Journal oflmmunological Methods, 150( 19921 111-119
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$115.00
Artifacts and limitations of enzyme immunoassay A m a d e o J. Pesce ~ a n d J. G a b r i e l M i c h a e l b Departments of " Pathology and Laboratory Medicine, and b Mok, cnlar Genetics. Unirersity of Cincinnati Colk,ge of Medicine. Cincbmati, OH 45267-0714, USA
(Accepted 12 February 19921 Key words: Enzyme immunoassay;Artifact; Limitation; Interference; Cross-reactivity;Homogeneous; Surface effect
Introduction Enzyme immunoassays have been so successful that literally thousands of publications dealing with these techniques appeared in the literature over the past 25 years. An industry valued using over one billion dollars per year in reagents has been developed, and hundreds of millions of tests are being done each year on patients evaluating their clinical condition. The advent of the enzyme immunoassay made possible the development of homogeneous immunoassays which have enabled automation in the hospital laboratory with consequent reduction in cost and improved precision. As with any of techniques, limitations and artifacts have been reported. Many of these are common to all types of immunoassay systems regardless of the method of detection, others are specific to enzyme immunoassay. Included among the common assay systems are some unique observations which are not initially obvious, but are based on the fundamental physicochemical properties of the system. The antibody, enzyme, antigen and clinical matrix all have singular properties which occasionally contribute to artifacts and limitations (Nickloff, 1981; Weber et al., 1990).
Correspondence to: A.J. Pesce, Department of Toxicology, University of Cincinnati College of Medicine, Goodman Street, Cincinnati, OH 45267-0714, USA (Tel.: 513-558-5986; Fax: 513-558-41761.
If the antibody-antigen reaction has a binding constant of 10-12 M -~ and it is possible to measure 1% of the reaction with a precision of 1%, then the limit of sensitivity would be 10-12 M × 0.01 × 0.01 or 10 -16 M. However, as Ekins (Jackson and Ekins 1986; Ekins 19911 has argued, if the system cannot distinguish between 0 and 10-~3 M, thcn the limit of sensitivity (minimal detectable difference) is 10-13 M. In addition if one defines sensitivity as molecules measured, then a technique which uses 0.01 ml of a te~t solution is 100 times more sensitive than one which requires 1 ml. Thus, assay sensitivity is a function of analyte concentration, detector sensitivity, assay imprecision, and reaction test volume. Ekins has proposed that the equation which defines maximal sensitivity includes the relationship of sigma/K, where K is the affinity constant of the antibody and sigma the experimental error of measurement. Ekins has developed mathematical arguments which show that the factor limiting sensitivity in 'non-competitive' immunoassay is the non-specific binding whereas for 'competitive' binding the precision or experimental error is limiting. The limiting affinity of the antibody in most test systems appears to be 10-~2 M-~. Many antibodies used in enzyme immunoassays have poorer affinity constants. One anomaly confuses the above theoretical argument. It was presumed that antibody and
112 antigen react in a fashion typical of the law of mass action. As the concentration of the reactants is increased, the reaction moves towards combination and as the system is diluted the reaction tends towards dissociation. However, it ~,as been observed in the heterogeneous systems 9f radio- and enzyme immunoassays, particularly those in which a solid phase is used that increasing concentration results in less reactivity. This is often termed the 'hook' effect (Rodbard et al., 1978; Ryall et al., 1982; Hoffman et al., 1984; Wolf and Brem, 1991). In this circumstance, increasing the concentration of antigen (or antibody) in the system results in less reactivity or binding by antibody. In order to obviate this occurrence, assays should be designed, so that antibody or antigen excess does not occur or that the test is performed at two dilutions to establish if linearity is maintained. Limitations and artifacts due to differences between solid-phase and solution based immunoassays Much of the early quantitative and qualitative measurement of immunoehemical reactions such as the precipitin reaction (Kabat and Mayer, 1961), radial immunodiffusion (Mancini et al., 1964), Farr assay (Wold et al., 1968), light scattering procedures (Gitlin and Edelhoch, 1951), were observed in solution. With the advent of ELISA (Engvall and Perlmann, 1971) more assays have used a solid phase (Kemeny and Chantler, 1988; Kemeny and Chailacombe,1988). Because of surface phenomena this resulted in both quantitative and qualitative differences between the two systems (Table I). Variances in quantitation between
assay techniques often revolve around the fact that steric and diffusion considerations limit the reaction on the surface compared to the same reaction in solution. The conditions, quantitation and limitations of chemical and binding reactions in solution have been worked out throughout the decades, so that there are good estimates of the amount of antibody and antigen involved in the reaction. This is exemplified by quantitative precipitation reaction in which the precipitate of essentially pure antigen and antibody can be measured using protein assays (Kabat and Mayer, 1961; Kawai, 1981). However, in the case of enzyme immunoassays on a solid surface, there is need for an absolute standard, (1) because of the problems of denaturation and changes in the molecular structure when .the molecules are bound to the surface, (2) because of the changes in antibody and antigen valence, and (3) because of steric constraints. Therefore, some method of standardization, usually achieved by isolation and quantitation of the reactive components involved has to be performed before the reaction can be quantified. Valence changes In the case of antibody, steric hindrance may prevent both combining sites of the lgG from reacting with the antigen. The valence of the antibody which is traditionally given or assumed to be 2 may in fact become 1 (Pesce et al., 1983), thus effecting quantification, since values obtained in solution often cannot be directly compared to those obtained from a solid surface. It is not feasible to directly translate the quantitative precipitin antibody measurement where valence
TABLE ! LIMITATIONAND DIFFERENCESBETWEENSOLID-PHASEAND SOLUTION-BASEDIMMUNOASSAYS Type of Change Antigenvalence Antibodyvalence Antigen denaturation Antigen-antibodyaffinity Diffusionrate limitation Polyclonalvs. monoclonal Competingspecificantibodies Nonlinear quantitalion of Farr assay
Cause Solid-phase steric restriction Solid-phase steric restriction Solid-phase surface tension Assay formulation Solid-phase steric restriction Quantitative and epitope changes Limitingsurface antigen-bindingsites Poisson distribution of anti':,odyreaction to antigenwith repeatingdeterminants
113 is 2 and which occurs in a three-dimensional lattice to antibody reaction on a solid surface. Isolation of the antibody by affinity absorption chromatography is often the only viable approach to quantify the reaction. Another example of valence change has been observed when antigen reactivity is examined. In the case of bovine serum albumin, the valence of the molecule in solution is estimated to be 5 while that on the solid phase is 1 (Pesce, 1983). Surface effects When proteins or D N A are absorbed to a plastic surface (Aarden et al., 1976a,b,c; Pesce et ai., 1978), antigen changes caused by surface effects include the conversion of native epitopes to denatured forms. In addition, when simple absorption is used, all of the surface binding sites must be covered by gelatin or other proteins or matrix to be certain that non-specific binding is minimized (Engvall and Perlmann, 1971, 1972; Engvall et al., 1971). Nonionic detergents are also incorporated into many buffers used in solidphase immunoassays (Engvall a~:d Perlmann, 1971, 1972; Engvail et al., 1971). In the case of proteins which have lipid moieties, delipidation may be required before the protein can be appropriately bound to the surface (Stein et al., 1986). The use of delipidation may denature the protein, creating a series of new epitopes which may not be the same as those that are to be measured in patients. Consequently the test antigen may also require a series of delipidation reactions before it can be measured as the same molecule present on the plastic surface. The possibility of a selective reaction with the plastic surface may result in adsorption of certain subclasses of antigen, i.e., denatured versus native or in protein mixtures, the selection of those molecules with preferred adsorption characteristics. Thus, the quantitation and determination of the nature of the bound protein or macromolecule is essential before the reaction can be judged truly quantitative. There can be considerable difference in the apparent affinity of antigen for antibody depending upon which is attached to the surface. For example, when antibody is attached, there is only
a small difference between human albumin and its fragments when measured by an inhibition assay. In contrast, when antigen is placed on the surface, there is a 10,000-fold preference for the undegraded molecule. A quantitative difference is observed when the reaction of monoclonal antibodies is compared to polyclonal ones. Polycional antibodies can react with many epitopes on a complex antigen surface such as a protein, whereas monoclonal antibodies are restricted to one epitope on proteins which do not have repeating sequences. Thus, the quantitation of the monoelonal antibody in a solid phase or a heterogeneous system is also quite different than the same molecule in solution (Pesce et ai., 1983). Another common difference between a solution and a solid surface is that surface sites can be saturated, thus limiting the amount ef either antigen or antibody available for react~ . Unlike the reactions of antigen and antibody in a homogeneous solution in which the antigen and antibody select for each other and then precipitate, the presence of non-reactive material can strongly influence the subsequent reactions. In many cases the surface binds all the proteins in a non-discriminate manner (Pesee et ai., !977). As a consequence, after coating much of the potentially reactive but excess antigen or antibody is not bound to the surface and is removed. Thus, if an antiserum has 1% of the proteins present as specific antibody, only 1% of the material on the surface will be specifically reactive. The remaining 99% of the material will become an inert matrix. When the test reaction is performed, a large amount of non-specific binding relative to the specific binding may be observed because of the 99:1 ratio of non-specific proteins to specific antibody. Diffusion limits the reaction on a solid surface compared to the same reaction in solution (Nygren and Stenberg, 1985; Stenberg and Nygren, 1985, 1988; Stenberg et a1.,1986, 1988; Nygren et al., 1987). The rate of reaction can be enhanced by mixing or by the use of sonic mixing devices (Fig. 1) (Watson and Boraker, 1990)
Common interferences These may be divided into exogenous and endogenous causes (Table II). The exogenous causes
114 are those which arc due to effects before the test reaction occurs, while endogenous causes are due to those inherent in the biological test solution. In the list of exogenous interferences are included the sample collection agents such as anticoagulants (e.g., heparin, EDTA,), the gels used to separate serum from clotted red cells etc. (Weber et al., 1990). These effects can be observed by subjecting control solutions to the test procedure and evaluating recovery. Avoidance of the interferent is the usual course of action. Standards made in pure solution or in a different matrix (animal rather than human) are often not suitable for biological fluids such as plasma. Values obtained by these standards must be compared to those obtained by reference or definitive methods. The solid surface of any immunoadsorbant assay cannot be assumed to be a passive passenger to the test reaction. Changes in the surface occur when proteins or other molecules are bound. It is also possible that when a molecule is absorbed to a plastic surface, the basic physical chemical properties of the plastic surface are altered so that one does not have a specific protein bound to a surface but rather a surface
with unique new physical properties. In particular, the binding of polycationic proteins results in a surface not unlike a D E A E ion exchange resin (Pesce et al., 1986). In this particular circumstance, there will be non-specific binding by any polyanionic protein or other type of molecule. Thus, the binding of particular molecules to a surface may result in the inadvertent creation of a different type of binding property. The absorption and d~sorption of antigen from the surface is a time- and surface-dependent phenomenon. It has been demonstrated that this is time-dependent (Pesce et al., 1977) and the type of surface or treatment of the surface also affects the amount and stability of the bound protein (Urbanek, 1985). In addition, the surface itself may have binding sites which can attract components of the test system thus interfering with quantitation. In the development of any solid-phase immunoassay it is necessary to test and when necessary add reagents to minimize or eliminate non-specific binding. Clinical application has shown that there are a number of unique properties of the test system, particularly the serum or plasma matrix or the
TABLE il INTERFERENCES COMMON TO IMMUNOASSAYSAND METHOD OF ELIMINATION Type of interference Exogenous h~terference Collectingand samplepreparation anticoagulants,samplestorage, drugs, tubes, plasma separatinggels
Resolutionmethod Test for interferenceand not use
Compare to definitiveor referencemethod
Changes in solid phase surfacebindingdue to coatingmolecule
Add reagentsto reduce non-specificbinding
Incompletesaturation of solid-phasebindingsites for antigen, antibodye~
Add blottingreagentsto reduce non-specificbinding
Endogenous interference Hyperlipidemia
Add animal sera tt~ test reagent
latrogenic inducedantibodies Rheumatoid factor
Test for anti-murineantibody,add mouse IgG Use Fab antibodies
Using test panel, not singletest
Dilution, add EDTA, use Fab
Cross-reactingsubstance Competingimmunospecificantibodiesto analyte
More specificantibody,separation Patient history,pre-separationstep
Enhoncement of ELISA by Acoustic Stirrin( ooo
Welhs Not Stirred
1 000 o 0.500 r - 99G
Antibody Concen(rotion (ng/mL)
Fig. 1. Example of rate limiting diffusion in EI.ISA using a bovine lgG system. A Sonata ADA-I acoustic instrument was used to stir the contents of Nunc Maxi-sorb fiat-bottom microweU plates. The acoustic driver oscillates the array of 96 probes linearly at 60 Hz. All incubations were for 30 rain at 21°C. Wells were coated with bovine IgG (1 /~g/ml, 200 t~l/well) in carbonate coating buffer, pH 9.6 with or without acoustic stirring (the unstirred plate was nevertheless incubated with a set of probes in place to stimulate the displacement of the meniscus). After coating, both plates were washed with PBS-Tween 20 using a Denley Microwash IV plate washer. The acoustic probes were washed twice on the Sonata in a container filled with 30 ml PBS. Rabbit anti-bovine lgG, previously diluted, was pipened to appropriate wells and the acoustic probes returned for 30 min, with or without stirring. The plates were then treated with a solution of alkaline phosphatase-labelled goat anti-rabbit lgG (1:4000) in a similar fashion. After the last wash, the plates were deveYo;~ed with p-nitrophenyl phosphate (1 mg/ml in diethanolamine buffer, pH 9.8) for 30 min without mixing and without the probes in place. Chromophore development was terminated with 50/zl 3 N NaOH. The plates were stirred to uniformly distribute the chromophore, then read in an automated microplate reader (EL309, Biotek Instruments, Winooski, VT) using wavelengths of 405 and 650 nm. Courtesy of D. Boraker Chromogen Inc. antigen to be tested which can result in e r r o n e o u s values ( W e b e r et ai., 1990). R e p o r t e d e n d o g e n o u s interferences in immunoassays include hyperlipidemia ( R a s h et al., 1980; Fritz and Bunker 1982), free fatty acids ( M e n d e l et al., 1986; Liewandahl et al., 1987; Csako et ai., 1989), and a variety of antigen or antibody reactions. Most often dilution can reduce or minimize the contribution o f hyperlipidemia. H u m a n antibodies to the animal test antibodies employed in the reaction are defined as heterophilic antibodies (Howanitz, 1982; Highton and Hessian, 1984; Boscato and Stuart, 1986,
1988; Zweig et al., 1988; O'Farelly et al., 1989; Rhys et al., 1989). Their reaction can often be minimized by adding sera from the animal species to the reaction formulation. W h e n mouse monoclonal antibodies are used in an assay system, sera from patients who have developed antibody to mouse immunoglobulin may react with the monoclonal antibodies in the test system. Such patients include those w h o have developed a natural antibody to mouse proteins through such events as rodent bites and t r e a t m e n t with monoclonal antibody (Morton et ai., 1988). in particular, patients t r e a t e d with OKT-3 develop antibodies to mouse lgG (Schroeder et al., 1990). ~r, this case the mouse antibody levels must be monitored before additional therapy or testing is performed. Current formulation of many monoclonal reagents use an additional amount of mouse immunoglobulin to prevent any competitive reaction in the system to minimize this problem. O t h e r workers have p r o p o s e d absorption techniques to minimize the problem ( N e w m a n et al., 1989). R h e u m a t o i d factor is an example of a commonly occurring reactive moiety which can interfere with an enzyme i m m u n o a s s a y system (Maiolini and Masseyeff, 1975). In this particular circumstance, the rheumatoid factor can react with the Fc portion of the antibody antigen complex resulting in ~ddition of m o r e h u m a n IgG to the reaction, making any h u m a n lgG antibody a p p e a r in greater amounts. This artifact can be overcome by designing a system in which the rheumatoid factor cannot react with the antigenantibody complex by using Fab test antibodies or by removing the rheumatoid factor.
Antibody competition Some patients develop antibodies to the test analyte. Reports o f thyroxine-binding thyroglobulin indicate that these interfere in the measurem e n t of these respective antigens (Bhagat et al., 1983; Kastrup et al., 1985) In some diseases, there can be competition b e t w e e n subclasses o f antibodies. This is particularly true w h e n measuring the atopic antibody IgE. Patients who are allergic usually have IgG as well as IgE antibodies, and they c o m p e t e for the same antigen. Thus, determinations of antigenspecific IgE require that some consideration be
given to quantifying the reaction when a significant amount of lgG with the same specificity is present. This can often be done by diluting the patient's serum until the amount of surface antigen presented to the test solution is not limiting, or until the amount of antigen present exceeds the amount of potentially reactive antibody of all classes (Kemeny, 1986). However, even this test procedure is subject to considerable interpretation, most often resulting a linear decrease in color yield. Care must be taken so that this linear range is wiihin the sensitivity of the assay.
Endogenous antigen cross-reactivity Finally, when some molecules such as the drug digoxin are to be measured, one cannot presume that even though the antibody for this reaction does not interact with any of the manufactured drugs which can be present in this patient, that the assay is specific. In the case of digoxin, there is an endogenous-like substance which can interact with digoxin antibodies (Valdes, 1985; Graves, 1986; Longeiich et al., 1988; Sadradeh et al., 1988) and ~or many years, erroneous values have been generated in many clinical laboratories throughout the world. With thc identification that this endogenous substance did exist, it was possible to obviate this endogenous reaction (Skogen et al., 1987). !n addition to naturally occurring molecules which can react like drugs, the drugs themselves generate metabolites, and there is
considerable difficulty with immunoassays to specifically measure the parent compound. A most recent example is the measurement of cyclosporine (Schroeder et al., 1989) Thus, because of the subtle changes which occur as drugs metabolize, the problem of monitoring cross-reactivity may be difficult in clinical situations (Schroeder, 1991).
Enzyme interference and limitations Interferences specific for enzyme immunoassays include exogenous and endogenous ones as well as those specific for the measurement of enzyme activity (Murachi, 1981; Weber et al., 1990) (Table Ill). Exogenous inhibitors include azide in the buffers used to prepare the substrate for the peroxidase reaction. Endogenous interferences also include the presence of enzymes in the test samples which may convert the substrate and give false quantitative values (Pesce, 1981). Moreover, substrate or product which give a positive reaction, and naturally occurring substances such as high concentrations of lipids, hemoglobin or bilirubin may interfere with the quantitation of reactions which use spectral methods. In most cases these interferences can be minimized by using a proper sample blank, dilution, or kinetic enzyme measurement. Interferences or inaccuracies commonly encountered in the measurement of enzyme activity include those typically observed for any enzyme such as (1) temperature,
TABLE ill INTERFERENCES SPECIFIC FOR ENZYME IMMUNOASSAYSAND METHOD OF ELIMINATION Type of interference Exogenous interference Enzyme inhibitor Endogenous interference Endogenous enzyme Endogenous substrate Spectral due to lipids, hemoglobin Drugs which inhibit enzyme activity
Measurement of enzyme acticity Temperature Substrate reaction on solid phase Non-linear kinetics Limited sensitivity Substrate depletion
Resolution method Test reagents for enzyme inhibition,eliminate Proper sample blank Proper sample blank Dilution, kinetic enzyme measurements Dilution? Temperature control Proper mixing Use initial reaction rate Longer reaction time Set maximumproduct formation limits
117 (2) diffusion limiting reaction on a solid phase, (3) non-linear kinetics, (4) small differences in values b e t w e e n calibration samples, particularly w h e n inhibition m e t h o d s are used, (5) substrate depletion. Each of these interferences require specific attention to details to eliminate or minimize them. F o r example, edge effects have been observed on microtiter plates due to the lack of t e m p e r a t u r e control leading to significant variations between inner and o u t e r well values (Oliver, 1981). T h e presence of the solid surface limits the diffusion of substrate and p r o d u c t c o m p a r e d to a homogen e o u s reaction. This limitation can be readily overcome by p r o p e r mixing the substrate w h e n the enzyme is immobilized to a surface (Magnotti et al., 1989). N o n l i n e a r kinetics can often be minimized by using the initial reaction rate (Pesce, 1981). In contrast, limited sensitivity can often be overcome by longer incubation times, or in the ease of competitive reactions by better precision of the assay p r o c e d u r e (Ekins, 1991). Detection of substrate depletion can often be accomplished by setting limits on the a m o u n t of product formed.
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