Endogenous Heterophile and Human anti-animal Antibodies Mode of Action with Respect to ELISA Interference

Defining characteristics of heterophilic antibodies

Heterophilic antibodies (HA) were formally defined as broad specificity, low affinity, IgM isotype antibodies that originate as a result of the maturation of the humoral branch of the immune system. Their appearance was not tied to an immunization event with external antigens. There was also a need to correlate the formation of slightly higher affinity rheumatoid factor (RF) IgM antibodies with this same early immune system maturation process. It wasn’t until the mid-80s that researchers started to take notice of the high frequency of false positive serum samples in sandwich ELISA formats [1]. This positive and negative interference was attributed to the presence of low affinity, polyspecific, natural heterophilic antibodies in serum and plasma samples [2, 3]. As more attention was directed to this issue and with advances in immuno-detection technology, it became apparent that IgG was the predominant heterophile antibody isotype [4]. This indicated that enough somatic mutation activity must have occurred to justify a class/isotype change-over at some time point to IgG.

Early in 2000, HA interference discrimination techniques advanced to a point where a new definition of HA was constructed. HA were split out into 2 categories, natural antibodies, and autoimmune antibodies [5]. The natural antibody category includes a natural poly-specific group, a natural Idiotypic group, and a natural RF group designation. The autoimmune antibody classification includes an autoimmune polyspecific group as well as an autoimmune RF and idiotype group designation. It is understood that a series of somatic mutations in B-cells leads to the conversion of low affinity IgM class/isotype expression into higher affinity multi-class/isotype B-cell outputs [6]. Suffice to say that the more somatic mutations that occur as a result of contact with self-antigen, the less polyspecific and higher affinity the antibody expression becomes. Autoimmune antibodies occur as the result of a very low number of somatic mutations. While still polyspecific in their targeting specificity, they possess higher binding affinity properties albeit still falling into the low affinity category. This type of autoimmune antibody is responsible for RF and anti-DNA chronic disease pathologies. It is reasonable to conclude that the natural antibody category of heterophilic antibodies are responsible for a majority of the heterophilic interference in two site (sandwich format) ELISA. Much of this is directed at anti-idiotype targets.

Characteristics defining HAMA and HAAA group antibodies

This section seeks to address both the common and differing features of this category of human assay interference antibodies. Improper nomenclature defining what constitutes a HA has made the process of differentiating heterophilic vs human anti-mouse antibody (HAMA) vs human anti-animal antibody (HAAA) a much more challenging task. A cursory glance at antibody interference discussions in the literature would seem to classify all types of antibody-based immunoassay interference as falling under the heterophilic antibody (HA) umbrella. Further examination however, indicates there are clear differences in the origin and composition of each of these three antibody driven assay interference sources.

As detailed above, HAs have the properties of being 1) poly-specific, 2) low affinity, 3) result from a clonal expansion of germ-line B-cells triggered by exposure to a self-antigen environment. These types of antibodies did not arise from exposure to an external source antigen trigger. Human anti-animal antibodies (HAAA) which are often sub-classified as human anti-mouse antibody (HAMA) when the interference is known to originate from prior exposure to mouse IgG, should not be classified or categorized within the heterophilic antibody grouping.

Human serum antibodies falling within the HAAA or HAMA classification grouping exhibit multiple features not associated with HA category antibodies. Some prominent differentiating features of the HAAA or HAMA include, their presence at much higher serum titers, greater target specificity, and greatly amplified binding affinity characteristics. These characteristics could only arise following a significant number of somatic mutation events and are further supported by their multi-isotype IgG, IgA, IgM, and even IgE isotypes expression [7]. Due to their higher serum titer levels and binding kinetics, patient samples containing HAAA and HAMA antibodies clearly present a much greater risk-level for sandwich ELISA interference.

Basis for heterophile/HAAA/HAMA sourced ELISA interference

Heterophile driven immunoassay interference problems are the inevitable outcome of the humoral immune system maturation processes. Natural germ line B-cell antibodies possess the potential to produce over 107 antibody variable region binding sites [6]. Initially their antibody output product is much more limited with respect to variable region amino acid diversity. This binding site limitation is overcome by the ability of these primal antibodies to concurrently bind multiple antigen epitopes per antigen encounter. It has also been established by X-ray diffraction techniques that these early antibodies are capable of configurational adjustment to accommodate a much broader pool of antigen binding opportunities [8]. There seems to be a consensus opinion that these early, weak binding, poly-specific antibodies be designated as natural antibodies whose clonal expansion is antibody-idiotype (antigen) driven. Ultimately this natural antibody population forms the basis for most of the HA cross reactivity activity leading to the topic of this tech review update. It should also be noted that the autoimmune antibody category of natural antibodies, those antibodies associated with autoimmune and chronic disease pathologies, are also activated by self-antigen. These differ from the natural antibodies by the fact that they come about after a slightly larger number of somatic mutations. As such, they exhibit slightly higher binding affinities and a more limited degree of polyspecificity. This antibody population can also contribute to the overall impact of HA facilitated immunoassay interference.

Human anti-animal (immunoglobulin) antibody (HAAA) mediated assay interference represents a more troubling problem. These antibodies, to also include the HAMA designation of HAAA, are the product of a bona fide foreign antigen stimulation event. They occur at high titer levels and for all practical purposes, represent an internal high affinity polyclonal antibody population. Although relatively specific for their animal Ig targets, they can and will cross react quite nicely with other animal source Ig isotypes [9]. Their ability to cause cross-reactive 2 point immunoassay interference within ELISA sandwich formats is greatly elevated relative to HA associated interference events.

HA and HAAA/HAMA initiated ELISA interference problems

Antibody sandwich ELISA along with other 2-point immunoassays are the most vulnerable to HA and HAAA/HAMA interference issues. As a rule, a vast majority of sample and assay diluents contain some form of serum or serum plus EDTA. Due to the low binding affinity and titer of the natural antibodies, the mere presence of these heterogeneous proteins in the sample/assay diluents seems to make HA interference a non-factor. In contrast, the presence of HAAA and HAMA in certain respective serum samples represent a significant problem. Having the characteristics of high binding affinity and high serum titer levels, addressing assay interference issues originating from HAAA/HAMA will require the incorporation of a much more heterogeneous serum diluent additive formulation.

Although sandwich ELISAs are subject to multiple sources of assay interference, interference resulting from the presence of HA and HAAA/HAMA antibodies in serum samples is mostly confined to their binding to the functional assay components. This can involve antibody binding to capture antibodies, “up” antibodies, and signal antibody if the “up” antibody is not directly labeled with an enzyme or fluorophore (ex HRP or Alexa Fluor, respectively). False positive interference events can occur when HA or HAAA or HAMA present in serum/plasma samples, bind to solid phase capture antibody and subsequently binds to the “up” or sandwich antibody or sandwich antibody-HRP. Thus, in the absence of target analyte in the serum/plasma sample, a false positive signal is still generated. In a different scenario, the problematic serum antibodies bind to solid phase capture antibody. This can result in a steric interference event where the ability of the capture antibody to bind analyte is diminished or possibly eliminated. False positives can also occur in more elaborate high throughput assay formats. These assays may use goat, rabbit, or other non-mouse sourced polyclonal as the capture antibody in conjunction with a mouse mono-signal antibody. High throughput potential is enabled by the ability to simultaneously combine antigen capture steps in serum samples with the “up” mono-signal antibody incubation step. This assay strategy combines a more streamlined assay protocol with greatly reduced turn-around times. However, when in the presence of serum samples containing endogenous HAMA type antibodies, there is an elevated probability of a false negative interference outcome. In this case, the serum-based HAMA targets the mono-HRP or “up”/signal generation antibody. This can lead to steric inhibition of the mono-HRP analyte binding process with the solid phase captured-analyte target. When an assay includes a mouse monoclonal capture antibody combined with a goat polyclonal “up”/signal antibody, the presence of endogenous HAAA antibodies can result in an artificially reduced or completely inhibited signal output for those HAAA-containing samples.

In summary, bad things happen when patient sample antibodies participate in the workings of the immunoassay format.

Human heterophile/HAAA/HAMA antibody frequency

Publications containing global projections of the frequency of HAAA/HAMA in the healthy human population appear to be a rare commodity. A scattering of publications provide HAAA/HAMA percentage frequencies within their small-number sampling population. Unfortunately, these HAAA/ HAMA immunoassay interference topics have not merited much in the way of extensive frequency survey analysis. In contrast, the probability of encountering HA interference is essentially 100%. If one subscribes to the premise that natural anti-idiotype antibodies are the net result of humoral immune system maturation processes, every human serum sample should hold the potential for HA interference [10].

HA HAAA HAMA ELISA Interference Summary

  • Heterophilic antibodies (HA) should never include the human anti-mouse antibody (HAMA) specificity or human anti-animal antibody (HAAA) specificity categories of endogenous cross-reactive ELISA interference agents.
  • Heterophilic antibodies are ubiquitous in terms of their presence in the human population.
  • HAAA that includes the HAMA heterogeneous specificity category of ELISA interference agents represent a true anamnestic humoral response from exposure to non-self-classification antigens. Usually the non-self HAAA/HAMA inducing agents consist of mouse or bovine ɣ-globulin (BGG) as well as BSA.
  • The HAAA/HAMA interfering antibodies group, though considerably more specific for their stimulating antigen, still retain enough polyspecific tendencies to facilitate elevated cross-reactivity with different animal origin ɣ-globulin proteins. Unlike the HA category of endogenous antibodies, HAAA/HAMA possess high binding affinity properties and are expressed at high titer levels. Because of these very different attributes as compared to the HA category of interfering agents, these HAAA category antibodies represent a serious ELISA interference problem.
  • Heterophilic antibodies are polyspecific low affinity antibodies arising from maturation of germ-line B-cell antibody production efforts. They arise from the natural stimulation of B-cells to idiotype epitope targets. No external non-self-antigen exposure required. Because of their low epitope binding affinity constants, they are non-factors in most immunoassay formats where the sample or assay diluents contain a mixture of animal proteins.
  • When operating within a permissive assay matrix environment, HA and the HAAA/HAMA category of interfering antibody agents cross-link assay performance-critical components. In the presence of sample origin anti-mouse IgG, an ELISA plate adsorbed monoclonal IgG can be crosslinked to mouse monoclonal IgG-HRP. This creates false positive signal in the absence of any assay targeting analyte. In a slightly different scenario where the “up” antibody is a goat poly-HRP and the ELISA plate adsorbed capture IgG is of mouse origin, the presence of HAMA in the serum or plasma sample could lead to steric inhibition of the ELISA plate well adsorbed capture IgG. Steric inhibition of mouse capture IgG could lead to significantly reduced or even negative plate-well signal output. In a different ELISA interference scenario, the presence of low percentage quantities of BGG in the monoclonal capture coating IgG can lead to false positive outcomes. Should the human serum/plasma samples instead contain HAAA with a specificity for BGG, should the HRP labeled “up” antibody be of goat origin, cross-linking of contaminating BGG on plate well surfaces can crosslink the goat IgG-HRP signal antibody to the plate well surface. Why, because there can occur, varying levels of cross-reactive epitopes on the goat IgG molecule. End result, more false positive type interference.



  1. Boscato, L.M. and M.C. Stuart, Heterophilic antibodies: a problem for all immunoassays. Clin Chem, 1988. 34(1): p. 27-33.
  2. Levinson, S.S., Antibody multispecificity in immunoassay interference. Clin Biochem, 1992. 25(2): p. 77-87.
  3. Bouvet, J.P. and G. Dighiero, From natural polyreactive autoantibodies to a la carte monoreactive antibodies to infectious agents: is it a small world after all? Infect Immun, 1998. 66(1): p. 1-4.
  4. Avrameas, S. and T. Ternynck, Natural autoantibodies: the other side of the immune system. Res Immunol, 1995. 146(4-5): p. 235-48.
  5. Levinson, S.S. and J.J. Miller, Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clin Chim Acta, 2002. 325(1-2): p. 1-15.
  6. Tonegawa, S., Somatic generation of antibody diversity. Nature, 1983. 302(5909): p. 575-81.
  7. Kricka, L.J., Human anti-animal antibody interferences in immunological assays. Clin Chem, 1999. 45(7): p. 942-56.
  8. Wedemayer, G.J., et al., Structural insights into the evolution of an antibody combining site. Science, 1997. 276(5319): p. 1665-9.
  9. Andersen, D.C., et al., High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies. J Immunoassay Immunochem, 2004. 25(1): p. 17-30.
  10. Hennig, C., L. Rink, and H. Kirchner, Evidence for presence of IgG4 anti-immunoglobulin autoantibodies in all human beings. Lancet, 2000. 355(9215): p. 1617-8.