ELISA has been the gold standard for 50 years. Here's where SPR outperforms it — real-time kinetics, label-free detection, and superior sensitivity for low-affinity interactions.
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. The main advantages of ELISA are high sensitivity and specificity. However, a sandwich ELISA has notable disadvantages: it is laborious, time-consuming, requires expensive reagents and labeled antibodies, and provides only endpoint data with no kinetic information.
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 an SPR experiment, a plane-polarized, monochromatic incident light shines upon a material of high refractive index in total internal reflection conditions. If there is a change in refractive index due to analytes binding to surface-immobilized receptors, the SPR response signal changes.
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. The manual injection mode in SPR leads to affinity data by determining the equilibrium dissociation constant (KD) from the sensorgrams. The pump-assisted mode yields both affinity and kinetic data by determining the rates of association and dissociation.
Faster Results with SPR
In the ELISA protocol, washing, incubation, blocking, and 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. Furthermore, washing and other sensor preparation steps can be done quickly inside the SPR instrument itself. Most importantly, results can be obtained by SPR much faster (e.g. in minutes to hours) compared to ELISA (hours to days).
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). One result of HAHA may be induced autoimmunity, whose side effects can be severe life-threatening conditions.
In one study by Lofgren et al., the researchers found that SPR identified 4.1% positive patients vs. 0.3% by ELISA. The highest affinity antibodies were detected with a higher sensitivity with ELISA, while SPR detected the low affinity antibodies with a higher sensitivity. 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 KD interactions.
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, allowing patients to be better monitored by physicians even if they do not display any clinical symptoms.
Affinité Instruments P4SPR™
Affinité's 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.