Introduction
ELISA, which stands for enzyme-linked immunosorbent assay, has been the gold standard for the quantification and detection of antibodies, peptides, proteins, and other biomolecules for the past 50 years. There are 3 main types of ELISA assays: direct, indirect, and sandwich. All of these approaches rely on a secondary reaction that generates a measurable signal to be detected by either a standard absorbance plate reader, spectrophotometer, fluorometer, or a luminometer. The main advantages of ELISA are high sensitivity and specificity. Take a standard sandwich ELISA assay, for instance. The use of avidin or streptavidin chemistry allows multiple enzymes (e.g. horseradish peroxidase) to be bound to the detection antibody, which leads to signal amplification of the biomolecule of interest. However, a sandwich ELISA has the following disadvantages:
Has long washing and incubation steps – takes hours to days to get results
Need to choose capture and detection antibodies wisely to prevent cross-reactivity
Need for labels or enzymes and substrates for indirect detection
Does not provide kinetic data – it has an endpoint detection
May wash away any low-affinity interactions of interest
Surface plasmon resonance (SPR), on the other hand, is an optical detection technique which is equally as sensitive and specific [1-3] that can address these issues. This blog will highlight the main advantages of SPR technology (Fig. 1) over sandwich ELISA (Fig. 2).
Figure 1. Schematics of an SPR experiment.
Fig. 2. Schematics of an ELISA experiment.
SPR as a Label-Free, Real-Time Technique
SPR is an optical, label-free technique because there is no requirement for a label. Detection is achieved by a change in refractive index at the sensing interface. In a SPR experiment, a plane-polarized, monochromatic incident light shines upon a material of high refractive index (RI) (usually a glass prism) in total internal reflection conditions. If there is a change in refractive index due to analytes binding to to surface-immobilized receptors, the SPR response signal would change. More details can be found in
Furthermore, SPR can be collected in real-time to determine both kinetics and affinity information. ELISA, on the other hand, is an endpoint detection method which can only provide affinity data. Both modes of the SPR experiment involve collecting sensorgram data (SPR response with time) for different concentrations of the analytes. There are two types of SPR setups, which are manual and pump-assisted. The manual injection mode leads only to affinity data by determining the equilibrium dissociation constant (Kᴅ) from the sensorgrams. The pump-assisted mode yields both affinity and kinetic data by determining the rates of association and dissociation to obtain Kᴅ. Please see
for more details.
Faster Results with SPR
In the ELISA protocol, washing, incubation, blocking, signal generation steps all require extra time and manual steps. In contrast, SPR bypasses the need for a secondary reaction step (no second antibody and detection steps are needed) as it directly detects any binding through a change in refractive index, which would change the SPR response. Furthermore, washing and any other sensor preparation steps can done quickly inside the SPR instrument itself. Sample injection and washing steps can be achieved by manual injections or through a pump. Most importantly, results can be obtained by SPR much faster (e.g. in minutes to hours) compared to ELISA (hours to days). Here is a detailed comparison between a colourimetric sandwich ELISA protocol and an SPR protocol (for concentration determination).
Advantages of SPR in Detection of Low-Affinity Interactions
Sometimes low-affinity interactions are more relevant than high-affinity ones for a particular application. One example relates to patient safety. An article by Nechansky reviews the studies that compared ELISA and SPR for the detection of human anti-human antibody (HAHA) response towards therapeutic monoclonal antibodies (mAbs) [4]. One result of HAHA may be induced autoimmunity, whose side effects can be severe life-threatening conditions. Therefore, it is important to monitor the level of immune response in terms of neutralizing antibodies (IgG and IgM) concentrations. In one study by Lofgren et al., the researchers found that SPR identified 4.1% positive patients vs. 0.3% by ELISA [5]. Of all the antibodies tested of various Kᴅ values, ranging from 8.1 x 10-10 M to 1.1 x 10-6 M, the highest affinity antibodies were detected with a higher sensitivity with ELISA. On the other hand, SPR detected the low affinity antibodies with a higher sensitivity [5]. This was likely due to the fact that low affinity antibodies were lost during repeated washing steps in ELISA, whereas SPR was able to collect data in real-time even for low Kᴅ interactions [4].
The low affinity antibodies are indicators of early onset autoimmunity and can evolve into higher affinity antibodies by a process called affinity maturation. To ensure patient safety, SPR should be used as a more sensitive method to detect low affinity antibodies [4] which allow patients to be better monitored by physicians, even if they do not display any clinical symptoms. In short, SPR enables early detection of patients with onset HAHA response.
Summary of Comparison Between ELISA and SPR
Affinité Instruments P4SPR™
Affinité Instruments’ P4SPR is an excellent SPR instrument that can provide high quality, real-time data to suit your research needs. It requires no labels or secondary reactions and reduces a significant amount of precious research time compared to performing an ELISA assay. It can also detect low affinity interactions with a higher sensitivity than ELISA due to real-time monitoring.
We help life science labs and biotech companies to do rapid assay development and characterization. Feel free to reach out to us about the expertise we offer at info@affiniteinstruments.com
Thank you for reading and watch out for additional blogs discussing SPR related topics such as wavelength vs. angle interrogation, planar SPR vs. nanoparticle localized SPR, and multiple channel SPR.
References
[1] Hana Vaisocherová, Vitor M. Faca, Allen D. Taylor, Samir Hanash, Shaoyi Jiang. Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera. Biosens. Bioelectron. 24, 2143-2148 (2009).
[2] Katrina Campbell, Anne-Catherine Huet, Caroline Charlier, Cowan Higgins, Philippe Delahaut, Christopher T. Elliott. Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins. J. Chromatogr. B, 877, 4079–4089 (2009).
[3] Marlen Zschätzsch, Paul Ritter, Anja Henseleit, Klaus Wiehler, Sven Malik, Thomas Bley, Thomas Walther, Elke Boschke. Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy. Eng. Life Sci., 19, 681-690 (2019).
[4] Andreas Nechansky. HAHA – nothing to laugh about. Measuring the immunogenicity (human anti-human antibody response) induced by humanized monoclonal antibodies applying ELISA and SPR technology. J. Pharm. Biomed. Anal., 51, 252-254 (2010).
[5] J.A. Lofgren, S. Dhandapani, J.J. Pennucci, C.M. Abbott, D.T. Mytych, A. Kaliyaperumal, S.J. Swanson, M.C. Mullenix, Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of Panitumumab, J. Immunol., 178, 7467–7472 (2007).