present because there are no memory cells for IgM. As a B-cell surface immunoglobulin, IgM exists as a monomer and functions as a receptor for antigens. The surface IgM is structurally different in the Fc region from the secreted form since it must bind through the membrane. Surface IgM binds directly as an integral membrane protein; it does not bind to an IgM Fc receptor like IgE does. A deficiency of IgM makes the host extremely susceptible to septicemia or other forms of sepsis. Being a large molecule (900 kD), the majority (80 per cent) of IgM exists within the vascular space. IgD Immunoglobulin D (IgD) is primarily found on the surface of B lymphocytes where it functions as a receptor for antigen. IgD does not bind complement or cells through the Fc receptor. A small amount of IgD is secreted accounting for about 0.25 per cent of the total serum immunoglobulins (Vladutiu, 2000). With use of sensitive assays, IgD has been found in all of the mammalian and avian species tested and is conserved across species, which suggests an evolutionary advantage (Blattner and Tucker, 1984). However, the physiologic function of serum IgD is not clear; it has even been thought that IgD might have no function (Blattner and Tucker, 1984). This was investigated in animals treated with anti-lgD and, more recently, in IgD-deficient mice. In one model of IgD knockout mice, there was only a slight decrease in the number of B cells in the periphery and immunoglobulin isotypes were almost normal (Nitschke et al., 1993). In another model, there was a delayed affinity maturation during T-celldependent antigen response (Roes and Rajewsky, 1993). IgD may also have a regulatory role by enhancing a protective antibody response of the IgM, IgG or IgA isotype, or by interfering with viral replication (Moskophidis et al., 1997). IgD can also participate in the generation and maintenance of B-cell memory and might have an important role in the transition from a stage of susceptibility to induction of B-cell tolerance to one of responsiveness (Vladutiu, 2000). In mice, IgD may substitute for some functions of IgM when IgM is absent. Studies in IgM-deficient IgM-/- mice reveal that B cells with surface expression of IgM were replaced by B cells with surface expression of IgD.

Immunization of IgM-/- mice revealed an IgD immune response in place of the now absent IgM response, although with a delayed increase in antibody concentration as compared to normal (Orinska et al., 2002). Finally, IgD is a potent inducer of TNF-a, IL-1/3 and IL-1 receptor antagonist (Drenth et al., 1996). IgD also induces release of IL-6, IL-10 and leukemia inhibitory factor from peripheral blood mononuclear cells.

Immunoglobulin E (IgE) exists as a monomer and has an extra domain in the constant region. IgE does not fix complement and is the least common serum immunoglobulin since it binds with a high affinity to the specific Fc receptors on basophils and mast cells even before interacting with antigen (Gould et al., 2003). Binding of the antigen to IgE on the cells results in immediate release of various mediators, including histamine, serotonin, leukotrienes and others. This class of antibody is responsible for Type I hypersensitivity reactions or immediate hypersensitivity reactions such as hay fever, asthma, hives and anaphylaxis. Although IgE is commonly involved in allergic reactions, the main function of IgE seems to be to protect the host against invading parasites (Gould et al., 2003). Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. IgE is distributed throughout the body, although cells synthesizing IgE are found predominantly in association with mucosal tissues and, like IgA, this class of antibody is particularly effective at mucosal surfaces. However, IgE is not found in breast milk, and only in very low amounts in other secretions such as saliva. IgE is found to increase significantly in response to parasitic infection. Of the five classes of immunoglobulins, IgG, IgA and IgM are the main components found in serum. Measurement of serum levels of these immunoglobulins is essential for the diagnosis and monitoring of primary and secondary immunodeficiencies and lymphoid malignancies. In addition, elevated levels of polyclonal immunoglobulins may occur in autoimmune diseases, liver diseases and chronic infections. Since total serum IgE is significantly raised in only 50-75 per cent of patients with asthma, the routine use of serum IgE analysis is limited.

IMMUNOGLOBULIN DEFICIENCIES Immunodeficiencies affecting either antibody production or cell-mediated immunity are commonly subdivided in two groups - primary and secondary. The primary immunodeficiency diseases are the naturally occurring defects that increase susceptibility to infections. The

Immunoglobulin Titers and Immunoglobulin Subtypes

disorders constitute a spectrum of more than 80 different innate defects in the immune system (Cooper et al., 2003). The overall incidence of these Immunodeficiency diseases is estimated at approximately 1 in 10 000, excluding selective immunoglobulin A deficiency. There may be as many as 500 000 cases in the USA, of which about 50 000 cases are diagnosed each year. Common primary immunodeficiencies include disorders of antibody immunity, affecting B-cell differentiation and/or antibody production, isolated T-cell abnormalities, combined B- and T-cell defects, phagocytic disorders and complement deficiencies (Cooper et al., 2003). The majority of immune defects involve antibody production; these immune deficiencies are found more often in adults than infants and children (Ballow, 2002). The immune system can also be adversely affected secondarily by a variety of conditions, such as malnutrition, malignancy, metabolic diseases, infections, as well as immunosuppressive drugs or radiation. Antibody deficiencies need to be excluded in patients who have recurrent, severe or unusual pyogenic infections. The commonest sites of infection are the upper and lower respiratory tracts and the infections caused by capsulated bacterial pathogens like Streptococcus pneumoniae and Haemophilus influenzae. Some patients also develop diarrhea and malabsorption due to bacterial overgrowth in the small intestine or chronic infection with Giardia, Campylobacter or Cryptosporidium (Puck, 1997; lUIS Scientific Committee, 1999).

Immunoglobulin A deficiency IgA deficiency is the most frequent primary immunodeficiency, with an average prevalence of 1:400-1:700. IgA deficiency is defined as a serum IgA concentration of less than 7 mg/dl with normal serum IgM and IgG levels (Table 12.3). Both IgA subclasses, IgAI and lgA2, are usually markedly reduced or absent, although isolated deficiencies of each subclass have been described. In general, an lgA2 deficiency would be expected to lead to more serious infections than an IgAI deficiency, due to the stronger protease susceptibility of the IgAI molecule. Approximately 50 per cent of individuals with selective IgA deficiency are clinically asymptomatic. IgA deficient patients usually have sino-pulmonary infections, involvement of the gastrointestinal tract with giardiasis and nodular lymphoid hyperplasia. An increased frequency of autoimmune disorders has also been associated with IgA deficiency, including arthritis, a lupus-like illness, autoimmune endocrinopathies, chronic active hepatitis, ulcerative colitis, Crohn's disease, a sprue-like disease and autoimmune hematologic disorders (Ballow, 2002). Selective IgA deficiency is also strongly associated with atopy. IgA deficiencies are often found in association with other immune abnormalities, including ataxia-telangiectasia and IgG subclass deficiencies. The occurrence of IgA

Table 12.3 Common levels of immunoglobulins in immunodeficiency diseases* Diseases

IgG

IgA

IgE

IgD

Selective IgA deficiency Selective IgG deficiency Selective Ig class deficiency X-linked agammaglobulinemia Hyper-IgM syndrome C o m m o n variable immunodeficiency Ataxia telangiectasia Wiskott-Aldrich syndrome 'Bare-lymphocyte syndrome'(MHC deficiencies) SCID

n ivL n/i

isl n n n/i n n n/i n/i n/i

n/i

ii

IgM

ii a n/T

si

i

T

-li i n/t n/i

i-l i i n/T i i

n/i n i ii

i i i n/T n/i

i i

i i

i i

i i

* n, normal levels; i , decreased levels; T, increased levels.

deficiency in both male and female patients and its clustering in families suggest an autosomal inheritance. The pathogenesis of IgA deficiency is still unknown, although abnormalities in immunoglobulin class switching and the cytokines involved in isotype switching have been implicated. The pathogenesis of IgA deficiency may share a common cause with common variable immunodeficiency because these two disorders share many immunological aspects. Immunoglobulin G deficiency A deficiency of total IgG will generally result in serious infectious problems. IgG subclass deficiency also occurs and is a common feature in a number of primary as well as secondary immunodeficiency syndromes (Ballow, 2002). Since a decreased level of one IgG subclass may be accompanied by increased levels of one or more of the other subclasses, the total IgG level may be within the normal range. Therefore, evaluation of IgG subclass levels is important, even when the total IgG level is within or only slightly below the reference range of healthy individuals (Meulenbroek and Zeijiemaker, 1996). As IgGI comprises up to 70 per cent of the total IgG serum concentration, patients with lgG1 deficiency usually have reduction in total IgG. Interest in IgG subclass deficiencies arose from a report of a family with recurrent sinopulmonary infections who had normal serum IgG, IgA and IgM concentration but reduced lgG2 and lgG4 levels. These patients were shown to be unable to produce antibodies to polysaccharide antigens but have normal antibody responses to the protein antigens. Deficiencies can occur in single or multiple IgG subclasses (Morell, 1994). Deficiencies of IgG subclasses can be subdivided in different groups. A complete deficiency of one or more subclasses, caused by deletions in chromosome 14 loci, is rare (Bottaro et al., 1989). Most abnormalities are based upon regulatory defects resulting in decreased levels rather than a total lack of one or more immunoglobulin subclasses. lgG2 deficiency may occur

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

either as an isolated finding or in association with IgA or lgG4 deficiency. Among the combined IgG subclass deficiencies, an lgG2/lgG4 deficiency predominates (Jefferis and Kumararatne, 1990). The relevance of selective lgG4 deficiency is uncertain, as approximately up to 15 per cent of the general population have lgG4 concentrations in serum below the limit of detection. Interestingly, the genes encoding lgG2, lgG4 and IgA are closely linked and this combined deficiency is due to a regulation defect of the 'downstream switch' in the heavy chain loci (Meulenbroek and Zeijiemaker, 1996). It seems that the expression of the immunoglobulins whose genes are located downstream in the C(H) region, such as lgG4 and IgE, requires more help from Th2 cells compared to the upstream isotypes, IgGI and lgG3 (Mayumi et al., 1983; Bich-Thuy and Revillard, 1984). lgG4 and lgG2 deficiencies are often found to be associated with a predominant T-cell defect, such as in ataxia telangiectasia, AIDS and immune reconstitution after bone marrow transplantation. It has been noticed that patients with IgGI and/or lgG3 deficiency are more likely to have more chronic and recurrent infections of the lower respiratory tract, whereas patients with lgG2 and/or lgG4 deficiency are more likely to have sinusitis and otitis (Herrod, 1993a,b). Immunoglobulin M deficiency Selective IgM deficiency implies correct gene rearrangements in B cells and might be caused by the failure of polysaccharide recognition systems. In this case, a certain decrease in the lgG2 and lgG4 subclasses would be also anticipated, because of their specificity for bacterial polysaccharide antigens. Patients with a combined X-linked Wiskott-Aldrich deficiency syndrome display low or absent serum IgM levels, while IgG, IgA and IgE levels may be normal or elevated (Thrasher, 2002). WASP patients are unable to respond to polysaccharide antigens and thus isoagglutinins are absent. Immunoglobulin D deficiency IgD deficiency is a defect of humoral immunity that is characterized by abnormally low serum levels of this immunoglobulin (Alper et al., 2003). It has been reported that approximately 11 per cent of 371 American Red Cross blood donors and 6 per cent of 1529 study subjects had low or undetectable IgD levels (<0.002 mg/ml). In the study group, a number of the individuals with low IgD had rheumatologic disease (e.g. juvenile rheumatoid arthritis, lupus, psoriatic arthritis, vasculitis), but the frequency of low IgD within groups of patients with each disease did not differ from the normal controls using chi-square analysis. In another study, using a cutoff of 2.15 lU/ml, assays of 245 healthy adults and 301 healthy children revealed that approximately 13 per cent of each group had low levels of IgD. Unfortunately, little is known

about the normal function of IgD and few clinical signs or symptoms are associated with its absence. Individuals with low or absent levels of IgD do not appear unusually predisposed to infections. Other immunodeficiencies associated with hypogammaglobulinemia Common variable immunodeficiency

(CVID)

Common variable immunodeficiency, also known as adult-onset hypogammaglobulinemia, acquired hypogammaglobulinemia or dysgammaglobulinemia, is a heterogeneous group of disorders involving both B-cell and T-cell immune function (Ballow, 2002). The average age of onset of symptoms is 25 years and the average age at diagnosis is 28 years. CVID is characterized by recurrent bacterial infections (recurrent otitis, chronic sinusitis and recurrent pneumonia), decreased serum immunoglobulin levels, especially IgG and IgA and abnormal antibody responses to specific immunization (Fischer, 2004). Most individuals with CVID are panhypogammaglobulinemic, but some produce substantial amounts of IgM. Although markedly diminished, the serum Ig levels are usually higher than those found in patients with X-linked agammaglobulinemia. Autoimmune disorders occur frequently in patients with CVID (approximately 22 per cent of patients) and include rheumatoid arthritis, autoimmune hematologic disorders, such as hemolytic anemia, idiopathic thrombocytopenic purpura and pernicious anemia, autoimmune neurologic diseases, such as Guillain-Barre syndrome, chronic active hepatitis often related to hepatitis C virus and autoimmune endocrinopathies, particularly involving the thyroid gland (Etzioni, 2003).

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Figure 12.1 Evaluation of gammaglobulins by total protein electrophoresis in serum specimens. Normal range of immunoglobulins is shown in samples 1, 2 and 6. Samples 3 and 5 demonstrate a polyclonal increase in immunoglobulin levels, which might be associated with infections, autoimmune diseases or certain liver abnormalities. Sample 4 is an example of hypogammaglobulinemia, which may be a sign of primary or secondary immunodeficiency or light chain myeloma. The automated SPIFE 3000 electrophoresis system was used for serum protein separation and staining (Helena Laboratories, Beaumont, TX).

Immunoglobulin Titers and Immunoglobulin Subtypes

Immunodeficiency

with hyper-IgM

During the last few years, a series of primary immune deficiencies characterized by a defect of class switch recombination and the inability to produce immunoglobulins other than IgM or IgD have been described. Some of these defects are collectively referred to as hyper-IgM immunodeficiency (HIGM) syndrome. This syndrome mainly affects males (55-65 per cent) and is characterized by decreased serum levels of IgG, IgA and IgE, but elevated IgM and accompanied with severe recurrent bacterial infections. Patients with HIGM syndrome can also develop autoimmune disorders (Etzioni, 2003). Coombs positive hemolytic anemia, inflammatory bowel disease, hepatitis, seronegative arthritis, hypothyroidism and discoid lupus erythematosus have all been reported in HIGM patients. A variety of serum autoantibodies have been detected as well, including anti-erythrocyte, anti-erythropoietin, antiplatelet, anti-smooth muscle, anti-cardiolipin, anti-Ro, anti-RNP, antinuclearand anti-thyroid antibodies. HIGM type 1 (HIGM1) is inherited as an X-linked trait (Gaspar et al., 2002). The disease is caused by defective expression of CD40 ligand (CD40L, GDI54), a molecule predominantly expressed by activated CD4+ T cells. CD40-CD40L interaction is essential for final maturation of B cells after their activation by antigen and supports class switch recombination. Immunological features include very low serum levels of IgG and IgA, with normal to increased IgM. The number and distribution of T-cell subsets are normal, as are proliferative responses to mitogens. Patients with HIGM1 syndrome are clinically and immunologically indistinguishable from subjects carrying genetic defects of CD40 (a molecule constitutively expressed by B lymphocytes, monocytes, dendritic cells and by endothelial and some epithelial cells) that results in an autosomal recessive form of HIGM, which has been recently described and termed HIGM type 3 (Puck, 1994). In contrast, patients with HIGM type 2 (HIGM2) present with a distinct clinical phenotype characterized by enlargement of tonsils and lymph nodes and recurrent bacterial sino-pulmonary infections, without increased susceptibility to opportunistic pathogens. Pathologic examination of lymph nodes reveals aberrantly large germinal centers, a finding that clearly distinguishes HIGM2 from HIGM1 and HIGM3. In addition to the characteristic immunoglobulin profile similar to HIGM1 and HIGM3, patients with HIGM2 show a lack of switched IgDnegative B cells and a profound defect of somatic hypermutation. The association of both somatic hypermutation and class switch recombination deficiency is explained by mutations in the recently cloned activation-induced cytidine deaminase (AID) gene that plays a critical role in both processes. A fourth form of HIGM affects males and is characterized by the association of hypogammaglobulinemia with hypohydrotic ectodermal dysplasia (HIGM-ED). Similarly to other forms of HIGM, HIGM-ED is not a pure humoral

deficiency since defects of T and natural killer (NK) cells have been also reported (Imai et al., 2003). X-linked aggammaglobulinemia

(XLA)

X-linked aggamnnaglobulinemia, also called Bruton's agammaglobulinemia, or congenital agammaglobulinemia, occurs with a frequency of about 1 per 3-6 million. Total absence or marked deficiency of serum immunoglobulins, extremely low percentages (<2 per cent) or absent B cells, but normal or even increased numbers of pro-B cell in the bone marrow due to the block of their maturation, are typical findings in XLA (Fischer, 2004). The upper respiratory tract is the most common site of infection. Other types of infection include pyoderma, chronic conjunctivitis, gastroenteritis, arthritis and meningitis-encephalitis. Capsulated bacteria, particularly H. influenzae and S. pneumoniae, are commonly associated with these infections. Arthritis and a dermatomyositis-like syndrome can also occur in patients with XLA, although autoimmune disorders do not seem to be a frequent problem in patients with XLA (Ballow, 2002).

HYPERIMMUNOGLOBULINEMIA Increased levels of serum immunoglobulin may be a consequence of upregulated synthesis or lowered catabolism. Distinctively, hypergammaglobulinemia may arise from monoclonal, polyclonal or oligoclonal pathological or physiological processes. For instance, monoclonal increase of immunoglobulins commonly results from a malignant or premalignant clonal B or plasma cell proliferation, while a polyclonal increase in serum antibodies could be the consequence of autoimmune or inflammatory reactions. Increase of total IgG, IgA and IgM can be detected in patients with hepatitis or cirrhosis, rheumatoid arthritis and systemic lupus erythematosus. In nnost infections the first antibodies to appear will be of the IgM class, while those of the IgG class will be produced later. In general, microbial protein antigens will mainly evoke antibody responses of the lgG1 and lgG3 subclasses, with a minor contribution of lgG2 and lgG4. On the other hand, polysaccharide antigens will predominantly induce lgG2 antibodies. Monoclonal gammopathy Numerous terms such as monoclonal gammopathy (MG), paraprotein, M-component or M-protein are applied to describe the immunoglobulins overproduced by a single clone of B cells. The term M-protein is generally preferred, because it does not imply structural characteristics of the molecule other than its homogeneous nature and does mean that the patient exhibits a pathological condition. M-protein corresponds to the observation of

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

an electrophoretically restricted migration pattern at serum protein electrophoresis, but does not reflect a particular cellular mechanism involved in the clonal production of the immunoglobulin. In an attempt to unify the various names mentioned above, the general term 'immunoglobulinopathy' has been proposed for clonal overproduction of immunoglobulins. The search for an M-component may be of relevance in patients presenting unexplained peripheral neuropathy, carpal tunnel syndrome, nephrotic syndrome, renal insufficiency, cardiomyopathy, refractory heart failure, orthostatic hypotension, acquired von Willebrand's disease, or malabsorption, because all these abnormalities have been associated with MG. The detection of a single overproduced immunoglobulin is classically associated with B-cell lymphoproliferative disorders, like multiple myeloma, Waldenstrom's macroglobulinemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia and amyloidosis, but it can also be observed in the absence of a malignant disease (Chaibi et al., 2002). The latter situation is known as monoclonal gammopathy of undetermined significance (MGUS). B-cell lymphoproliferative disorders should be suspected instead of MGUS in patients with M-protein associated with unexplained anemia, back pain, weakness or fatigue, osteolytic lesions, fractures, osteopenia, hypercalcemia, renal insufficiency, proteinuria, diffuse failure of reabsorption in the proximal renal tubule resulting in glycosuria, generalized aminoaciduria and hypophosphatemia (acquired Fanconi's syndrome), or with the history of recurrent bacterial infections. Plasma cell neoplasms usually manifest as disseminated bone marrow lesions (multiple myeloma) and

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infrequently as solitary extramedullary tumors classified as plasmacytomas. Malignant plasma cells usually secrete monoclonal IgG (60 per cent) or IgA (20 per cent) and rarely IgD (1-2 per cent) (Ho et al., 2002). There is a quantitative increase in the immunoglobulin concentration in the blood of multiple myeloma patients, but all of the immunoglobulins have identical specificity and therefore do not contribute to immune defense. The concentration of normal immunoglobulins is usually decreased due to dysregulation of lymphopoiesis and B-cell differentiation. The term 'monoclonal gammopathy of undetermined significance' denotes the presence of a monoclonal protein in patients without evidence of plasma or B-cell proliferative disorders (Kyle and Rajkumar, 2003). This type of gammopathy is relatively common, with a prevalence of about 0.15 per cent of the general population. However, occurrences rise with age as MGUS has been found in approximately 1 per cent of persons older than 50 years, 3 per cent of persons older than 70 years and 4 per cent of individuals over 80 years old (Chaibi et al., 2002). Monoclonal gammopathy of undetermined significance may be associated with various disorders, including non-B-cell hematological malignancies, autoimmune diseases, secondary immunodeficiency states, von Willebrand disease, connective tissue diseases and neurologic disorders. A significant proportion of patients with MGUS will develop multiple myeloma or related disorders during a long-term follow-up. In addition, there are a number of unusual clinical findings that are due to the specific binding properties of the MG. These particular antibodies can lead to severe clinical problems in the absence of signs of malignancy and were named 'perverse' MG.

Serum Proteins Hospital # Patient name Age :89 Sex :M Institution Sample 14 11-28-2001

Accession Specimen 1 Physician Total prof

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37.97 2.62 8.61 8.94 41.87 34.87

1

g/dl Range 5.30 0.30 1.00 1.50 1.80

g/dl 3.38 0.23 0.77 0.80 3.73+ 3.10

3.00 0.10 0.40 0.70 0.60

8.90+

6.00

8.30

Figure 12.2 Detection of monoclonal gammopathies in serum specimens by total protein electrophoresis, A. Samples 14, 15 and 17 are examples of monoclonal gammopathy visible as the presence of significant band (Mprotein) in gamma region. B. Densitometric analysis of sample 14 reveals the concentration of monoclonal protein in serum specimen. Windows-based QuickScan 2000 flat-bed densitometry (Helena Laboratories, Beaumont, TX) was used to image visible electrophoretic analytes on the agarose gel.

Immunoglobulin Titers and Immunoglobulin Subtypes

Biclonal^ triclonal and oligoclonal gammopathies

M

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Biclonal gammapathies occur in more than 5 per cent of patients with MG and are characterized by the presence of two homogeneous plasma immunoglobulins. The biclonal gammopathy may result from proliferation of two clones of plasma cells, each producing an unrelated monoclonal protein or from the production of two M-proteins by one clone of plasma cells (Ho et al., 2002). Triclonal gammopathies or oligoclonal gammopathies have been also reported. The identification of multiple clonal immunoglobulins largely depends on the sensitivity of the technique utilized. Although it has been proposed that oligoclonal gammopathies should be considered to form a separate category of immunoglobulinopathies, studies in healthy volunteers, patients and animal models confirm that oligoclonal gammopathies and MG usually result from different pathophysiolological processes. MG is most frequently observed in patients with a malignant disease involving a single B-cell clone (Ho et al., 2002), whereas oligoclonal gammopathies are often detected in patients without overt B-cell malignancies. Immunoglobulin oligoclonality may reflect a T-cell/B-cell imbalance, a selective antigenic pressure, or both. In all cases, however, identification of an immunoglobulinopathy may have important clinical implications.

1j

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Figure 12.3 Analysis of monoclonal immunoglobulins in serum specimens by serum electrophoresis with immunofixation. Using specific antibodies against heavy and light chains of immunoglobulins allows detection of the nature of monoclonal immunoglobulins after electrophoretic separation of serum proteins on agarose gels. Immunofixation analysis of sample 14 (see Figure 9.2) revealed the presence of monoclonal IgM/k. The automated SPIFE 3000 electrophoresis system was used for serum protein separation, staining and development (Helena Laboratories, Beaumont, TX).

were also evident, notably in those patients having a malignant form of lymphoproliferative disorder. Monoclonal heavy chain disease

Immunoglobulin M paraproteins IgM paraproteins are IgM antibodies derived from the same B-cell clone and hence have identical immunochemical properties and functional activity (RobertsThomson et al., 2002). They are identified as monoclonal 7 or /3 bands on serum protein electrophoresis with immunofixation having JJL heavy chain and K or A light chain antigenicity. Circulating IgM paraprotein in large quantities is an essential feature of Waldenstrom's macroglobulinemia (Ghobrial et al., 2003) but may also occur in other B-cell lymphoproliferative disorders or as a 'benign' finding, particularly in the elderly. It is important that the clinical distinction is made between these possibilities, as Waldenstrom's macroglobulinemia and similar lymphoproliferative disorders with IgM paraproteinemia are potentially life-threatening disorders with unique clinico-pathological features. IgM paraproteins accounted for - 2 0 per cent of all new paraproteins detected. IgM paraproteinemia occurs more commonly in males and its frequency increases with age (Roberts-Thomson et al., 2002). Approximately 30 per cent of IgM paraproteins are associated with B-cell lymphoproliferative disorders with the remainder being identified as monoclonal IgM gammopathies of uncertain significance. IgM paraproteins frequently exist in multiple molecular forms, the pentamer being the dominant species in all patients but decamers also being observed in most patients, particularly those with high levels of IgM (Ghobrial et al., 2003). Monomeric and oligomeric IgM

Heavy chain disease (HCD) is a rare disorder characterized by the secretion of monoclonal proteins consisting of incomplete heavy chains of the immunoglobulins. Gamma-HCD is associated with the increased synthesis and release of monoclonal y chains of IgG and accompanied by lymphoadenopathy and osteolysis. Alpha-HCD, or Mediterranean lymphoma, is characterized by the secretion of a chains of IgA by mucosal plasma cells in the intestines. This results in the development of progressive malabsorption syndrome, diarrhea and abdominal pain. Mu-HCD occurs in conjunction with lymphoproliferative diseases, particularly B-CLL

Light chain disorders Bence Jones plasmacytomas are characterized by the exclusive secretion of light chains, which can be detected in serum and urine (Bence Jones proteins) in different concentrations depending on the stage of disease progression (Stone, 2002). Protein electrophoresis of serum obtained from these patients often demonstrates marked decrease of total immunoglobulins (secondary antibody deficiency) due to replacement of hematopoietic precursors in the bone marrow with myeloma cells and thus deficient lymphopoiesis. Production of light chains is also associated with primary, non-hereditary light chain (AL) amyloidosis, which is characterized by the deposition of fibrils from fragments of immunoglobulin light chains (Buxbaum, 2004). AL occurs in plasma cell disorders, especially

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

multiple myeloma or Waldenstrom's macroglobulinemia. Amyloidosis most commonly induces renal symptoms, although cardiac and gastrointestinal involvement is also typically observed. Hyper-lgD syndrome A definite increase in serum IgD was found in patients with Hodgkin's disease several months after splenectomy (Corte et al., 1978). A correlation with the histologic type was observed; e.g. patients in the lymphocyte-depleted group had extremely high levels of IgD. Increased concentrations of serum IgD were found in other diseases and conditions, e.g. in children with kwashiorkor (Rowe et al., 1968) and after allogeneic bone marrow transplantation (Mizuma et al., 1987). High values of serum IgD were also reported in children following chemotherapy for malignancies (Azuma et a!., 1991), in some patients with hyperparathyroidism (Papadopoulos and Frieri, 1984) and in cigarette smokers (Bahna et al., 1983). IgD was also increased (>100U/ml) in many children with periodic fever, aphthous stomatitis, pharyngitis and adenopathy syndrome (Padeh et al, 1999). The new syndrome - hyperimmunoglobulinemia D syndrome (HIDS) - was described in 1984 by Van der Meer et al. and seems to have boosted the measurement of serum IgD in various patients in the hope of detecting more patients with HIDS and also of finding increased IgD in other conditions (Vladutiu, 2000). HIDS is the only entity for which the measurement of polyclonal IgD is necessary for diagnosis. This syndrome is characterized by recurrent febrile attacks accompanied by abdominal pain, arthralgia, headache and skin lesions. HIDS is probably inherited as an autosomal recessive trait and mutations in the mevalonate kinase gene were shown to cause HIDS (Houten et al., 1999). It has been proposed that patients exhibit an uncontrolled type 3 hypersensitivity reaction, possibly with involvement of IgD-containing complexes (Boom et al., 1990).

ANALYSIS OF SPECIFIC ANTIBODIES IN SERUM Evaluation of the serum levels of specific antibodies is essential for diagnosis of certain immunodeficiency diseases, many autoimmune disorders, allergies and a number of infectious diseases. Specific antibodies in immunodeficiencies The ability to produce specific antibodies against defined antigens is the most sensitive method of detecting certain malfunctions of antibody production. The incidence of specific antibody defects in patients with normal immunoglobulins is unknown (Bird, 1991). However, in the presence of history suggestive of immunodeficiency without gross deficiency of total serum immunoglobulins.

detection of specific antibodies before and after immunization should be assessed. The levels of specific antibodies are usually determined using a panel of protein and carbohydrate antigens. This includes (i) thymusdependent antigens tetanus toxoid, diphtheria toxoid, influenza vaccine and Salk vaccine (inactivated polio) and (ii) thymus-independent antigens, such as E. coli LPS, pneumococcal polysaccharide, H. influenzae type b polyribose phosphate and meningococcal polysaccharide type A and C (Bird, 1991). If initial levels of specific antibodies are low, the patient should be immunized with the appropriate antigen and the antibody response should be reassessed in 3-4 weeks. However, immunization with live vaccines is contraindicated in patients with suspected immunodeficiency. Specific antibodies in autoimmune diseases Autoimmune diseases are conditions in which structural or functional damage to organs and tissues results from immunological mechanisms mediated by autoantibodies or autoreactive cells. The autoantibody in organ-specific autoimmune diseases is directed predominantly at particular target organs or tissues, which are also the site of immunopathology. This includes autoimmune thyroiditis (Hashimoto's thyroiditis and Graves' disease), autoimmune gastritis, autoimmune hepatitis (chronic active hepatitis and primary biliary cirrhosis) and other autoimmune diseases of skin (bullous pemphigoid), Gl tract (celiac disease), CNS (multiple sclerosis), muscles (myasthenia gravis), kidney (Goodpasture's syndrome), pancreas (insulin-dependent diabetes mellitus) and erythrocytes (autoimmune hemolytic anemia). In non-organ specific autoimmune diseases, which include systemic lupus erythematosus, rheumatoid arthritis and the vasculitides, the autoantibodies are not organ restricted and present in many different tissues. Autoantibodies can be grouped into primary antibodies, which are directly pathogenic, and secondary antibodies, which recognize intracellular antigens. Secondary antibodies are usually not involved in the pathogenesis of many conditions, but can serve as useful diagnostic markers of different autoimmune diseases (Bird, 1991). The ease of detection of antibodies recognizing a variety of tissue and intracellular antigens has led to the description of a wide range of autoantibody specificities. The list of autoantibodies and corresponding antigens is constantly growing. For instance, rheumatoid factor (RF) for many years served as the only serological factor associated with rheumatoid arthritis (RA). RF consists naturally occurring autoantibodies with specificity for the Fc region of IgG (Moore and Dorner, 1993). A number of other autoantibodies associated with RA have been described, most of which remain largely experimental tools. Furthermore, no single antibody marker is sufficiently sensitive to be used solely in the diagnosis of RA as was concluded on the basis of their sensitivity range (Marcelletti

Immunoglobulin Titers and Immunoglobulin Subtypes

and Nakamura, 2003). However, a few markers are highly specific for RA suggesting that a diagnostic panel consisting of two or three markers may achieve sufficient sensitivity. Of particular interest are those autoantibodies that recognize citrullinated forms of native proteins. Antikeratin and antiperinuclear factor antibodies have been tested for several years and were found to be highly specific for RA (van Boekel et al., 2002). New studies indicate that these antibodies recognize an epitope that includes the deimidated form of arginine called citrulline (Schellekens et al., 1998). Cyclic peptides containing citrulline (CCP) have been used to develop a highly sensitive and specific assay for the differential diagnosis of RA. Subsequent clinical studies demonstrated predictive value of the anti-CCP plus RF combination suggesting that the disease could potentially be identified solely on the basis of serology (Marcelletti and Nakamura, 2003). Determination of tissue-specific autoantibodies is of great value for disease diagnosis, monitoring of therapy and prediction of the disease progression. For instance, Hashimoto's thyroiditis involves antibodies to various intracellular antigens, including thyroglobulin and thyroid peroxidase. Hyperthyroidism in Graves' disease is due to antibodies to the thyrotropin receptor that cause overstimulation of the gland (Reiterer and Borkenstein, 2003). Autoimmune liver diseases are associated with an autoimmune attack on hepatocytes and bile duct epithelial cells mediated by antibodies recognizing a sialoglycoprotein receptor and 2-oxo acid dehydrogenase enzyme complexes on mitochondrial membranes. Autoantibodies to myelin basic protein and myelin-associated glucoprotein are found in multiple sclerosis and polyneuropathy, respectively (Lutton et al., 2004). Goodpasture's syndrome is characterized by the presence of autoantibody to glomerular, renal tubular and alveolar basement membranes, resulting primarily in injury to the glomerulus that can rapidly progress to renal failure (Jara et al., 2003). Autoantibody-mediated damage to the acetylcholine receptors in skeletal muscles leads to the progressive muscle weakness and is the pathogenetic basis of myasthenia gravis (Vincent et al., 2003). A diagnosis of autpimmune disease implies that the autoimmune response is a significant component of the disease process. In most instances the initiating events of autoimmune disease are not understood and autoantibodies are an effect of disease, not a cause. Even though the cause may not be known, the presence of autoantibodies can be clinically useful and their titers may have prognostic value. Specific antibodies in infectious diseases Medical importance of serological, i.e. antibody-based test in the diagnosis of infectious diseases is evident when 1 the microorganism cannot be cultured, e.g. syphilis and hepatitis A, B and C

2 the microorganism is too dangerous to culture, e.g. rickettsial diseases 3 culture techniques are not readily available, e.g. HIV and EBV 4 the microorganism takes too long to grow, e.g. Mycoplasma (Gaur et al., 1994). For instance, infection with Treponema pallidum provokes a complex antibody response and serological tests for syphilis are based on the detection of one or more of these antibodies. No 'perfect' single test is available at present and although more than 200 methods have been reported, only a few are routinely used. Detection of antibodies of two types are currently used for screening and diagnostic purposes: (i) non-treponemal antibodies, or regain, which react with lipid antigens and (ii) treponemal antibodies, which react with I pallidum and closely related strains (Young, 1998). Infection with hepatitis B virus (HBV) stimulates strong immunological responses to a number of HBV antigens. Acute and chronic phases of the disease are discernible based on specific serologic profile. In acute HBV hepatitis, HBsAg, HBeAg and HBcAb (antibody to the core protein) may be detected in the blood during viral replication. In chronic hepatitis, the development of HBeAb (antibody to the e antigen) indicates that the disease is no longer infectious. During a 'window' period, IgM HBcAb may be the only detectable serologic marker. Serology tests are commonly used in both the diagnosis of different infections and in screening for specific immunity (Gaur et al., 1994). For instance, primary infection is indicated by the presence of specific IgM antibodies or by a fourfold rise in specific IgG antibody titers between acute- and convalescent-phase samples.

QUANTITATIVE I M M U N O G L O B U L I N S Quantitative measurement of serum, urine or CSF immunoglobulins is used in the workup of both immunodeficiency states and some lymphoproliferative disorders. If an abnormality in humoral immunity is suspected, initial evaluation should include determination of IgG, IgA and IgM levels. The IgA level is especially helpful in that IgA concentrations in serum are low in almost all permanent types of agammaglobulinemia and in selective IgA deficiency IgA measurement yields information about the body's resistance to mucosal infection as well as information related to specific diseases, such as myeloma and infection. Decreased levels are associated with immune deficiencies, protein losing conditions, non-lgA myelomas, mucosal infection and otitis media. Increased levels are associated with IgA myelomas, alcohol-related cirrhosis, chronic infection, active rheumatic disease and malabsorption syndrome. IgA deficiency may be associated with an anaphylactic reaction to blood products containing IgA and with development of antibodies to animal proteins.

Popovic Petar, Diane Dubois, Bruce S. Rabin and Michael R. Shurin

IgE level assessment would be appropriate in patients with suspected atopy, Wiskott-Aldrich syndronne or suspected hyperimnnunoglobulin E syndronne (Ennanuel, 2003). Serunn IgE concentrations are also increased in parasitic infestations and bronchopulnnonary aspergillosis. Since total serum IgE is markedly elevated in about one half of patients with asthma, measurement of total IgE is not essential in any allergic conditions (Haeney, 1991). However, total serum IgE levels can be of value in infants with a positive family history of atopic disease and troublesome symptoms of suspected allergic origin, since levels are normally very low under the age of 2 years (Haeney, 1991). On the contrary, detection of antigen-specific IgE antibodies is of a great importance for patients with asthma. There are several available techniques to detect antigen-specific IgE in serum. These are radioallergosorbent test (RAST), radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA) (Dolen, 2003). IgG subclass testing should not be used for screening patients for suspected immunodeficiency, but should be reserved for patients who lack specific antibody responses to antigenic challenge yet have slightly low or normal immunoglobulin serum concentrations. Occasionally serum and urine protein electrophoresis may be helpful in evaluating patients with polyclonal or oligoclonal gammapathies and immunodeficiencies (Keren et al., 1999). Serum protein electrophoresis (SPE) is a technique in which molecules are separated on the basis of their size and electrical charge. SPE profile is traditionally divided into five regions: albumin, a- 1, a- 2, j8 and 7 regions. IgG, IgM, IgD and IgE migrate in the y-globulin region, while IgA migrates as a broad band in the j8 and y regions. Additional evaluation of serum immunoglobulins is conducted if the SPE results reveal a monoclonal component, or if there is a significant quantitative abnormality of serum immunoglobulins. Another method of evaluating immunoglobulins in serum is immunofixation electrophoresis (IFE). In IFE, serum specimens are electrophoresed first and then specific antibodies recognizing y, a, JJL, 8 and e heavy chains and K and A light chains are applied directly to the gel. The antibody-antigen complexes are formed and can be visualized by staining. Polyclonal immunoglobulins are pointed out by diffuse staining, whereas monoclonal bands are reflected by narrow and intensely stained bands. Lack of staining indicates immunodeficiencies of one or more immunoglobulin classes (Keren, 1999). Multiple immunological methods are also available for the quantitation of immunoglobulin levels. These methods can be classified into two general classes. The first group involves evaluation of physical properties from immune complexes generated by serum immunoglobulins and specific antibodies, such as light scattering, precipitation in gels, or agglutination of different particles. The second group involves assessment of serum immunoglobulins using specifically labeled antibodies. Three commonly used procedures include rate

nephelometry, radial immunodiffusion (RID) in the first group and ELISA in the second group. In the RID procedure, an antigen solution is placed in a well cut in the agarose gel containing specific antibodies. As the antigen diffuses into the gel, it reacts with the antibody forming a precipitin ring and its diameter is proportional to the antigen concentration. Nephelometry is based on the detection of light scattered from antigenantibody complexes formed in a cuvette where serum samples are mixed with a specific antibody solution. Nephelometry is automated, fast and accurate. Detection of autoantibodies is usually done by ELISA for known specific antigens or by immunofluorescence for initial screening of different groups of autoantibodies. For instance, known antigen is attached to a slide in form of cell lines or tissue with a known pattern of antigen distribution. The patient's serum is added and if it contains antibody against the antigen, it will remain fixed to it on the slide and can be detected on addition of a fluorescent-dye-labeled antibody to human IgG and examination by fluorescent microscopy. This method is commonly used to detect nuclear, gastric parietal cell, mitochondrial, smooth muscle and reticulin autoantibodies when frozen sections from a composite block of several tissues, rat kidney, liver, and stomach served as a substrate. Antinuclear antibodies (ANA) can be detected by the same technique using human epithelial cell line HEp-2 as a substrate.

CONCLUSIONS Immunoglobulins play a key role in host defense against infections. Thus evaluation of the titers of each immunoglobulin subclasses as well as the levels of specific antibodies is essential for diagnosis of many immunological diseases. Similarly analysis of the presence and titer of a number of specific antibodies is widely used for prognosis and diagnosis of a variety of autoimmune diseases and allergies. However, immunoglobulins serve not only as a diagnostic tool, but could be also used for different therapeutic modalities. For instance, replacement therapy with y-globulin in patients with immunodeficiencies, passive immunization for prophylaxis of some infectious diseases or neutralization of toxins or poisons and antibody-based immunotherapeutic approaches for cancer patients.

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Immunoglobulin Titers and Immunoglobulin Subtypes

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Schellekens, G.A., de Jong, B.A., van den Hoogen, F.H.J., Van de Putte, LB. and van Venrooij, W.J. (1998). Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 101, 273-281. Schur, PH. (1987). IgG subclasses - a review. Ann Allergy 58, 89-96, 99. Stahl, D. and Sibrowski, W. (2003). Regulation of the immune response by natural IgM: lessons from warm autoimmune hemolytic anemia. Curr Pharm Des 9,1871-1880. Stone, M.J. (2002). Myeloma and macroglobulinemia: what are the criteria for diagnosis? Clin Lymphoma 3, 23-25. Thrasher, A.J. (2002). WASp in immune-system organization and function. Nat Rev Immunol 2, 635-646. van Boekel, M.A.M., Vossenaar, E.R., van den Hoogen, FH.J. and van Venrooij, W.J. (2002). Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthrit Res 4, 87-93. Van de Perre, P (2003). Transfer of antibody via mother's milk. Vaccine 27, 3374-3376. Vincent, A., McConville, J., Farrugia, M.E. et al. (2003). Antibodies in myasthenia gravis and related disorders. Ann NY Acad Sci 998, 324-335. Vladutiu, A.O. (2000). Immunoglobulin D: properties, measurement, and clinical relevance. Clin Diagn Lab Immunol 7,131-140. Young, H. (1998). Syphilis. Serology. Dermatol Clin 16, 691-698.