Clin Biochem, Vol. 25, pp. 77-87, 1992

0009-9120192 $5.00 + .00 Copyright © 1992 The Canadian Society of Clinical Chemists.

Printed in the USA. All rights reserved.

Antibody Multispecificity in Immunoassay Interference STANLEY

S. L E V I N S O N

Department of Pathology, University of Louisville, and Laboratory Service, Department Veterans Administration Affairs Medical Center, Louisville, KY 40202, USA

Recent findings indicate that many endogenous antibodies exhibit multispecificity. These antibodies exhibit a potential for interference with immunoassays. Antibodies that interfere with immunoassays have been called heterophile or heterophilic antibodies. The purpose of this review is: (1) to identify the nature of heterophile antibodies; (2) to delineate the processes that produce them; (3) to examine the mechanisms by which these antibodies cause interference; and (4) to explore how this information can be used to reduce immunoassay interference. In addition to producing specific antibodies, the process of antibody production gives rise to rudimentary antibodies that are polyspecific; e.g., the antigen-combining site has an affinity for antigens of different chemical composition. This process also generates idiotypic antibodies containing cross-reactive idiotopes. These antibodies along with rheumatoid factors, which are themselves polyspecifio and rich in cross-reactive idiotopes, are inherent parts of the process of antibody production, and exhibit multispecificity. Mechanisms by which these antibodies cause immunoassay interference are outlined. These properties of antibodies may have substantial consequence in directing future assays toward greater clinical predictive value.

K E Y W O R D S : antibody diversity; polyspecific antibodies; rheumatoid factor; idiotopes; cross-reactive idiotepes; internal-image idiotopes; immunoregulatory network; heterophile antibodies; immunoassay, interference; irnmunoassay, specificity.

Introduction

of

toimmune disease and immunoregulation, a great deal of n e w information has become available regarding the production of endogenous antibodies which exhibit multispecific binding properties. I These antibodies have a great potential for test interference. The purpose of this review is to explore the meaning of this new information as it relates to interference with immunological testing, and to use concepts derived from this information to propose explanations accounting for this interference. Classically, we think of antibodies as molecules that react with antigens by way of a precise fit. Behring and Kitasato (1) were the first to demonstrate this specificity, over 100 years ago, when they showed that tetanus antitoxin cannot neutralize diphtheria toxin. The classical theory of antibodyantigen interaction suggests that each antibody has a great affinity for a particular antigen, and that each unique antigenic region will bind only complementary antibody. Such a unique region is called the epitope. Although cross-reactions with antigens of very similar structure do occur, the affinity for a cross-reactive substance is much less than for the specific antigen. Such an antibody may be described as monospecific.

ver the past few years, m a n y articles (several of

Owhich will be cited here) have appeared in the clinical chemistry literature discussing interfer-

ences with immunological testing due to endogenous heterophile antibodies. Although these effects have been well described, and some modes of interference have been uncovered, less attention has been directed toward the nature of these antibodies and the immunological mechanisms producing them. As a result of research into mechanisms involved in au-

Correspondence: Stanley S. Levinson, Laboratory Service, Department of Veterans Administration Affairs Medical Center, 800 Zorn Avenue, Louisville, KY 402061466, USA. Manuscript received February 27, 1991; revised July 8, 1991 and November 19, 1991; accepted December 2, 1991.

CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

IMultispecific antibodies are antibodies with more than one binding site.These antibodies may be polyspecific,or they may be idiotypic antibodies. Polyspecific antibodies are those that bind via the antigen-combining site with antigens of different chemical composition, which may have some structural homology (la-5). These may include antibodies with a single antigen-combining sitethat cross-react with antigens that may be similar in structure or charge, or antibodies that have more than one antigencombining site,which can bind to very different types of antigens (also,see Glossary). Idiotypic antibodies are antibodies that contain an antigenic determinant in the Fab region to which other antibodies can bind (anti-idiotypic antibodies). According to Jerne (38), distinguishing between recognizing and being recognized at the molecular level becomes meaningless, therefore, idiotypic antibodies are multispecific and exhibit more than one binding site. This concept will be explored further in the text.

77

LEVINSON

While many antibodies exhibit a high affinity for a specific antigen, even to the exclusion of all others, accumulating evidence indicates that other antibodies show a good deal of nonspecificity, with substantial affinities or binding capabilities for multiple molecules. The premise of this review is that, like antibody specificity, antibody multispecificity is a fundamental product of the process of antibody diversity: the process by which antibodies can be produced against all antigens. Besides giving rise to specific antibodies, this process inherently produces polyspecific antibodies, idiotypic antibodies, and rheumatoid factors (RF), molecules with substantial potential for multispecificity which can interfere with immunological testing. Modes of interference may be due to one or a combination of these factors. Here, the following concepts will be considered: (1) the nature of heterophile antibodies; (2) the processes of antibody diversity and the production of antibodies that exhibit multispecific characteristics; (3) the nature of polyspecific antibodies, idiotypic antibodies, and RF; (4) proposed mechanisms by which these antibodies cause assay interference; and (5) the application of this information to identify and reduce immunoassay interference.

Heterophile antibodies Heterophile antibodies are a group of poorly defined antibodies that react with many molecules. Historically, these antibodies are sheep cell agglutinins associated with mononucleosis. More recently, antibodies that interfere with immunoassays have been classified as heterophile or heterophilic antibodies (6-10). Some antibodies that interfere with immunoassays are monospecific, produced in response to immunizations (i.e., such as anti-animal antibodies in animal handlers and persons treated with animal globulins for immunodiagnostic or therapeutic purposes), while others are characterized by substantial nonspecificity, developed in response to no clear immunogen. By definition, the first group of antibodies should not be called heterophile, because they are welldefined antibodies against specific immunoglobulins that caused the immunization. Although the distinction may not always be apparent in a practical sense, this review focuses on the nature of the poorly defined group of antibodies, which are heterophilic antibodies in the true sense. These antibodies, which are associated with autoimmune and other inflammatory diseases, are also found to a lesser extent in apparently normal people. Heterophilic antibodies may cause interferences by immunoglobulin aggregation, binding the capture antibody to the detection antibody as a result of RF (Fab-Fc) reactions (9,11), or due to idiotypic antibody (Fab-Fab) interactions (8,12). It has been suggested that, under appropriate conditions, aggregation may occur by an Fc-Fc-dependent mechanism (13). They also cause interferences due to 78

polyspecific antibodies binding to capture antigens or neighboring antigens (14-20).

Antibody diversity and the production of multispecific antibodies Antibodies are multichained molecules. Although there are five classes of antibodies, each basic antibody unit consists of four chains: two heavy chains, providing class specificity, and two light chains, K and k. Each whole antibody consists of a constant, carboxyl terminal end (Fc) and variable, amino terminal end (Fab). The amino terminal end of both heavy and light chains contain regions that vary in amino acid sequence in accordance with the B-cell clones from which they are derived. The variable amino terminal contains the antigen combining site, while the Fc portion binds to plasma proteins (such as complement and fibronectin, cell Fc receptors) and contains the heavy-chain class determinants. Much of antibody diversity can be explained on a genetic basis. The heavy-chain variable region is coded for by three separate regions: V, D, and J. The light chain by two genes: V and J. The heavy-chain genes are on chromosome 14, z light-chain on chromosome 2, and k light-chain on chromosome 22. There are about 1000 different V, 10 different D, and four different J heavy-chain genes. There are about 200 V and six J light-chain genes. The chains are synthesized independently and then assembled within the cytoplasm. This process allows for combinations of gene products giving rise to more than 5 × 107 different antibodies with different variable end-antigen combining sites (21). This explains how antibodies can be produced against millions of different antigens. In this way, the genome can direct the synthesis of rudimentary forms of all antibodies. As antigenically driven cells divide, somatic mutations occur at a surprisingly high rate, creating an opportunity for even more antibody diversity (22). B-cells that contain surface receptors which best fit the antigen are encouraged to multiply, while those with a lesser fit are not (22). This provides a means whereby B-cells producing antibodies with greater specificity can proliferate. It follows that antibodies generated early in this process may show a large degree of multispecificity with broad reactivity for many different types of molecules, while those produced later will be selected to show greater specificity. The broad reactivities of polyspecific antibodies may play an important role in normal immunity as antibodies which are useful in the initial defense against multiple bacterial and viral antigens. The cells producing these antibodies may mutate into producers of high-affinity antibodies with increased specificity as the immune response proceeds (23). Although not alone in this respect, autoantibodies exhibit substantial polyspecificity (la-5). The molecular basis for polyspecific binding may be due to the antigen-combining site being much CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

A N T I B O D Y MULTISPECIFICITY IN I M M U N O A S S A Y I N T E R F E R E N C E

larger than that needed to bind to a single epitope, and, therefore, it may potentially be able to bind to other ligands as well (5). It appears that both conformational epitopes and charge interactions may be important in this binding (3,4). The nature of multispecific antibodies POLYSPECIFIC ANTIBODIES

Anti-DNA antibodies These are polyspecific. Although the basic premise in developing monoclonal technology is to produce large amounts of highly specifc antibodies easily, much of the data first identifying polyspecificity in anti-DNA antibodies were obtained from studies with monoclonal antibodies. Mouse monoclonal anti-DNA antibodies were shown to bind to a variety of polynucleotides and phospholipids, including cardiolipin (la). Human monoclonal anti-DNA antibodies were shown to exhibit similar binding properties (2). Monoclonal anti-DNA antibodies can bind to membrane components other than Fc or C3 receptors. Earlier studies suggested that the binding was due to the cross-reaction of antibodies with structurally similar phosphodiester groups in polynucleotides of DNA and phospholipids in membranes. It soon became apparent that monoclonal anti-DNA antibodies bind to a host of other antigens. Polyclonal anti-DNA antibodies bind to proteoglycans (i.e., hyaluronic acid and chondroitin sulfate), suggesting that they recognize repeating negativelycharged units as epitopes (24). Mouse monoclonal and human endogenous anti-DNA antibodies bind with: the cytoskeletal protein vimentin (25); certain polypeptides, called lupus-associated membrane proteins (14,26); trinitrophenyl derivatives (27,28); glycolipids isolated from mycobacterial cell walls

(29); and membranes with a hydrophobic-aromatic structure and a negatively charged structure in close proximity (30). Table i illustrates some polyspecific h u m a n antibodies. Antibodies other than anti-DNA Although anti-DNA antibodies have been the most studied, as indicated in Table 1, other antibody types have also been shown to be polyspecific.These include antibodies against mycobacterium and cardiolipin. M a n y of these antibodies also bind to D N A and, therefore, m a y be classified as anti-DNA antibodies. It is unclear which is the primary antigen for polyspecific antibodies. In this regard, it is interesting that while polynucleotides are strong immunogens, D N A is a poor immunogen, and it is conceivable that it m a y be a bystander antigen (see Glossary). IDIOTYPES O N ANTIBODIES

Private and cross-reactive idiotopes Besides an antigen-combining site, the variable region of an immunoglobulin contains segments that are themselves antigenic. The determinants making up this antigenic region are called idiotopes (Id), collectively referred to as idiotype. Some Id lie within the antigen-combining site of the antibody, while others are outside it. As the process of antibody diversity proceeds, somatic mutation causes some idiotopes to be modified. As a result, two types of Id have been recognized: private Id and crossreactive Id (CRI), the latter also being called comm o n or public Id (see Glossary). Private Id represent a clonally derived, structurally unique variable region, and they are not found on antibodies with different antigen-combining site specificities.CRI are

TABLE 1 Polyspecific Human Antibodies Primary Specificity

Other Specificities

DNA DNA DNA DNA M. tuberculosis (TB)

Proteoglycans Cytoskeletal proteins Human cell-surface protein Cardiolipin ssDNA, dsDNA, Polynucleotides, cardiolipin TNP, myosin, tubulin, actin, albumin dsDNA ssDNA, thyroglobulin, insulin, tetanus toxoid, LPS Mitochondria, ssDNA, cytoskeletal proteins, acetylcholine receptor Actin, DNA, myosin, TNP, tubulin, albumin

DNA Cytoskeletal protein IgG-Fc M. leprae Kidney

Clonality

Class

(Reference No.) Year

Polyclonal Monoclonal Monoclonal Polyclonal Monoclonal

NS IgM IgG IgG IgG

(24) 1984 (25) 1984 (26) 1984 (76) 1984 (29) 1986

Polyclonal

IgM

(77) 1987

Monoclonal Monoclonal

IgM IgM-RF

(78) 1988 (5) 1988

Monoelonal

IgM

(79) 1988

Polyclonal

IgA

(28) 1990

NS, not specified; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; TNP, trinitrophenyl derivatives; LPS, lipopolysaccharide. CLINICALBIOCHEMISTRY,VOLUME25, APRIL1992

79

LEVINSON diversity in variable regions of antibodies, J e r n e reasoned that there should be antibodies whose variable region should have a "lock-and-key" complem e n t a r i t y to the three-dimensional s t r u c t u r e of other immunoglobulins (38). If antibody 1 (Ab 1) is directed against an immunogen, and Ab 2 is directed against the variable region of Ab 1 (Ab 1 is serving as an Id), then Ab 2 will have a three-dimensional structure similar to the original immunogen. The v a r i a b l e r e g i o n s on Ab 2 m a y be t h e t h r e e dimensional image of the original immunogen. Ab 2 may be called an auto-anti-idiotypic antibody (39). Ab 3, with activity against Ab 2 is an auto-anti-antiidiotypic antibody. The structure of Ab 3 is the same as Ab 1, so these antibodies are indistinguishable for practical purposes. This sequence is illustrated in Figure 1. The variable region of an antibody displays several equivalent binding sites: a set of idiotopes and a set of combining sites. J e r n e notes that at this three-dimensional molecular level, the distinction between recognizing and being recognized becomes meaningless so that, in essence, every antibody molecule is multispecific (38). J e r n e and others suggest that the vast majority of endogenous antibodies are not antibodies against foreign substances but antibodies against other antibodies (Id and anti-Id), and that these idiotypic antibodies form a regulatory network within the immune system (38,39). Under normal circumstances this network acts to preserve an immunological equilibrium. When foreign antigens penetrate an or-

found on antibodies that contain different antigencombining sites,but exhibit similar antigenic properties within the idiotype. CRI have been shown to have some structural homology (31-34). This comm o n characteristic m a y indicate derivation from the same germ lines, with somatic mutation causing progression of the antigen-combining site toward diverse specificities(35). As such, like polyspecific antibodies, antibodies exhibiting m a n y CRI m a y represent a more rudimentary form that has not substantially progressed from the original germ line. Although normal antibodies, without self-directed activity, exhibit CRI, autoantibodies, including anti-nuclear, anti-DNA, anti-cardiolipin, and RF, appear to be especially rich in CRI. With the advent of hybridoma technology, it became possible to identify CRI using specific monoclonal antibodies. Shoenfeld et al. (29) demonstrated a substantial degree of idiotypic sharing, not only among monoclonal anti-DNA autoantibodies from the same patient, but also in anti-DNA antibodies from different patients. Table 2 illustrates some human CRI that have been identified on monoclonal and endogenous polyclonal antibodies. The same CRI m a y be found on different antibodies within the same individual, different individuals, and different animals (36,37). Internal image Id Antibodies with activity against idiotopes are called anti-idiotypic antibodies. Given the enormous

TABLE 2 Cross-Reactive Idiotopes

Reference No.

Year

IgM-RF Ig IgM-RF

44 64 35

1984 1985 1986

IgG Ig Ig IgG

68 80 81 82

1987 1987 1987 1987

IgG/IgM/IgA IgG/IgM IgG

83 84 85

1987 1987 1988

IgM-RF Ig

86 87

1989 1989

88 89

1989 1990

4B4

IgM-RF IgM-RF, IgG, IgA Ig

90

1990

T44

IgG

91

1990

Antibodya

Idiotope

Anti-Fc Anti-DNA Anti-Fc

Bla b 16/6b PSL2, PSL3, 17.109 Y2 AMId 16/6 MOR-h2, MOR-h3 8.12 32/15 ADP-1

Anti-Sm Anti-DNA Anti-DNA SLE

Anti-DNA Anti-DNA Anti-DNA/ anti-Poly Anti-Fc Anti-DNA Anti-Fc Anti-Fc Anti-p24 gag of HIV-1 IVIg

IdRQ IdGN2, IdGN1 17.109 6B6.6b

Class

aAll idiotopes were found on endogenous human polyclonal antibodies. bThree of the idiotopes were also found on human monoclonal antibodies. IVIg, Ig for therapeutic use; Sin, Smith antigen. 80

CLINICAL BIOCHEMISTRY,VOLUME 25, APRIL 1992

ANTIBODY MULTISPECIFICITY IN IMMUNOASSAY INTERFERENCE Antigen a

Ab la

Ab la

©)

Ab 2a

(0-Ab

3a(

Ab 3b

Antlgen b

Ab lio

At) Ib

Ab 2b

Figure 1 - - Internal image of idiotypic antibodies. Each antibody serves as an antigen for the next antibody. Ab la, from germ line 1, is the antigen for Ab 2a. Ab 3a is an antibody against Ab 2a, and has exactly the same threedimensional structure as Ab la. It is the internal image of the original antigen (antigen a). Ab lb, from germ line lb, is an antigen for Ab 2b. Ab 3b is an antibody against the antigen Ab 2b, and is the internal image of Ab lb. ganism, the network acts to isolate them (38). There is now substantial endogenous anti-idiotypic antibodies against normal and auto-antibodies immune response (40-43).

and eliminate evidence that can be formed as part of the

RHEUMATOID FACTORS

RF are autoantibodies that bind to multiple antigenic determinants on the Fc portion of IgG. The Fc binding of RF is not polyspecific in a strict sense since it binds to specific epitopes. Nevertheless, the tendency to bind IgG causes self-aggregation of immunoglobulins with different antigen specificities, which m a y facilitate the binding of different antigens by a single entity composed of a macromolecular complex of antibodies (immune complexes). Such a complex could act like a polyspecific antibody. Besides, like other autoantibodies, subsets of RF have themselves been shown to be polyspecific, binding to ssDNA, thyroglobulin, insulin, tetanus toxoid, lipopolysaccharide, and DNA histone (5,44,45). B e c a u s e m o s t l a b o r a t o r y a g g l u t i n a t i o n techniques detect only pentarneric 19S IgM-RF, R F of the IgM class only is normally measured. Studies indicate that most patients who are positive for IgMR F also exhibit R F of the IgG and/or IgA classes,and that, although a majority of patients with seronegative rheumatoid arthritis do not exhibit IgM-RF, they do possess IgG-RF (46,47).The exact physiological role of R F in the normal i m m u n e response is unknown. Yet, it is known that R F is produced in CLINICAL BIOCHEMISTRY,VOLUME 25, APRIL 1992

response to m a n y bacterial and viral infections, and is a part of the normal secondary i m m u n e response (48,49). Recent findings indicate that the synthesis of RF, like that of other antibodies that exhibit multispecificity,m a y be directed by a segment of the V gene that is not highly diversified from the primordial germline, and thus m a y represent a propagation of antibodies expressed in early B-cell ontogenesis (50). There is also evidence that at least some R F bear the internal image of the Fc T-binding protein of herpes simplex virus, streptococci, or other infectious agents, and it has been speculated that R F m a y be generated as anti-idiotype antibodies (51,52). RF m a y be important in the clearance of antigens, including immunoglobulins, because of properties for self-association and aggregation (53,54). Selfassociation of immunoglobulins (via F a b - F c binding) leads to the formation of immune complexes which are cleared more rapidly by white blood cells than monomeric molecules. RF m a y also be important in immune regulation. It has been demonstrated that large RF-containing immune complexes activate B cells to a greater extent than smaller complexes (55). T cells m a y recognize antigens in RF-containing immune complexes after processing by antigen-presenting cells (53). There is evidence that Id m a y function as such an immunoregulatory antigen (56,57), and it has been demonstrated that CRI are frequently associated with polyclonal RF (58,59). To what degree these factors m a y cause alterations in immunoregulation leading to autoimmune disease is unclear. The possibility that CRI are involved in immunoregulation is intriguing.

Mechanisms of interference by multispecific antibodies in immunoassays Assays using cells or cell fractions as the solidphase binder and two-site immunometric assays are extremely sensitive to heterophilic antibody interference (6,7,15-20,60,61). Competitive proteinbinding assays (RIA) m a y also be affected, although to a lesser extent (6). Polyspecific antibodies can interfere with immunoassays for antibodies by competing with a specific antibody for a capture antigen which was designed to bind the specificantibody, or by binding to a component other than the capture antigen (neighboring antigen). The latter mechanism is especially relevant when cellsor cell fractions are used for capturing the antibodies, because polyspecific antibodies have a great affinity for binding to components similar to those constituting membranes and cell structures other than specific receptors. Sources of interference for cell-type assays include both the aggregation properties of immunoglobulins as well as the propensity for polyspecific antibodies to bind to cell membranes. The work of m a n y investigators indicates that binding by polyspecific autoantibodies, especially anti-DNA antibodies, causes false eleva81

LEVINSON tions with the Raji-cellassay for circulating immune complexes (CIC) (14,17-20). It follows that assays using other types of cells such as platelets and red blood cells,or isolated cell fractions, m a y also suffer from this problem. Prince et al. (7) showed that interference with twosite immunometric assays is due to an endogenous h u m a n antibody causing immunoglobulin selfaggregation by binding to both the capture and detection antibodies. Figure 2 illustrates this mechanism. Both polyclonal and monoclonal antibodies are susceptible to the interference. Antibody neutralization has been used to identify this interference, and the addition of nonimmune globulin to assays to reduce it (6,7,54-63). Two-site immunometric tests for hepatitis B antigen in serum use an antibody-neutralization technique to identify false positives due to interfering antibodies (7). After an initial assay for antigen which gives a positive result, sera are preincubated with exogenous antibody against the viral antigen. The assay is then repeated. The exogenous antibody binds to the antigen (if present) and prevents its attachment to the solid-phase antibody when the asINTERFERENCE

SPECIFIC /~4 4

Antigen

Specific Antibody

~iiil.e__ So,la p.ase " ~ ~ i l ~ i i ~',"~:!:,:| ~::i::: ..............................

wash

f ~ %

Labelle¢l Specific Antibody

(

#, %

--m /

i

Figure 2 -- Interference with two sit jmmunoassay by endogenous antibodies. Left shows specific assay without interference. Right shows nouspecific assay with interference by heterophile antibody. Antibody (solid Fab) attached to the rectangle is the capture antibody (specific antibody). Antibody with dashed Fab represents the detection antibody. *Represents label attached to the detection antibody (labelled specific antibody). 82

say is repeated. When viral antigen is present, but the interfering antibody is not, the subsequent assay will show reduced activity, while it will not be reduced ifonly the interfering antibody is present (7). In order to avoid blocking heterophilic antibodies as well as viral antigen, neutralization antibody must be of human or chimpanzee origin, because reactive human heterophilic antibody does not react with antibody from these species. The neutralization mixture must contain high concentrations of antibodies against all viral subtypes. Sera which contain both the viral antigen and heterophilic antibody will give an equivocal result. Data indicate t h a t idiotypic antibodies are a source of immunoassay interference with two-site immunoassays. Heterophilic antibodies have been shown to be directed against epitopes residing on the Fab fragment (8,12). Also, F a b - F a b interactions are implicated by findings showing that heterophilic antibody interference cannot always be removed by incubation with nonimmune sera from the species used to produce the antibody which should remove RF (64,65). Although the exact nature of these F a b Fab interactions are not known, they may be due to human antibodies containing activities against CRI which are shared by other animals (37,66-68). Normally, such interactions would be expected to be weak, but data indicate that stronger binding leading to increased self aggregation of immunoglobulins may occur as the immune system becomes more activated (69). This type of interaction might be expected to facilitate aggregation of immunoglobulins that could interfere with immunoassays, not unlike the aggregation produced by RF, except with F a b Fab binding, rather than that of Fab-Fc. Not only have F a b - F a b interactions been shown to cause immunoassay interferences, but the epitope bound has been shown to be common to several species (8,12). It can be hypothesized that internal image antigens may be responsible for immunoassay interference. For example, a human serum may contain antibodies (Ab 1) against a ubiquitous bacteria or virus that infects animals as well as humans. The same serum m a y also contain an anti-Id (Ab 2) against A b 1. A n animal antibody m a y contain the same internal image Id (Ab 1). If these animal antibodies were used in an immunoassay, aggregation of the capture-antibody to the label-antibody, due to binding by h u m a n antibodies, could occur by h u m a n Ab 2 binding animal Ab 1. R F is another source of interference with two-site immunoassays. Although IgM-RF, like heterophilic antibodies, have been shown to cause anomalous activity in immunoassays (4,5),more often anomalous activity has been associated with IgG (6,7,60-63). Some of the interference m a y be due to IgG-RF, which is not measured in the usual sheep cell or latex R F assays for IgM. Although a role for R F in assay interference has long been known, the multitude of mechanisms by which R F can cause interference has probably been underestimated. Theoretically, R F can not only cause aggregation by binding CLINICAL BIOCHEMISTRY, VOLUME 25, APRIL 1992

A N T I B O D Y MULTISPECIFICITY IN I M M U N O A S S A Y I N T E R F E R E N C E

to Fc portions of other immunoglobulins, but because they are rich in CRI and exhibit a wide range of polyspecificities, they may be able to link Fc binding to F a b - F a b aggregation and to nonspecific competition with capture antigens. Although competitive protein-binding assays are less affected by heterophilic antibodies than two-site binding assays, a sensitive RIA for a-fetoprotein that used a large amount of patient serum (50% of the reaction solution), and a long incubation period gave a false positive when a serum with very high titers of heterophilic antibody was assayed (6). Also, sera with very high titers of heterophilic antibodies caused a reduced recovery with a RIA when doubleantibody techniques were used to separate the free and bound fractions, and increased recovery when a solid-phase system was used (6). The postulated mechanism of positive interference with solid-phase RIA is that antigenic sites are blocked by the binding of the interfering antibody (6). Negative interference with double-antibody techniques is postulated to occur by the production of an antibody complex prior to the addition of the second antibody causing an enhanced precipitation in affected samples (6).

Applications for improved immunoassay specificity The concepts discussed in this review explain a number of difficulties associated with the specificity of immunoassays for measuring antigens. They also help to explain poor correlations between antibody titers and disease activity. There is a continuing evolution of new, sensitive instrumentation. This, along with monoclonal technology, continues to provide more sensitive assays for identification and quantitation of very low concentrations of analytes in the clinical laboratory. As a result, the problems associated with heterophilic antibodies will increasingly plague these tests. Some explanations and solutions to these difficulties with immunoassay specificity are explored below. ANTIGEN TESTING

Nonimmune immunoglobulin from at least one species used to raise the reagent antibodies blocks the interfering antibody (6,7,60-63). Although this technique has been used most commonly to reduce interference, the addition of excess nonimmune species-specific immunoglobulin does not always completely eliminate interference (62,63). Idiotypic F a b - F a b interactions explain this p h e n o m e n o n (8,11,12). The number of equivocal results may be reduced by using Fab fragments as the capture antibody, rather t h a n whole iramunoglobulins. This will eliminate the effects of RF (70), but not F a b Fab interferences. Pretreatment of serum with polyethylene glycol 6000 (130 g/L) has been used to precipitate endogenous immunoglobulins, thereby removing all antibodies including idiotypic antibodies, without precipitating the antigen (71). Rarely, when CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

the antigen also precipitates with polyethylene glycol, heating at 90 °C also eliminates anomalous activity by denaturing the antibodies (71). In the future, development of new ultrasensitive immunological tests for identifying low concentrations of infectious antigens, such as hepatitis C, hepatitis delta, and B. burgdorferi, (the causative agent of Lyme disease) must be anticipated. If potential interfering antibodies are not removed or denatured by pretreatments, an antibedy-neutralization step must be performed for confirmation, as is done for hepatitis B virus and, more recently, for h u m a n immunodeficiency virus. ANTIBODY TESTING

Antibody multispecificity may explain anomalies in assays for immunoglobulins, such as those for anti-receptor antibodies in patients with receptor autoimmune diseases where disease activity correlates poorly with antibody titers (72). Because of polyspecificity, antibodies which are not major contributors to the disease process, with minor affinity for the antigen of interest and stronger affinities for other antigens, may appear concomitantly and in varying concentrations along with the punitive antibodies (see Glossary) which exhibit a stronger affinity for the pertinent antigen. For example, in Graves' disease, the poor correlation between disease activity and thyrotropin-receptor antibody titers has been speculated to be due to the presence of antibodies with varying affinities and concentrations for the pertinent receptor antigens in different stages of the disease (72). These antibodies may include those that block binding as well as those that bind (72). New assays for antibodies include those for antiendothelial cell and anti-neutrophil cytoplasmic antigens, which appear to be important in identifying and monitoring vasculitis (73), and the anti-nuclear antibodies Sm, SS-A/Ro, SS-B/La, RNP, and DNA (74,75). It must be expected here, as was described above for anti-receptor antibodies, that populations of polyspecific antibodies with various binding capacities will complicate the clinical correlations between the test results and the disease. The interference caused by polyspecific antibodies can be reduced by using highly purified receptor antigens to reduce nonspecific binding sites. These are now available due to the emergence of recombinant DNA technologies.

Conclusion Growing evidence indicates that antibodies exhibit the characteristic of multispecificity. The purpose of this review has been to explore this characteristic as a cause for assay interference. Alternatively, one might argue that it is still unclear to what extent these antibodies are responsible for the interferences, because m a n y studies identifying multispecific characteristics in antibodies have been performed with monoclonal antibodies which may exhibit peculiarities as compared to endogenously 83

LEVINSON produced antibodies. Besides, studies identifying multispecific properties in polyclonal antibodies have used extremely sensitive E L I S A and RIA techniques that m a y overestimate the importance of the antibodies. Also, although the structural basis for multispecificity has been postulated, experimental confirmation has not yet been achieved. Therefore, it is possible that multispecificity is due to an aberration in binding caused by factors such as charge and shape, rather than chemical specificity. Nevertheless, in a practical way, it makes little difference whether the binding is chemical in a strict sense or due to hydrogen bonding, hydrophobic bonding, charge interactions, three-dimensional, or other fits.Regardless of the structural basis, should these properties of antibodies ultimately prove to be common, they will largely explain immunoassay interference by endogenous heterophilic immunoglobulins. It is clear that present assays will benefit from modifications that remove or identify such antibodies. It is also evident that, because of the ubiquitous nature of this process, newer techniques measuring very low concentrations of specific antibodies will be more affected. Increased knowledge about the mechanisms of antibody multispecificity is not only important for the future understanding of autoimmune disease and immunoregulation, but is important for designing exact means for improving immunoassay specificity.

Acknowledgement I thank Kathy Levinson for helping to revise this manuscript.

Glossary Ab 1: Antibody directed against an antigen other than Id. Ab 2: Anti-Id. Ab 3: Anti-anti-Id. ANA: Antinuclear antibodies, antibodies against nuclear antigens. Bystander antigen: antigen which did not cause the immune response, but against which the response is directed. CIC: Circulating immune complexes, free immune complexes in plasma. CRI: Cross-reactive Id, Id with structural homology and are common to several different antibodies even though they may have different antigen-combining specificities. D: Gene coding for a part of the variable region of Fab. Epitope: The antigenic determinant to which an antibody binds. Fab: The variable region of an immunoglobulin, conraining the antigen-combining site and Id. Fab-Fab: Aggregation of Fab regions of immunoglobulins. Fab-Fc: Aggregation of two immunoglobulins as a result of R F activity. 84

Fc: The constant region of an i m m u n o g l o b u l i n which gives class specificity. Heterophile antibodies: A group of antibodies exhibiting non.specificity which react with heterogeneous antigens. Id: Idiotope, antigenic determinant of Fab, collectively referred to as idiotype. J: Gene coding for a portion of the variable region of Fab. Polyspecific antibody: An antibody that combines via its antigen-combining site with antigens of different chemical composition, which may have some structural homology. Private Id: Unique Id found on antibodies derived from a single clone, containing the same antigencombining site. Punitive antibody: An antibody t h a t is directed against a tissue leading to tissue destruction. RF: Rheumatoid factor, an antibody that binds to the Fc region of IgG. RIA: Radioimmunoassay, type of competitive protein-binding assay. RNP: Ribonuclear protein, a nuclear antigen. Sixteen/six (16/6): A CRI that correlates with disease activity. SLE: Systemic lupus erythematosus. Sm (Smith antigen): A nuclear antigen. SS-A Ro (Sjogren's syndrome antigen-A/Robert antigen): A nuclear antigen. SS-B/La (Sjogren's syndrome antigen-B): A nuclear antigen. ssDNA: Single-stranded DNA. V: Gene coding for a portion of the variable region of Fab. Variable: Region of Fab containing antigen-combining site and Id. References

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