Overcoming the challenges of ELISA with surface plasmon resonance (SPR)

Abstract

Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) are widely used techniques for biomolecular interaction and quantification analysis. While ELISA has been used in research labs for decades, it has limitations, including endpoint detection, extensive washing steps, and the need for labeled reagents. SPR is a label-free and real-time technique that overcomes many of these challenges by providing kinetic data, higher sensitivity for low-affinity interactions, and faster, automated workflows. Recent studies have demonstrated SPR’s superior performance in immunogenicity assessments, virology, oncology, and drug discovery and development. This white paper explores the key advantages of SPR over ELISA and highlights its potential to enhance biomolecular analysis across various research applications.

1. Understanding ELISA and SPR

Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) are both highly sensitive and specific techniques used for detecting and quantifying biomolecules such as proteins and antibodies (1). Both methods provide robust and consistent results across many applications in the life science research and drug discovery sectors and can be utilized in a complementary manner.

1.1 ELISA

ELISA is a well-known technique used for detection and quantification of biomolecules including proteins, peptides, and antibodies (2). ELISAs utilize enzyme-linked antibodies to generate a measurable signal through colorimetric, fluorescent, or chemiluminescent reactions and requires the use of labeled antibodies and substrates. Workflows using ELISA include steps like coating, incubation, washing, blocking, and signal generation. This means that a single ELISA experiment can take hours or days to complete (2). These workflows also require significant manual labor, optimization, and validation which is extremely time and resource consuming. ELISA provides endpoint detection, which is useful for high-affinity interactions, but may miss low-affinity interactions due to washing away weakly bound proteins. Interactions may not reach steady state during the incubation period which can lead to an underestimation of affinity (Fig 1).

Fig 1. Schematic representation of the underestimation of KD that can occur when measuring at non-steady state. Here, the same dataset was evaluated at different time points to illustrate the importance of measuring at steady state.

1.2 SPR

SPR has recently emerged as a powerful tool for biomolecular analysis with applications spanning numerous areas of research and development in the life sciences and drug discovery (3). SPR is a label-free technique that detects changes in refractive index resulting from binding events on the sensor surface. The washing and sensor preparation steps are completed within the instrument and there’s no need for secondary detection molecules, which reduces the amount of manual labor necessary when compared to ELISA. These attributes allow SPR to provide real-time data in minutes to hours while detecting high and low affinity interactions (4). Additional kinetic data is useful as multiple interactions may have the same affinity but demonstrate different kinetics, which is highly relevant for research applications (Fig 2).

Fig 2. Representation of three interactions with the same affinity but different kinetics. This highlights the importance of kinetic data in addition to affinity measurements for characterizing biological interactions in research applications.

Biacore™ SPR systems are SPR platforms tailored for automated, high-throughput, and high-sensitivity analyses. Biacore™ SPR systems can handle multiple interactions simultaneously through unattended runs, which allow for rapid screening and characterization of multiple biomolecules at once. For example, a binding screen of 384 samples can be completed in only four hours, and further methods can be queued up in the software with a total unattended run time of 72 hours. Biacore™ SPR systems utilize built-in autosamplers and plate handling, which eliminate the need for external robotic integration and software integration often necessary to automate ELISA.

1.3 Comparative Overview of ELISA vs. SPR

Both ELISA and SPR are valuable analytical tools that support the characterization and quantification of biomolecules for different research use cases. However, they have distinct differences that should be considered when selecting the most appropriate tool for your research needs. Table 1 provides an overview of the key similarities and differences between ELISA and SPR.

Table 1 Comparison between ELISA and SPR technology

Property	Biomolecule detection and qualification technique
	ELISA	SPR
Label requirement	Requires labeled antibodies and substrates.	Label-free.
Detection mechanism	Colorimetric, fluorescent, or chemiluminescent reactions based on enzyme-linked antibody reactions.	Detects changes in refractive index due to binding events at the sensor surface.
Experiment complexity	Multiple steps, relies on secondary reactions.	Bypasses secondary reactions.
Experiment length	Hours to days.	Minutes to hours.
Manual labor	Multiple manual wash steps unless automated by external robotics.	72-hour unattended run time, fully integrated automation.
Affinity	Primarily high-affinity interactions.	Both high and low affinity interactions.
Kinetics	Endpoint detection.	Real-time kinetic data.

2. Real world research applications: When SPR outperforms ELISA

2.1 Immunology

2.1.1 Assessing immunogenicity of panitumumab

In a study published in The Journal of Immunology, researchers used both ELISA and Biacore™ SPR systems to detect anti-panitumumab antibodies. The researcher’s goal was to evaluate the immunogenicity of panitumumab, a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody used in colorectal cancer treatment (5).

Here, the ELISA demonstrated a higher sensitivity, capable of detecting 10 ng/mL of anti-panitumumab antibodies, and tolerated a greater excess of the drug (aided by its design with an acid dissociation step to minimize drug interference). The ELISA detected developing antibody responses in a small percentage (0.3%) of subjects. Biacore™ SPR assay had a lower sensitivity (detecting 1 µg/mL of anti-panitumumab) and was less tolerant to the presence of the drug. However, it detected antibody responses in a significantly higher percentage (4.1%) of subjects.

Despite the ELISA's higher sensitivity and drug tolerance, the SPR assay detected more anti-panitumumab antibody-positive subjects. This discrepancy prompted further investigation using anti-panitumumab monoclonal antibodies with varying affinities. The study found that the SPR assay had greater sensitivity for detecting lower-affinity antibodies and was able to detect them even when higher molar ratios of drug to monoclonal antibody were present.

The ELISA's extended incubation and wash steps likely caused low-affinity antibodies to dissociate, leading to under detection. The SPR assay provided real-time analysis without extensive washing and was able to capture these transient interactions. Ultimately, SPR provided a more complete assessment of immunogenicity by detecting a broader range of anti-panitumumab antibodies, including those with lower affinities. This is relevant since low-affinity antibodies still play a role in immune responses and potentially impact drug efficacy or patient safety—making them important research targets.

2.1.2 Assessing immunogenicity of native or pegylated E. coli and Erwinia asparaginases in sera from patients with acute lymphoblastic leukemia

Asparaginases (ASNase) are vital to treat acute lymphoblastic leukemia (ALL). However, they can induce immune responses, forming anti-ASNase antibodies that lead to allergic reactions and impact treatment efficacy. Accurate antibody detection is crucial for research into this immune response (6). This study compared a standard ELISA with a Biacore™ SPR assay for detecting anti-ASNase IgG antibodies in the sera of ALL patients who experienced allergic reactions (7).

While both assays showed excellent linearity and sensitivity, the SPR assay demonstrated a tenfold higher sensitivity. The authors highlighted that the SPR could detect weak affinity antibodies at a tenfold lower concentration compared to ELISA, where the antibody is likely washed away. The ELISA’s washing steps, which are necessary to remove unbound reagents, could inadvertently remove low-affinity antibodies, leading to underestimation of the immune response. The real-time, label-free technique of SPR doesn't require extensive washing and preserves the detection of weaker interactions.

This study showed that the SPR technique is reliable, accurate, and more sensitive than the ELISA method. The ability of SPR to determine the subtype of antibody (IgG) and whether the antibody was neutralizing, which is not possible with ELISA, means that it can provide a more comprehensive assessment of immunogenicity. This information is invaluable in immunology research.

2.2 Virology

2.2.1 Ranking a large panel of anti-dengue virus non-structural 1 antibodies

Another study evaluated the use of different biosensor platforms, including Biacore™ SPR, alongside ELISA to rank and characterize anti-dengue virus non-structural 1 (NS1) antibodies (8). Dengue NS1 protein is an established serological marker for the early detection of dengue infection (9). The goal of this study was to compare these techniques for improving the sensitivity and specificity of dengue immunoassays for diagnostic and research purposes.

A capture ELISA was used to determine the lower limit of detection (LLOD) for selected anti-dengue NS1 antibodies, and SPR was used for kinetic analysis of the interaction between NS1 antigen and the selected anti-dengue NS1 antibodies (Fig 3). In this study, SPR provided detailed kinetic information via association and dissociation rates, that ELISA could not. This allowed researchers to gain insight into how the antibodies interacted with the antigen, rather than merely confirming their interaction.

Fig 3. Comparison of SPR determined percent activity of Abs between Abs of different affinity subset, 10 nM and 100 nM in RWG. The p-value above the dots represents the statistical difference between both subsets of mAbs (8).

This data helped to contextualize the ELISA results and revealed that affinity measurements alone are not sufficient to predict immunoassay performance. The kinetic information from SPR enabled a more nuanced understanding of the antibody-antigen interactions. The ability to measure the kinetic parameters provided a greater understanding of the function of the capture antibodies and showed weaknesses in using simple measures such as LLOD or simple affinity values.

2.2.2 Detecting hepatitis B surface antigen antibodies

Another study, by researchers Tam et al., used a Biacore SPR™ system to detect hepatitis B surface antigen antibodies (anti-HBs) using recombinant hepatitis B surface antigen (HBsAg) derived from Pichia pastoris (10). The performance of the optimized SPR assay was compared to a commercial ELISA for detecting anti-HBs in human serum. Accurate and rapid anti-HB detection is crucial for monitoring vaccine efficacy and identifying individuals with immunity, for example, in population health studies (11, 12).

In this study, the SPR assay demonstrated a wide dynamic range of detection (~0.00098–0.25 mg/L) for anti-HBs. The study found that SPR and ELISA had comparable abilities to detect anti-HBs in human serum samples, indicating that SPR is a viable alternative for antibody detection in real-world samples. In addition to this comparable detection ability, the authors highlighted that the SPR assay, with its multiple screening capacity, has an advantage over ELISA in terms of throughput, which is further improved through its capacity for full automation. This could be particularly useful in large-scale epidemiological studies. The optimized regeneration protocol allowed for multiple screening cycles with the same sensor chip, reducing research costs and increasing efficiency.

2.3 Oncology

2.3.1 Detecting activated leukocyte cell adhesion molecule in human sera from cancer patients and healthy controls

CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is a protein involved in cell adhesion and is reported to contribute to the pathophysiology of various cancers. Accurate detection of CD166/ALCAM levels in serum can aid in cancer research, diagnosis, and monitoring (13, 14). A study published in Biosensors and Bioelectronics compared SPR with ELISA for detecting CD166/ALCAM in human sera from cancer patients and healthy controls (15).

The study found that SPR had comparable sensitivity to ELISA and could detect low levels of CD166/ALCAM in serum samples, which could be useful in early cancer detection and monitoring disease progression. SPR can also provide real-time kinetic data on the binding interactions between CD166/ALCAM and its antibodies, which could help in the research and development of targeted therapies by allowing researchers to understand not just the presence but also the dynamics of the interaction. The authors also highlighted that the label-free nature of SPR is beneficial for reducing assay complexity and its high throughput capacity could be useful for screening blood samples for the discovery of candidate biomarkers.

2.4 Translational research

2.4.1 Detecting multiple sclerosis specific antibodies in sera

Current multiple sclerosis (MS) diagnostic methods, including magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis, are limited in terms of their appropriateness for use in research applications. Developing non-invasive, sensitive assays is required to support translational research in MS using patient samples (16, 17). To address this need, a study investigated the feasibility of using a glycopeptide-based biosensor with SPR to detect MS specific antibodies in patient serum (18). A previously validated ELISA was used as a reference point.

The researchers developed an SPR assay using a Biacore™ system. The glycopeptide, CSF114(Glc), was immobilized onto a sensor chip, and serum samples from healthy donors and MS patients were passed over the chip. The binding of antibodies in the serum to the immobilized glycopeptide was detected in real-time as a change in the SPR signal (Fig 4). The SPR assay required only 3 µL of patient serum per assay and showed within-assay and between-assay coefficients of variation below 10%. The sensor chip also demonstrated stability over multiple cycles. The authors showed that the specificity was similar for both methods, while the sensitivity was increased by 10% with SPR compared to ELISA.

Fig 4. Column scatter and mean values of anti-CSF114(Glc) antibodies in multiple sclerosis (MS, n=61) and blood donors (BD, n=60). The lines indicate the mean value of each group (18).

Compared to ELISA, SPR's advantages in direct measurement and real-time monitoring offered a more detailed and potentially more accurate assessment of antibody-antigen interactions. The SPR assay presents a promising approach for detecting MS specific antibodies, with the potential for further optimization and refinement for research purposes in the future.

2.5 Therapeutic drug and immunogenicity monitoring

2.5.1 Measuring serum concentrations of therapeutic antibodies and anti-drug antibodies

There is an unmet need for therapeutic drug and immunogenicity monitoring (TDIM) to guide therapy with biologics. This is particularly important for infliximab (an anti-TNFα antibody) due to high inter-individual variability in blood levels of the therapeutic, and >70% of patients experiencing a loss of response (19). Currently, ELISA is the most commonly used technique for this purpose due to its relative simplicity, but its multiple incubation and washing steps may affect the detection of low-affinity antibodies and reduce the accuracy and precision of measurements (20). To address this, the authors of a study published in Scientific Reports developed an SPR approach for measuring serum concentrations of infliximab and anti-infliximab antibodies in patients (19). The SPR signal provided immediate (within minutes) and simultaneous measurement of antibody concentrations based on calibration curves (Fig 5).

Fig 5. Effect of IFX on determination of ATI concentration. Aliquots of human serum from healthy volunteers were spiked with commercial ATI to the final concentrations indicated and with IFX or its vehicle. Serum aliquots were diluted, pretreated, and injected over immobilized IFX and IgG (the latter for evaluation of non-specific binding). The points indicate the specific SPR signal due to the binding of flowing ATI to immobilized IFX (19).

Not only does the use of SPR reduce concerns about the potential impact of washing steps on low-affinity antibody detection, but it also increases testing efficiency due to the faster workflow and simultaneous detection of therapeutic and anti-drug antibodies (ADAs). SPR addresses the key limitations of ELISA, driving researchers toward more accurate and efficient TDIM. In the future, it could be applied to other biotherapeutic applications, contributing to research that ultimately leads to better management of patients treated with these expensive medications.

The authors investigated this assay further in a follow-up study, in which they focused on a larger cohort of 76 patients and investigated how the SPR assay compared to ELISA in ADA detection (21). The key finding is that SPR detects ADAs with significantly faster dissociation rates (i.e., lower affinity) that are often missed by ELISA. This is attributed to ELISA's longer incubation and washing steps, which allow these low-affinity ADAs to dissociate while the high-affinity ADAs used for calibration remain bound, leading to inaccurate quantification. By monitoring binding in real-time, SPR mitigates this issue. The authors emphasize that this has important implications because it suggests ELISA, the most common TDIM technique, may be missing ADAs in some patients, potentially affecting treatment decisions. This highlights SPR's value in capturing a more complete picture of the immunogenic response to infliximab, enabling a better understanding of treatment success and failure for research purposes.

3. Key advantages of SPR over ELISA

The studies discussed above provide insights into the advantages of SPR compared to ELISA, which are summarized below.

• Label-free detection: SPR eliminates the need for enzyme-linked antibodies or fluorescent labels, which reduces potential assay interference and simplifies experimental design.
• Real-time kinetic analysis: Unlike ELISA, which provides only endpoint measurements, SPR captures real-time interaction kinetics, which allows researchers to assess binding affinities, association, and dissociation rates. This is critical for detecting both strong and weak interactions for research applications.
• Higher sensitivity for low-affinity interactions: SPR can detect transient or low-affinity interactions that may be missed by ELISA due to extensive washing steps. For example, in an immunogenicity study, SPR detected a significantly higher percentage (4.1%) of anti-drug antibody-positive subjects compared to ELISA (0.3%) due to its ability to capture low-affinity antibodies, highlighting its potential to provide more accurate results (5).
• Faster workflow and automation: SPR integrates washing and sensor preparation steps within the instrument, reducing hands-on time. Experiments that take hours to days with ELISA can be completed in minutes to hours with SPR, making it ideal for high-throughput applications.
• Broader dynamic range and sensitivity: SPR has demonstrated up to tenfold higher sensitivity compared to ELISA in detecting antibodies, such as in the study in ALL patients (7). This increased sensitivity allows for earlier detection and better quantification of biomolecules.
• Multiplexing capability: Advanced SPR systems, such as Biacore™ instruments, allow simultaneous analysis of multiple interactions in a single run. This enhances throughput and efficiency, particularly in large-scale studies like vaccine efficacy monitoring or population health studies (10).
• Reusability of sensor chips: SPR enables multiple analysis cycles using the same sensor chip, significantly reducing assay costs and improving research sustainability compared to single-use ELISA plates.
• Greater biological relevance: Studies have shown that SPR provides a more complete assessment of therapeutic antibody and anti-drug antibody interactions (19, 21). It detects a wider range of immune responses, including lower-affinity antibodies often missed by ELISA, which can impact research supporting treatment decisions and patient safety.

4. Conclusion

SPR is a powerful alternative to ELISA, with significant advantages in terms of sensitivity, real-time kinetic analysis, and automation. By enabling the detection of both high and low affinity interactions without the need for labels, SPR provides a more comprehensive understanding of biomolecular interactions. As demonstrated in numerous studies, its superior performance in immunogenicity assessments, diagnostics, and therapeutic drug monitoring makes it an invaluable tool in modern biomedical research applications.

Ready to replace time and resource consuming ELISAs with fast, accurate, kinetic SPR assays? Contact one of our assay experts today to learn more about how Biacore™ SPR systems could help you optimize your workflows.

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