Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing

Highlights

• Review of cross-reactivity in single-plex and multiplex immunoassays.

• Analysis of why cross-reactivity can disproportionally affects multiplex immunoassays.

• Review of eight approaches and their effectiveness in mitigating cross-reactivity.

• Hints for the development of large scale and high sensitivity multiplex immunoassays.

Immunoassays are indispensable for research and clinical analysis, and following the emergence of the omics paradigm, multiplexing of immunoassays is more needed than ever. Cross-reactivity (CR) in multiplexed immunoassays has been unexpectedly difficult to mitigate, preventing scaling up of multiplexing, limiting assay performance, and resulting in inaccurate and even false results, and wrong conclusions. Here, we review CR and its consequences in single and dual antibody single-plex and multiplex assays. We establish a distinction between sample-driven and reagent-driven CR, and describe how it affects the performance of antibody microarrays. Next, we review and evaluate various platforms aimed at mitigating CR, including SOMAmers and protein fractionation-bead assays, as well as dual Ab methods including (i) conventional multiplex assays, (ii) proximity ligation assays, (iii) immuno-mass spectrometry, (iv) sequential multiplex analyte capture, (v) antibody colocalization microarrays and (vi) force discrimination assays.

Introduction

Cross-reactivity (CR) to non-target proteins is ubiquitous and widespread for antibodies (Abs) [1, 2•], and together with a lack of Abs against many targets, arguably the biggest obstacle in establishing high performance and large scale multiplexed immunoassays. Unless CR is adequately addressed and suppressed, or at least mitigated, it can be devastating to the performance and reliability of immunoassays. CR is not a mainstream scientific area of research, and rather seen as an impediment, and hence only receives little attention compared to that devoted to the developments of new assay technologies and methods, such as antibody microarrays and high sensitivity assays. Ironically, progress in the development and application of novel assay technologies is often stumped by CR.

Immunoassays depend on an affinity binder — a polyclonal or a monoclonal Ab, a recombinant binder, an aptamer, or a receptor — that binds a target protein with high specificity and affinity. The binding is transduced and amplified into a detectable signal, and in the ideal scenario, the intensity of the signal is ratiometric with the concentration of target analyte. Improving immunoassays is predicated on the availability and quality of the affinity binders, and the need for more, better, and cheaper binders is widely recognized [3, 4]. CR of affinity binders can be tested using random peptide arrays for example, but CR to non-homologous amino-acid sequences was found to be widespread [5]. The suppression of CR is further complicated by the large parameter space of possible three dimensional conformations adopted by proteins [6]. The Human Protein Atlas has established a polyclonal Ab production pipeline with rigorous quality control standards, and in a Herculean effort, produced Abs against 15 000 of the ∼20 000 human proteins (Human Protein Atlas; URL: http://www.proteinatlas.org/). Yet, when Schwenk et al. evaluated a preselection of 11,000 affinity-purified, monospecific Abs, only 531 Abs produced a single band on a Western blot, indicating that ∼95% bound to proteins outside of the expected band [7]. Whereas some of the binding might be ascribed to protein isoforms, cleaved proteins, or post-translational modifications, much is likely caused by CR. Collectively, these studies underline that affinity binders often cross-react.

The challenge of scaling up assays and enhancing their sensitivity may thus be formulated as follows: how to produce a ‘perfect assay system’ while using ‘imperfect building blocks’, that is, cross-reacting affinity binders? Or, how should a multiplexed immunoassay with ultrahigh sensitivity while efficiently suppressing CR be designed? What is the trade-off, i.e. [2•, 8], how does multiplexing and CR affect assay performance and can it be predicted? To provide some answers to these questions, we first review and define CR in single Ab and dual Ab single-plex and multiplex assays. We review the strategies developed to mitigate CR over the last decade in multiplexed assays, and assess their robustness, scalability, and potential for ultrasensitive detection. Finally, we provide some suggestion for future studies and development.

Direct detection of binding can be accomplished by labeling the entire sample with biotin and incubating it with fluorescently labeled streptavidin (Figure 1a) or alternatively, by using label-free detection technologies that record a change in refractive index, mass, or conductivity at the surface [9]. However, signal arising from CR (and non-specific adsorption) is typically indistinguishable from the one arising from specific binding (Figure 1b) thus limiting the performance of this assay format. To clarify the language, we define this type of CR as sample-driven CR.

CR and non-specific binding has been studied extensively for single-plex assays, and whereas it may not be possible to eliminate it completely, it is fairly well understood and managed [10]. Dual Ab assays, also called sandwich assays, and often simply referred to as enzyme linked immunosorbent assays (ELISAs) embody an effective strategy to mitigate CR by binding two distinct epitopes on the same protein: a capture Ab (cAb) immobilizes and concentrates the analyte, while the simultaneous binding of a labeled detection Ab (dAb) transduces the binding into a detectable signal (Figure 1c). The strength of the sandwich assay stems from its tolerance to CR because a single CR (or non-specific binding) does not result in a detectable (false positive) signal (Figure 1d). Indeed, two simultaneous spurious binding events are required to lead to detectable CR, but the odds for it to occur are very low. This point highlights the importance to distinguish between CR that leads to false positive signals and CR that does not lead to a signal, and which can be tolerated, but should not be ignored. In all cases, CR can be further minimized by seeking affinity binders with high specificity and affinity, and by developing assays protocols that minimize CR. For example, binders with low dissociation constants (and low off-binding rates) have long been used, because they can withstand harsh wash steps in ELISA and other assays, while weakly bound and cross-reacting species are washed off [4].

Recently, the limit of detection (LOD) for sandwich assays was extended to aM and even zM [11, 12, 13•], even outperforming nucleic acid tests with PCR amplification [14]. High performance Abs and high signal amplification, which are sometimes combined with digital assay formats that tally single binding events, are the key to these advances. Sandwich assays have also been adopted in multiplexed immunoassays — comprising both antibody microarrays on chips and dispersed bead-based assays (also called bead arrays), but as described below, new types of CR arise as a consequence of adding the dAbs as a mixture.