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AFFINITY AND AVIDITY OF AUTOANTIBODIES
4
AFFINITY AND AVIDITY OF AUTOANTIBODIES ˇ Cˇ ∗† BORUT BOZI ∗ ˇ CU ˇ CNIK ˇ SASA TANJA KVEDER∗ BLAZˇ ROZMAN∗ ∗
Department of Rheumatology, University Medical Centre, Vodnikova 62, SI-1000 Ljubljana
†
University of Ljubljana, Faculty of Pharmacy, Chair of Clinical Biochemistry, Askerceva 7, SI-1000 Ljubljana
HISTORICAL NOTES AVIDITY AND AFFINITY CLINICAL UTILITY IN AUTOIMMUNE DISORDERS TAKE-HOME MESSAGES REFERENCES
ABSTRACT The basic knowledge and clinical applications of the detection of affinity/avidity of autoantibodies are relatively scarce. Surprisingly, there have been very few recent studies concerning this topic. While theoretical thermodynamics of antigen–antibody interactions are well known, their practical use in detection or determination of autoantibodies is deficient. Both, affinity and specificity for antigens may have important impact for selection of the detection assay. The major problem may be attributed to the lack of suitable measuring methods according to the Law of Mass Action. Selective nonorgan-specific autoimmune disorders have been chosen to demonstrate the currently existing controversies. Nevertheless, some recent information including new methods and sophisticated technologies gives some hope for future usefulness.
HISTORICAL NOTES Antibody affinity has been sporadically studied for almost half a century. In the 1970s, it was established that antibody affinity is a multi-genetically controlled parameter of the immune response and that such control is exerted by genetic mechanisms which are independent of those controlling antibody levels [1]. In the mid-1990s, the role of affinity and avidity in the pathogeneity of autoantibodies Autoantibodies, 2/e Copyright © 2007, Elsevier, B.V. All rights reserved.
21
22
AFFINITY AND AVIDITY OF AUTOANTIBODIES
remained unclear. In the 1990s, there have been surprisingly few studies to answer the question, does the diagnostic impact of antibodies correlate with the discrete binding pattern, avidity, molecular specificity or purely with titres? [2]
AVIDITY AND AFFINITY Definition The binding of antibody with its ligand is the most important feature for the detection of (auto)antibodies and for the determination of analytes by antibodies as reagents. Antibody–antigen intermolecular forces are relatively weak physical forces, which include (a) van der Waals or electrodynamic forces, (b) hydrogen bonding or polar forces, and (c) electrostatic forces. The energy of antibody–antigen binding is called either “affinity” or “avidity”. The specificity of antibodies is also associated with the binding energy. Antibody specificity for a given antigen is determined by its relative affinity. Antibodies with very high affinity to target a specific epitope can bind to similar epitopes with lower affinity. This important aspect is not fully implemented in the analytical strategies for characterizing antibodies. Despite their clear difference, the terms “affinity” and “avidity” are often indiscriminately used. Affinity is the force of binding of one antibody molecule’s paratope with its corresponding epitope on the antigen molecule and can be determined only by a single monovalent Fab fragment. It is the sum of all attractive forces resulting in increased binding strength and repulsive forces resulting in decreased binding strength. Affinity constant K can be described by thermodynamic terms: G = −RT ln K Where G is the standard free enthalpy or change in standard Gibbs’s free energy, T is the temperature, R is the gas constant and K is the equilibrium constant or constant of reaction. Avidity is the binding force between a multivalent antibody and a multivalent antigen. The measured binding energy between antibodies and their relevant, mostly complex antigens reflects the avidity of antibodies. Avidity cannot be described by thermodynamic terms, and is obtained by kinetic measurements. It is commonly indicative of the association constant, depending on the assay procedure employed. Ionic and hydrophobic interactions also increase the avidity energy, which can be tens, hundreds or even thousands of times higher than that of affinity.
Methods of Affinity Calculation Binding between antibodies and antigens is a reversible reaction and takes a few seconds to several hours to achieve its equilibrium. The reaction may be described by the Law of Mass Action with several assumptions. The most important are: the antibody must be monovalent, recognizing only one epitope on the antigen, thus having one affinity; both reagents must be pure (homogeneous regarding to binding sites) and in a solution; and the reaction must be in equilibrium without nonspecific binding to the walls of the reaction vessel. In practice, these assumptions are not completely met. Nevertheless, the Law of Mass Action does provide a useful basis for the theoretical assessment of antibody–antigen interactions [3,4]. The reaction
23
AVIDITY AND AFFINITY
between the monovalent antibody (Ab) and the monovalent antigen/hapten (H) can be written as follows: ka
Ab + H AbH kd
where ka is the association constant and kd is the dissociation constant. The equilibrium constant Keq , also called the “antibody affinity” according to previously mentioned assumptions, represents the concentration ratio of bound to unbound monovalent antibody and hapten: Keq = ka /kd = AbH/AbH The rearrangement using the calculation of free antibody concentration [Ab] from the total concentration of antibody Ab = Abt − AbH designates a linear relationship between the ratio of bound [AbH] to free hapten [H] and the concentration of bound hapten: AbH/H = Keq AbH − Keq Abt Graphic presentation of the equation gives the Scatchard plot, from which two parameters can be obtained, measuring only molar concentrations of the complex (= bound hapten) and molar concentrations of the free hapten (equilibrium constant as the slope, and total concentration of Ab as the intersection on the x-axis) (Figure 4.1). When binding of univalent hapten to multivalent antibody is measured, the Scatchard plot is useful after rearrangement of the Langmuir adsorption isotherm equation: d/n = KH/1 + KH where d represents the fraction of occupied binding sites and n represents antibody valence. The corresponding Scatchard equation is as follows: d/H = nK − dK It can be calculated with extrapolation of experimental data that K is equal to 1/[H], when half the antigen binding sites in bivalent antibody are hapten bound. Determining the most correct extrapolation on the Scatchard graph may be difficult; therefore reciprocally rearranged data are often used: 1/d = 1/nHK + 1/n
Bound/Free
Theoretical background is necessary using experimental data for calculation of the equilibrium constant (affinity). Linear graphs such as Scatchard or Langmuir are obtained only theoretically. In practice, particularly dealing with polyclonal
Slope = –K eq Bound
FIGURE 4.1 Scatchard plot.
Intercept = [Abt]
24
AFFINITY AND AVIDITY OF AUTOANTIBODIES
Bound/Free
Slope = –K eq high affinity
Slope = –K eq low affinity
Bound
Intercept = [Abt]
FIGURE 4.2 The Scatchard plot of mixed affinity antibodies.
patient sera, affinity heterogeneity (and consequently avidity heterogeneity) gives a curved line, making approximations impossible (Figure 4.2). Measurements and calculations of the average association constant may be useful in such situations.
Methods of Avidity/Affinity Measurement Controversies surrounding the role of antibodies’ avidity may be chiefly attributed to the lack of suitable measuring methods. Both affinity and specificity for antigens may have an important impact for the selection of a specific detection assay. There are basically three approaches: (a) solution phase, (b) solid phase and (c) the combination of both [5]. Solution-Phase Assays According to the Law of Mass Action, the very basic affinity determination comprehends methods in which an antigen–antibody interaction, as well as the separation of free molecules from the complex, always occurs in a solution: 1. The equilibrium dialysis to determine the affinity constant for the antibody directed to a small monovalent antigen, by measuring the concentration of free antigen (small antigen molecules separated from a larger antigen– antibody complex). 2. Ultrafiltration or ultracentrifugation to separate free and bound antibody, if antigens are large and multivalent. When sufficient quantities of the reagents are available and the antigen is much larger than the antibody, free antibody separated from the complex can be measured spectrophotometrically. When only small amounts of reagents are available, the amount of free antibody can be measured by enzyme-linked immunosorbent assay (ELISA) after separating free antibody from the complexes by centrifugation. 3. Equilibrium sedimentation to measure the concentration of labelled free antibody at the meniscus of the centrifuge tube when the size of an antigen is similar to that of an antibody molecule. 4. Labelling of antigen molecules to use various procedures to separate free labelled antigen from bound complexes: • precipitation of antibody with 50% ammonium sulphate or 15% polyethylene glycol • specific binding to protein A or to anti-immunoglobulin antibodies.
AVIDITY AND AFFINITY
25
5. Spectrofluorimetric methods (fluorescence quenching, fluorescence polarization, enhanced fluorescence) for antigen–antibody interaction such as that of a bound ligand. Solid-Phase Assays In solid-phase assays, a constant amount of antigen is usually adsorbed to a solid support, but its total concentration remains unknown: 1. ELISA: Varying amounts of an antibody are applied to the microtiter wells and after washing away free antibody, the amount of bound antibody is measured. Because proteins adsorbed to a plastic surface usually undergo some conformational changes it is questionable whether the value of equilibrium dissociation constant thus measured pertains to the interaction with an epitope in its native state. Therefore, it is preferable to capture the antigen on the plastic by pre-adsorbed antibodies and to measure K by a double-antibody sandwich assay format (capture ELISA). The commonly used ELISAs directly measure the interaction of an antibody with an immobilized antigen (or vice versa) and, therefore, do not permit the measurement of the true equilibrium dissociation constant since the antigen and the antibody are in separate phases. 2. Chaotropic ELISA: Increased concentrations of NaCl or other chaotropoic reagents have been shown to be useful in cases of polyclonal sera, where it is not possible to determine the exact association or dissociation constant [6]. Therefore, a relative average dissociation constant (relative avidity) is a good parameter for the evaluation of different polyvalent autoantibodies in patients’ sera. 3. Surface plasmon resonance: Measures macro-molecular interactions, including antigen–antibody interactions, in real time. It detects alterations in the refractive index of the medium surrounding the receptor, immobilized on a solid support, at the moment of ligand binding. It can be employed to determine kinetic parameters (association and dissociation rate constants), equilibrium binding constants, and concentration measurements. Combination of Solution-Phase and Solid-Phase Assays ELISA–equilibrium titration method combines solution-phase and solid-phase assays to benefit from both the equilibrium in a solution (according to the Law of Mass Action) and the simplified measurements of one of the reactants with an assay on a solid support. It measures a free antibody at the state of equilibrium in an antigen–antibody reaction mixture in a solution phase.
Biological Functions Antibody affinity is important: (a) in the activation of B-cells, (b) in the elimination of antigens and (c) in the regulation of the immune response. Measurements of autoantibodies depend on in vitro binding of autoantibodies to relevant antigens with the bias that in vitro conditions are similar to those in vivo. There are several important questions: Is the binding strength sufficient for in vivo binding? Is such binding strong enough to provoke a biological response (e.g. activation of B-cells)? Very low affinity autoantibodies can bind in vitro to their antigen under mild conditions (e.g. low salt concentration), but they do not bind in vivo under
26
AFFINITY AND AVIDITY OF AUTOANTIBODIES
physiological conditions. However, even the binding itself does not guarantee a biological consequence. Low affinity antibodies may not attach strongly enough to their antigen in order to be effective (equilibrium constant is low, the average time of bound molecules is short and the concentration of the complex is low). The efficiency of binding and provoking a biological effect of low affinity antibodies could be significantly increased by polyvalent binding. In the classical model of an immune response against infective agents during the early phase, IgM antibodies are produced, which possess low affinity, but their binding to the antigen is strong due to ten available binding sites (avidity). Due to class switching later on, bivalent IgG are produced compensating lower valency with higher affinity. The continuation of an antigen exposure results in the selection of clones that produce even higher affinity antibodies known as “affinity maturation”. The genes encoding the variable regions of antibodies undergo somatic hypermutations with the selection of B lymphocytes with higher affinity receptors. In autoimmune disorders, differences between primary and secondary immune responses are not recognizable. Most of the anti-self-normal repertoire includes IgM, which has weak affinity for self-antigens and is widely cross-reactive with multiple antigens. IgM can be present for decades without class switching. Pathogenic autoantibodies, usually IgG, have high affinity for self-antigens in the target tissue and restricted specificity. High avidity anti-self-antibodies can be created from many different Ig gene segments, but they tend to derive from a few preformed families of genes, suggesting derivation from an antigen-activated “mother cell”. In contrast to the infection model of the immune response, the question of affinity maturation in autoimmune disorders has not been solved. Patients with chronic autoimmune diseases produce (also) low affinity autoantibodies even in the later stage of the disease. Autoantibodies with very high affinity may form antibody–antigen complexes with optimal potential for tissue injury, whereas low affinity autoantibodies, perhaps with little or no deleterious effects, remain detectable in the circulation. In homeostatic regulation of immune response through idiotype—anti-idiotype network both affinity and concentration of autoantibodies are essential. The product of affinity constant and molar concentration are important for successful regulatory interactions and may be important for clinical utility of autoantibodies [4].
CLINICAL UTILITY IN AUTOIMMUNE DISORDERS Clinical utility of antibody presence depends on clinical sensitivity, clinical specificity and on the a priori probability of disease in the testing subject. Bayes theory combines these three parameters as a useful tool for diagnostic purposes. One might speculate loosely that organ-specific, autoantibody-associated disease, such as Goodpastures’ syndrome and myasthenia gravis, the avidity of the autoantibodies may play a critical role. Contrary to non-organ-specific, immune complexes– mediated disorders, low avidity autoantibodies may be equally pathogenic as high avidity autoantibodies. Several examples can illustrate the ongoing controversies.
Rheumatoid Arthritis Patients with rheumatoid arthritis (RA) produce a high proportion of IgM rheumatoid factors (RF) that react more avidly with human IgG (KD 10/−7M). RF in RA have undergone antigen-induced expansion and affinity maturation, together with
TAKE-HOME MESSAGES
27
recruitment of new classes of B cells. However, low-affinity polyspecific RF can be derived from RA patients as well. In RA, there is evidence for selective enrichment of high affinity clones in the synovium. Although it is unlikely that prolonged production of high-affinity RF is responsible for the joint involvement in RA, they probably exacerbate joint inflammation and promote immune dysregulation [7].
Systemic Lupus Erythematosus Anti–double stranded DNA (dsDNA) antibodies are the hallmark of systemic lupus erythematosus (SLE). In humans, anti-dsDNA antibodies have been reported in past studies to be of high avidity in patients with lupus glomerulonephritis compared to those without. Very high avidity anti-dsDNA was found in the IgG glomerular eluates from autopsy kidneys of patients with severe lupus nephritis. High avidity anti-dsDNA have high predictive value for the development of SLE if present in patients with manifestations compatible with SLE, who fulfill fewer than four ACR criteria. Anti-dsDNA antibodies strongly correlated with nephritis and disease activity, and appear to contribute to disease pathology through high avidity to DNA. Recently, the pathogenic potential of anti-dsDNA antibodies has been shown to depend on qualities other than pure affinity/avidity or specificity for DNA structures. Patients may have nephritis irrespective of whether or not they have high avidity antibodies as detected by Crithidia Luciliae immunofluorescence tests and with cloning vector pUC18 solution phase anti-dsDNA ELISA [8].
Antiphospholipid Syndrome Autoimmune antiphospholipid (aPL) autoantibodies, the laboratory hallmark for antiphospholipid syndrome (APS), require cofactors for binding to phospholipids. The avidity of aPL and its clinical significance have been evaluated in several studies [9]. Antibodies against 2-glycoprotein I (anti-2-GPI) have been studied extensively. In the past, the binding was generally believed to be of low affinity requiring high density of antigen coated on ELISA plates as well as bivalent autoantibodies. Recently, it was demonstrated that patients with APS with or without SLE may have anti-2-GPI of high or low or heterogeneous avidity. High avidity anti-2-GPI appear to be associated with thrombosis in APS, while in pure SLE low avidity anti-2-GPI may prevail. However, the demonstration of a clear pattern of the avidity of anti-2-GPI regarding the time of thrombotic events or pregnancy failure was equivocal. In general, anti-2-GPI avidity did not change substantially during disease course. Further on, recent results suggest that neither high density of anti2-GPI nor high avidity of the antibodies (or Fab fragments) alone were sufficient for the binding of anti-2-GPI to 2-GPI. Some conformational modifications and, consequently, exposed neoepitopes were required for the recognition of 2-GPI by polyclonal anti-2-GPI [10].
TAKE-HOME MESSAGES • Basic physiological and pathological reactions in autoimmune response depend on the discrete binding pattern, avidity, molecular specificity and antibody titres.
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AFFINITY AND AVIDITY OF AUTOANTIBODIES
• Affinity/avidity of autoantibodies has not been sufficiently studied and so far has failed to give decisive answers of their clinical utility. • The aspect of low/high affinity together with specificity of autoantibodies is not fully implemented in the analytical strategies for characterizing autoantibodies. • New approaches and advanced technologies may contribute to understanding important pathogenic implications of low/high affinity/avidity autoantibodies in the future.
REFERENCES 1. Steward MW. Antibody affinity: Immunogenetic aspects and relationship to immune complex disease. J Clin Pathol Suppl (R Coll Pathol) 1979; 13: 120–5. 2. Gharavi AE, Reiber H. Affinity and avidity of autoantibodies. In: Peter JB, Shoenfeld Y (eds) Autoantibodies, Elsevier, Amsterdam 1996, 13–23. 3. Davies C. Introduction to immunoassay principles. In: Wild C (ed.) The Immunoassay Handbook, Elsevier, Amsterdam 2005, 3–40. 4. Cruse JM, Lewis RE. Atlas of Immunology, 2nd edn. CRC Press, London 2004, 227–50. 5. van Regenmortel MHV, Azimzadeh A. Determination of antibody affinity. J Immunoassay 2000; 21(2–3): 211–34. ˇ cnik S, Kveder T, Rozman B. High avidity anti-anti-2-glycoprotein I antibodies in patients with 6. Cuˇ antiphospholipid syndrome. Ann Rheum Dis 2004; 63: 1478–82. 7. Firestein GS. Etiology and pathogenesis of Rheumatoid arthritis. In: Harris ED Jr, et al. (eds), Kelley’s Textbook of Rheumatology, Elsevier Science, USA 2005, 996–1042. 8. Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: The role of analytical diversity. Ann Rheum Dis 2004; 63(4): 386–94. ˇ cnik S, Kveder T, Rozman B. Avidity of anti-beta-2-glycoprotein I antibodies. Autoim9. Boˇziˇc B, Cuˇ munity Reviews 2005; 4: 303–308. ˇ cnik S, Kveder T, Rozman B, Boˇziˇc B. Binding of high-avidity anti-2-glycoprotein I antibodies. 10. Cuˇ Rheumatology (Basel), 2004; 43: 1353–6.
AFFINITY AND AVIDITY OF AUTOANTIBODIES ˇ Cˇ ∗† BORUT BOZI ∗ ˇ CU ˇ CNIK ˇ SASA TANJA KVEDER∗ BLAZˇ ROZMAN∗ ∗
Department of Rheumatology, University Medical Centre, Vodnikova 62, SI-1000 Ljubljana
†
University of Ljubljana, Faculty of Pharmacy, Chair of Clinical Biochemistry, Askerceva 7, SI-1000 Ljubljana
HISTORICAL NOTES AVIDITY AND AFFINITY CLINICAL UTILITY IN AUTOIMMUNE DISORDERS TAKE-HOME MESSAGES REFERENCES
ABSTRACT The basic knowledge and clinical applications of the detection of affinity/avidity of autoantibodies are relatively scarce. Surprisingly, there have been very few recent studies concerning this topic. While theoretical thermodynamics of antigen–antibody interactions are well known, their practical use in detection or determination of autoantibodies is deficient. Both, affinity and specificity for antigens may have important impact for selection of the detection assay. The major problem may be attributed to the lack of suitable measuring methods according to the Law of Mass Action. Selective nonorgan-specific autoimmune disorders have been chosen to demonstrate the currently existing controversies. Nevertheless, some recent information including new methods and sophisticated technologies gives some hope for future usefulness.
HISTORICAL NOTES Antibody affinity has been sporadically studied for almost half a century. In the 1970s, it was established that antibody affinity is a multi-genetically controlled parameter of the immune response and that such control is exerted by genetic mechanisms which are independent of those controlling antibody levels [1]. In the mid-1990s, the role of affinity and avidity in the pathogeneity of autoantibodies Autoantibodies, 2/e Copyright © 2007, Elsevier, B.V. All rights reserved.
21
22
AFFINITY AND AVIDITY OF AUTOANTIBODIES
remained unclear. In the 1990s, there have been surprisingly few studies to answer the question, does the diagnostic impact of antibodies correlate with the discrete binding pattern, avidity, molecular specificity or purely with titres? [2]
AVIDITY AND AFFINITY Definition The binding of antibody with its ligand is the most important feature for the detection of (auto)antibodies and for the determination of analytes by antibodies as reagents. Antibody–antigen intermolecular forces are relatively weak physical forces, which include (a) van der Waals or electrodynamic forces, (b) hydrogen bonding or polar forces, and (c) electrostatic forces. The energy of antibody–antigen binding is called either “affinity” or “avidity”. The specificity of antibodies is also associated with the binding energy. Antibody specificity for a given antigen is determined by its relative affinity. Antibodies with very high affinity to target a specific epitope can bind to similar epitopes with lower affinity. This important aspect is not fully implemented in the analytical strategies for characterizing antibodies. Despite their clear difference, the terms “affinity” and “avidity” are often indiscriminately used. Affinity is the force of binding of one antibody molecule’s paratope with its corresponding epitope on the antigen molecule and can be determined only by a single monovalent Fab fragment. It is the sum of all attractive forces resulting in increased binding strength and repulsive forces resulting in decreased binding strength. Affinity constant K can be described by thermodynamic terms: G = −RT ln K Where G is the standard free enthalpy or change in standard Gibbs’s free energy, T is the temperature, R is the gas constant and K is the equilibrium constant or constant of reaction. Avidity is the binding force between a multivalent antibody and a multivalent antigen. The measured binding energy between antibodies and their relevant, mostly complex antigens reflects the avidity of antibodies. Avidity cannot be described by thermodynamic terms, and is obtained by kinetic measurements. It is commonly indicative of the association constant, depending on the assay procedure employed. Ionic and hydrophobic interactions also increase the avidity energy, which can be tens, hundreds or even thousands of times higher than that of affinity.
Methods of Affinity Calculation Binding between antibodies and antigens is a reversible reaction and takes a few seconds to several hours to achieve its equilibrium. The reaction may be described by the Law of Mass Action with several assumptions. The most important are: the antibody must be monovalent, recognizing only one epitope on the antigen, thus having one affinity; both reagents must be pure (homogeneous regarding to binding sites) and in a solution; and the reaction must be in equilibrium without nonspecific binding to the walls of the reaction vessel. In practice, these assumptions are not completely met. Nevertheless, the Law of Mass Action does provide a useful basis for the theoretical assessment of antibody–antigen interactions [3,4]. The reaction
23
AVIDITY AND AFFINITY
between the monovalent antibody (Ab) and the monovalent antigen/hapten (H) can be written as follows: ka
Ab + H AbH kd
where ka is the association constant and kd is the dissociation constant. The equilibrium constant Keq , also called the “antibody affinity” according to previously mentioned assumptions, represents the concentration ratio of bound to unbound monovalent antibody and hapten: Keq = ka /kd = AbH/AbH The rearrangement using the calculation of free antibody concentration [Ab] from the total concentration of antibody Ab = Abt − AbH designates a linear relationship between the ratio of bound [AbH] to free hapten [H] and the concentration of bound hapten: AbH/H = Keq AbH − Keq Abt Graphic presentation of the equation gives the Scatchard plot, from which two parameters can be obtained, measuring only molar concentrations of the complex (= bound hapten) and molar concentrations of the free hapten (equilibrium constant as the slope, and total concentration of Ab as the intersection on the x-axis) (Figure 4.1). When binding of univalent hapten to multivalent antibody is measured, the Scatchard plot is useful after rearrangement of the Langmuir adsorption isotherm equation: d/n = KH/1 + KH where d represents the fraction of occupied binding sites and n represents antibody valence. The corresponding Scatchard equation is as follows: d/H = nK − dK It can be calculated with extrapolation of experimental data that K is equal to 1/[H], when half the antigen binding sites in bivalent antibody are hapten bound. Determining the most correct extrapolation on the Scatchard graph may be difficult; therefore reciprocally rearranged data are often used: 1/d = 1/nHK + 1/n
Bound/Free
Theoretical background is necessary using experimental data for calculation of the equilibrium constant (affinity). Linear graphs such as Scatchard or Langmuir are obtained only theoretically. In practice, particularly dealing with polyclonal
Slope = –K eq Bound
FIGURE 4.1 Scatchard plot.
Intercept = [Abt]
24
AFFINITY AND AVIDITY OF AUTOANTIBODIES
Bound/Free
Slope = –K eq high affinity
Slope = –K eq low affinity
Bound
Intercept = [Abt]
FIGURE 4.2 The Scatchard plot of mixed affinity antibodies.
patient sera, affinity heterogeneity (and consequently avidity heterogeneity) gives a curved line, making approximations impossible (Figure 4.2). Measurements and calculations of the average association constant may be useful in such situations.
Methods of Avidity/Affinity Measurement Controversies surrounding the role of antibodies’ avidity may be chiefly attributed to the lack of suitable measuring methods. Both affinity and specificity for antigens may have an important impact for the selection of a specific detection assay. There are basically three approaches: (a) solution phase, (b) solid phase and (c) the combination of both [5]. Solution-Phase Assays According to the Law of Mass Action, the very basic affinity determination comprehends methods in which an antigen–antibody interaction, as well as the separation of free molecules from the complex, always occurs in a solution: 1. The equilibrium dialysis to determine the affinity constant for the antibody directed to a small monovalent antigen, by measuring the concentration of free antigen (small antigen molecules separated from a larger antigen– antibody complex). 2. Ultrafiltration or ultracentrifugation to separate free and bound antibody, if antigens are large and multivalent. When sufficient quantities of the reagents are available and the antigen is much larger than the antibody, free antibody separated from the complex can be measured spectrophotometrically. When only small amounts of reagents are available, the amount of free antibody can be measured by enzyme-linked immunosorbent assay (ELISA) after separating free antibody from the complexes by centrifugation. 3. Equilibrium sedimentation to measure the concentration of labelled free antibody at the meniscus of the centrifuge tube when the size of an antigen is similar to that of an antibody molecule. 4. Labelling of antigen molecules to use various procedures to separate free labelled antigen from bound complexes: • precipitation of antibody with 50% ammonium sulphate or 15% polyethylene glycol • specific binding to protein A or to anti-immunoglobulin antibodies.
AVIDITY AND AFFINITY
25
5. Spectrofluorimetric methods (fluorescence quenching, fluorescence polarization, enhanced fluorescence) for antigen–antibody interaction such as that of a bound ligand. Solid-Phase Assays In solid-phase assays, a constant amount of antigen is usually adsorbed to a solid support, but its total concentration remains unknown: 1. ELISA: Varying amounts of an antibody are applied to the microtiter wells and after washing away free antibody, the amount of bound antibody is measured. Because proteins adsorbed to a plastic surface usually undergo some conformational changes it is questionable whether the value of equilibrium dissociation constant thus measured pertains to the interaction with an epitope in its native state. Therefore, it is preferable to capture the antigen on the plastic by pre-adsorbed antibodies and to measure K by a double-antibody sandwich assay format (capture ELISA). The commonly used ELISAs directly measure the interaction of an antibody with an immobilized antigen (or vice versa) and, therefore, do not permit the measurement of the true equilibrium dissociation constant since the antigen and the antibody are in separate phases. 2. Chaotropic ELISA: Increased concentrations of NaCl or other chaotropoic reagents have been shown to be useful in cases of polyclonal sera, where it is not possible to determine the exact association or dissociation constant [6]. Therefore, a relative average dissociation constant (relative avidity) is a good parameter for the evaluation of different polyvalent autoantibodies in patients’ sera. 3. Surface plasmon resonance: Measures macro-molecular interactions, including antigen–antibody interactions, in real time. It detects alterations in the refractive index of the medium surrounding the receptor, immobilized on a solid support, at the moment of ligand binding. It can be employed to determine kinetic parameters (association and dissociation rate constants), equilibrium binding constants, and concentration measurements. Combination of Solution-Phase and Solid-Phase Assays ELISA–equilibrium titration method combines solution-phase and solid-phase assays to benefit from both the equilibrium in a solution (according to the Law of Mass Action) and the simplified measurements of one of the reactants with an assay on a solid support. It measures a free antibody at the state of equilibrium in an antigen–antibody reaction mixture in a solution phase.
Biological Functions Antibody affinity is important: (a) in the activation of B-cells, (b) in the elimination of antigens and (c) in the regulation of the immune response. Measurements of autoantibodies depend on in vitro binding of autoantibodies to relevant antigens with the bias that in vitro conditions are similar to those in vivo. There are several important questions: Is the binding strength sufficient for in vivo binding? Is such binding strong enough to provoke a biological response (e.g. activation of B-cells)? Very low affinity autoantibodies can bind in vitro to their antigen under mild conditions (e.g. low salt concentration), but they do not bind in vivo under
26
AFFINITY AND AVIDITY OF AUTOANTIBODIES
physiological conditions. However, even the binding itself does not guarantee a biological consequence. Low affinity antibodies may not attach strongly enough to their antigen in order to be effective (equilibrium constant is low, the average time of bound molecules is short and the concentration of the complex is low). The efficiency of binding and provoking a biological effect of low affinity antibodies could be significantly increased by polyvalent binding. In the classical model of an immune response against infective agents during the early phase, IgM antibodies are produced, which possess low affinity, but their binding to the antigen is strong due to ten available binding sites (avidity). Due to class switching later on, bivalent IgG are produced compensating lower valency with higher affinity. The continuation of an antigen exposure results in the selection of clones that produce even higher affinity antibodies known as “affinity maturation”. The genes encoding the variable regions of antibodies undergo somatic hypermutations with the selection of B lymphocytes with higher affinity receptors. In autoimmune disorders, differences between primary and secondary immune responses are not recognizable. Most of the anti-self-normal repertoire includes IgM, which has weak affinity for self-antigens and is widely cross-reactive with multiple antigens. IgM can be present for decades without class switching. Pathogenic autoantibodies, usually IgG, have high affinity for self-antigens in the target tissue and restricted specificity. High avidity anti-self-antibodies can be created from many different Ig gene segments, but they tend to derive from a few preformed families of genes, suggesting derivation from an antigen-activated “mother cell”. In contrast to the infection model of the immune response, the question of affinity maturation in autoimmune disorders has not been solved. Patients with chronic autoimmune diseases produce (also) low affinity autoantibodies even in the later stage of the disease. Autoantibodies with very high affinity may form antibody–antigen complexes with optimal potential for tissue injury, whereas low affinity autoantibodies, perhaps with little or no deleterious effects, remain detectable in the circulation. In homeostatic regulation of immune response through idiotype—anti-idiotype network both affinity and concentration of autoantibodies are essential. The product of affinity constant and molar concentration are important for successful regulatory interactions and may be important for clinical utility of autoantibodies [4].
CLINICAL UTILITY IN AUTOIMMUNE DISORDERS Clinical utility of antibody presence depends on clinical sensitivity, clinical specificity and on the a priori probability of disease in the testing subject. Bayes theory combines these three parameters as a useful tool for diagnostic purposes. One might speculate loosely that organ-specific, autoantibody-associated disease, such as Goodpastures’ syndrome and myasthenia gravis, the avidity of the autoantibodies may play a critical role. Contrary to non-organ-specific, immune complexes– mediated disorders, low avidity autoantibodies may be equally pathogenic as high avidity autoantibodies. Several examples can illustrate the ongoing controversies.
Rheumatoid Arthritis Patients with rheumatoid arthritis (RA) produce a high proportion of IgM rheumatoid factors (RF) that react more avidly with human IgG (KD 10/−7M). RF in RA have undergone antigen-induced expansion and affinity maturation, together with
TAKE-HOME MESSAGES
27
recruitment of new classes of B cells. However, low-affinity polyspecific RF can be derived from RA patients as well. In RA, there is evidence for selective enrichment of high affinity clones in the synovium. Although it is unlikely that prolonged production of high-affinity RF is responsible for the joint involvement in RA, they probably exacerbate joint inflammation and promote immune dysregulation [7].
Systemic Lupus Erythematosus Anti–double stranded DNA (dsDNA) antibodies are the hallmark of systemic lupus erythematosus (SLE). In humans, anti-dsDNA antibodies have been reported in past studies to be of high avidity in patients with lupus glomerulonephritis compared to those without. Very high avidity anti-dsDNA was found in the IgG glomerular eluates from autopsy kidneys of patients with severe lupus nephritis. High avidity anti-dsDNA have high predictive value for the development of SLE if present in patients with manifestations compatible with SLE, who fulfill fewer than four ACR criteria. Anti-dsDNA antibodies strongly correlated with nephritis and disease activity, and appear to contribute to disease pathology through high avidity to DNA. Recently, the pathogenic potential of anti-dsDNA antibodies has been shown to depend on qualities other than pure affinity/avidity or specificity for DNA structures. Patients may have nephritis irrespective of whether or not they have high avidity antibodies as detected by Crithidia Luciliae immunofluorescence tests and with cloning vector pUC18 solution phase anti-dsDNA ELISA [8].
Antiphospholipid Syndrome Autoimmune antiphospholipid (aPL) autoantibodies, the laboratory hallmark for antiphospholipid syndrome (APS), require cofactors for binding to phospholipids. The avidity of aPL and its clinical significance have been evaluated in several studies [9]. Antibodies against 2-glycoprotein I (anti-2-GPI) have been studied extensively. In the past, the binding was generally believed to be of low affinity requiring high density of antigen coated on ELISA plates as well as bivalent autoantibodies. Recently, it was demonstrated that patients with APS with or without SLE may have anti-2-GPI of high or low or heterogeneous avidity. High avidity anti-2-GPI appear to be associated with thrombosis in APS, while in pure SLE low avidity anti-2-GPI may prevail. However, the demonstration of a clear pattern of the avidity of anti-2-GPI regarding the time of thrombotic events or pregnancy failure was equivocal. In general, anti-2-GPI avidity did not change substantially during disease course. Further on, recent results suggest that neither high density of anti2-GPI nor high avidity of the antibodies (or Fab fragments) alone were sufficient for the binding of anti-2-GPI to 2-GPI. Some conformational modifications and, consequently, exposed neoepitopes were required for the recognition of 2-GPI by polyclonal anti-2-GPI [10].
TAKE-HOME MESSAGES • Basic physiological and pathological reactions in autoimmune response depend on the discrete binding pattern, avidity, molecular specificity and antibody titres.
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AFFINITY AND AVIDITY OF AUTOANTIBODIES
• Affinity/avidity of autoantibodies has not been sufficiently studied and so far has failed to give decisive answers of their clinical utility. • The aspect of low/high affinity together with specificity of autoantibodies is not fully implemented in the analytical strategies for characterizing autoantibodies. • New approaches and advanced technologies may contribute to understanding important pathogenic implications of low/high affinity/avidity autoantibodies in the future.
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