TY - JOUR
AU - Ismail, Adel, AA
AB - Interference in immunoassays by circulating endogenous antibodies is as old as the technique itself. Its mechanisms have remained poorly studied, however, and its detrimental effects are underestimated despite numerous reports highlighting its serious consequences (1)(2)(3)(4)(5)(6)(7)(8)(9). One reason for this situation is that immunoassays are moving targets. Assay protocols and reagents, even from the same provider and for the same analyte, may change every few years. Even when research on a method is published, it may be perceived as method-dependent and obsolete by the time of publication as the technique may have been altered in the interim. Defining the precise mechanisms of interference by endogenous antibodies has been challenging because of variation in the phenomena produced by the antibodies. Endogenous antibodies may increase readings in some assays but decrease the results in others (4)(5). Interfering antibodies may be identified by nonlinearity in some assays but show perfect linearity on serial dilution in others (5). Interference from some antibodies may be blocked by commercially available “blocking reagents”, but interferences from other antibodies are not (5). Repeat analyses by other methods may identify samples with interfering antibodies, with the comparison assay yielding gross disparity, but in other cases good agreement may be seen between two methods (4)(5). The purpose of this report is to attempt to highlight some of the potential underlying mechanisms of interference from endogenous antibodies and to show that these mechanisms suggest that this interference is and will remain an insidious, variable, and unpredictable problem. This is arguably true for all serum measurements by immunoassay, irrespective of assay design or format, and a radical approach may be needed to solve this long-standing problem. The common “sandwich” immunoassays use two reagent antibodies, a capture antibody and a second antibody coupled to a signal transducer such as a fluorescent or chemiluminescent agent. The capture antibody is immobilized on a solid phase, and the signaling antibody may be added simultaneously in some assays or sequentially after a washing step in others. The washing step does not eliminate interference as it inevitably requires a compromise between removal of undesirable/nonspecific binding agents and affecting the physical binding of the antigen to the antibody. In this report, however, the discussion will focus on two reactions, the binding of the antigen to capture antibody and the second immunologic reaction with the signaling antibody. The following are some relevant basic points that may help in understanding the very complex and unpredictable nature of immunologic binding reactions. The first point is that the concentration of reagent antibodies in an assay cocktail is always fixed/constant, whereas the titer of interfering endogenous antibodies varies from one patient to another. Furthermore, interfering antibodies can have a wide range of avidities to the antigen compared with the reagent antibodies (i.e., higher, comparable, or lower). Sample dilutions, if performed, would further change the relative ratios of reagent and interfering antibodies. This could lead to numerous outcomes ranging from persistently but variably false lower results if the titer of low-avidity interfering antibodies remains excessive even after dilution, to an increase in the measured analyte concentrations when the titer of high-avidity interfering antibodies becomes increasingly insignificant on dilution. The second point is that in the majority of immunoassays, the capture reagent antibody is monoclonal but the signaling antibody may be either monoclonal or polyclonal. Endogenous interfering antibodies are generally polyclonal. The binding affinity of an antigen to a monoclonal antibody (i.e., the association constant, or Ka, which is a ratio of two rates, the association and dissociation rates) tends to be uniform, whereas in an immunologic reaction involving polyclonal antibodies (reagent or interfering antibodies), the Ka or avidity is a mean value of binding constants of each of the antibody populations. Immunologic reactions involving polyclonal antibodies are therefore more variable and complex. The third point is that a high titer of even low-avidity endogenous interfering antibodies could exert a significant adverse effect in the commonly used, fully automated, short-incubation, nonequilibrium immunoassays. A shorter reaction time in a nonequilibrium assay may therefore increase susceptibility to interference. The magnitude of interference is greater the higher the avidity of interfering antibodies. The fourth point is that interfering antibodies may mimic the antigen itself, mimic the reagent antibodies, or mimic both, e.g., in idiotype network immune response (10)(11)(12). The magnitude of immunoassay interference would be dependent on the assay format and the relative affinities/avidities of interfering antibodies and their titer. Some interfering antibodies do not recognize either the antigen or the reagent antibodies as entities for interaction, but they may recognize the “antigen-antibody bound complex” (i.e., metatope) and bind to it (13), thus altering the immunoassay kinetics. The fifth point is that an antigen may have more than one epitope, and an antibody may also have more than one paratope (14)(15)(16). The normal binding reaction may therefore involve more than one binding site on the antigen and/or on the antibody, although in equilibrium immunoassays, preferential binding commonly occurs for the epitope-paratope pair with the greatest Ka. The presence of interfering antibodies could disrupt this binding reaction, and the magnitude of disruption would be dependent on factors such as the titer of interfering antibodies, their avidities and reaction times, and the location(s) of antibody binding site(s). The site(s) of binding of interfering antibodies on the capture antibodies could lead to blocking of the binding to the antigen (partially or completely), giving falsely low results. Alternatively, it could increase the binding with signaling antibodies by binding to a distal site on the capture antibody but reacting with the signaling antibody, giving a falsely higher result with the latter. This form of interference could occur in all immunoassays that use signaling antibodies and is likely to show linearity on dilution. The variations in the nature of interfering antibodies in terms of class (IgG, IgM, or IgA), subclass (e.g., IgG1, -2, or -3), titer and affinities/avidities, and the multiplicity of epitopes/paratopes are only examples that highlight the complexity and unpredictability of binding reactions when potentially interfering antibodies are present. The above discussion is neither prescriptive nor comprehensive but illustrates inherent immunologic interactions that could give rise to numerous scenarios and permutations of interference. Changes in assay protocols and/or use of higher quality reagents, although potentially helpful, are unlikely to eliminate this form of interference. Because it is almost impossible to predict a priori the presence of interference, we have considered the possibility of removing endogenous immunoglobulins before immunoassays. This approach could eradicate almost entirely this potential and insidious source of interference if endogenous immunoglobulin-free serum samples were to be used. We have examined the potential use of polyethylene glycol (PEG), a reagent used and known to remove immunoglobulins before some immunoassays (17). Our basic findings were encouraging (18)(19). Treatment of serum at room temperature with PEG at a final concentration of 125 g/L precipitated IgM and IgG and 70% of IgA over a wide range of their serum concentrations without any adverse effect on the sensitivity, imprecision, or accuracy of the tested follicle-stimulating hormone, prolactin, and α-fetoprotein immunoassays (18), nor was there appreciable coprecipitation of many other tested analytes, such as thyroxine, testosterone, estradiol, and cortisol, but 35–40% of luteinizing hormone and thyroid-stimulating hormone was lost (19). Because the action of PEG is rapid and the chemical is inexpensive and reacts under mild conditions, its wide potential application and optimization could enhance the accuracy of immunoassays for many serum analytes without significant impediment to automation, throughput, or operating costs. However, incorporating such an additional step may necessitate reengineering of immunoassay analyzers to allow online centrifugation, increasing the capital cost. Our approach of using PEG to remove immunoglobulins is only one simple approach, which suggests that improving the accuracy of immunoassay is not an insurmountable task. It warrants further studies, which if successful, could greatly enhance the clinical utility of this valuable technology as an analytical tool. 1 Cole LA, Rinne KM, Shahabi S, Omrani A. False-positive hCG assay results leading to unnecessary surgery and chemotherapy and needless occurrences of diabetes and coma. Clin Chem 1999 ; 45 : 313 -314. 2 Covensky M, Laterza O, Pfeifer JD, Farkas-Stallasi T, Scott MG. An IgM lambda antibody to Escherichia coli produces false positive results in multiple immunometric assays. Clin Chem 2000 ; 46 : 1157 -1161. 3 Bjemer J, Nustad K, Norum LF, Olsen KH, Bomer OP. Immunometric assay interference: incidence and prevention. Clin Chem 2002 ; 48 : 613 -621. 4 Marks V. False positive immunoassay results: a multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries. Clin Chem 2002 ; 48 : 2008 -2016. 5 Ismail AAA, Walker PL, Barth JH, Lewandowski KC, Jones R, Burr WA. Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid stimulating hormone and gonadotropin results. Clin Chem 2002 ; 48 : 2023 -2029. 6 Levinson SS. Antibody multi-specificity in immunoassay interference. Clin Biochem 1992 ; 25 : 77 -87. 7 Kricka LJ. Human anti-animal antibody interference in immunological assays. Clin Chem 1999 ; 45 : 942 -956. 8 Kricka LJ. Interference in immunoassays—still a threat. Clin Chem 2000 ; 46 : 1037 -1038. 9 Ismail AAA, Walker PL, Cawood ML, Barth JH. Interference in immunoassay is an under-estimated problem. Ann Clin Biochem 2002 ; 39 : 366 -373. 10 Pan Y, Yuhasz SC, Amzel LM. Anti-idiotypic antibodies: biological function and structural studies. Exp Biol Med J 1995 ; 9 : 43 -49. 11 Weir DM, Stewart J. Antigens and antigen recognition. Immunology 1997 : 69 Churchill Livingstone Edinburgh. . 12 Fields BA, Goldbaum FA, Ysem X, Poljak RJ, Mariuzza RA. Molecular basis of antigen mimicry by anti-idiotope. Nature 1995 ; 374 : 739 -742. 13 Voss EW, Jr. Significance and properties of the autoanti-metatype immune response. Mol Immunol 1993 ; 30 : 949 -951. 14 Wingren C, Hansson UB, Magnusson CGM, Ohlin M. Antigen-binding sites dominate the surface properties of IgG antibodies. Mol Immunol 1995 ; 32 : 819 -827. 15 Braden BC, Poljak RJ. Structural features of the reactions between antibodies and protein antigens. FASEB J 1995 ; 9 : 9 -16. 16 Kramer A, Keitel T, Winkler K, Stocklein W, Hohne W, Schneider-Mergener J. Molecular basis for the binding promiscuity of an anti-p24 (HIV-1) monoclonal antibody. Cell 1997 ; 91 : 799 -809. 17 Ismail AAA, Walker PL, Fahie-Wilson MN, Jassam N, Barth JH. Prolactin and macroprolactin: a case report oh hyperprolactinaemia highlighting the interpretation of discrepant results. Ann Clin Biochem 2003 ; 40 : 298 -300. 18 Jassam NF, Ismail AAA, Cawood M, Walker PL, Barth JH. Validation of PEG as a pre-analytical step for immunoassay of AFP, FSH and prolactin. Proceedings of the ACB national meeting 2003 : 82 Association of Clinical Biochemists Manchester, UK. London. . 19 Ismail AAA. Interfering antibodies in immunoassay. Proceedings of the ACB national meeting 2003 : 18 -19 Association of Clinical Biochemists Manchester, UK. London. . © 2005 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Radical Approach Is Needed to Eliminate Interference from Endogenous Antibodies in Immunoassays
JF - Clinical Chemistry
DO - 10.1373/clinchem.2004.042523
DA - 2005-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-radical-approach-is-needed-to-eliminate-interference-from-endogenous-kZF03TVwh0
SP - 25
VL - 51
IS - 1
DP - DeepDyve
ER -