Introduction
From virus detection to pregnancy test, lateral flow assays (LFA) have revolutionized biological testing. It requires only a few drops of biological fluids like saliva, blood or urine to achieve the detection of biomarkers within 30 minutes [1]. Disposable and with a long shelf-life, LFA are the perfect point-of-care test that can be deployed in rural areas, public spaces like schools and airports, and even at home. During the COVID-19 pandemic, LFA proved to be an effective tool against the spread of the disease. However, despite the rapid commercialization of LFA, numerous ones proved to be inaccurate causing harm and confusion. Quick access to high quality LFA against new virus variants or other viral threats remains important to maintaining productive global economic activities. To meet the rising demand, LFA developers need better analytical tools to accelerate a development process that currently takes more than a year and costs hundreds of thousands of dollars.
Most LFA developers rely on Enzyme-Linked Immunosorbent Assay (ELISA) throughout feasibility and development processes. Slow, labour-intensive, and expensive, ELISA provides limited insights as this complex method translates poorly to the more simplistic LFA format. On the other hand, Surface plasmon resonance (SPR) is a type of biosensor format much closer to LFA. Label-free SPR provides direct binding results for any biomolecular interactions in real-time (Fig. 1). The Affinité SPR platform has the distinct advantage of having multichannels that test various assay conditions on a single chip. Besides obtaining affinity information in under 2 h as opposed to hours to a day for ELISA, SPR also provides kinetic information, which is key to better understand the response time and specificity of a developed LFA. Therefore, SPR is an excellent alternative technique for the development of LFAs.
Figure 1. A general setup scheme for SPR experiments where a complex biological sample is used. The upper-left inset depicts the 4-channel configuration of Affinité's SPR sensor chip. Each channel can accommodate a different antibody-antigen pair.
The advantages of using Affinité's portable SPR platform for the development of LFA include:
Adapt antibody immobilization strategy as model for LFA development
Real-time tracking of immobilization steps and binding interactions
Affinité SPR can analyze the same complex biological samples as LFA
Simultaneous SPR screening of antibody-antigen pairs and KD determination required by LFA
Similar reaction time window between Affinité SPR and LFA
More details of how SPR can speed up LFA development can be found below.
1. Immobilization of Antibodies
Immobilization of both types of recognition elements are well established for conventional SPR sensor chips. Proteins can be immobilized using biotin-streptavidin linkages or through NHS-EDC chemistry. Affinité also offers a surface coating called Afficoat™ that serves as a linker to enable carbodiimide conjugation to form covalent amide bonds between proteins and the surface linker’s carboxyl groups. It can also form metal-ligand coordination bonds between his-tagged proteins and nickel-modified surfaces. Immobilizing capture probes via high affinity or covalent linkages on SPR sensor chips rather than by passive adsorption on ELISA well plates is a better model for the development of LFA, as proteins have more degrees of freedom for binding interactions, and a constant flow of running buffer can be added during sample injection by using a pump to study the kinetic interaction. Furthermore, passive adsorption may lead to lower binding interactions by denaturing proteins and deactivating their binding sites.
2. Tracking of Immobilization and Protein Binding in Real-Time
Another advantage of using SPR instead of ELISA to optimize LFA is the real-time tracking of surface modification and immobilization steps and subsequent binding interactions between proteins (Fig. 2). In ELISA, the only evidence that proves that there is binding is the endpoint absorbance values of a true binding interaction. It would be difficult to troubleshoot which step in ELISA has gone wrong or requires optimization.
Figure 2. Top Panel: Full sensorgram of a typical SPR experiment in which the successful immobilization of the linker, antibody, and blocking agent onto an sensor chip in Affinité's P4SPR instrument could be observed in real-time. Bottom panel: Close-up view of the sensorgram between the yellow and red vertical cursor bars. Protein solutions were injected at increasing concentrations and the corresponding increase in SPR shifts could be seen.
3. Use of Complex Samples
LFAs are designed to test complex samples such as urine, saliva, sweat, serum, plasma, and whole blood. An analytical tool that is used to help develop LFA should not be easily encumbered by components in these biological samples and should not require much sample preparation. Conventional SPR is an optical technique that does not rely on a direct optical path through the sample, but rather the light path goes through a prism (Kretschmann configuration) (Fig. 1). The change in refractive index on the surface of the gold film on the prism induces changes in the SPR signal. Therefore, conventional SPR can easily handle complex samples for LFA development. In contrast, the use of complex samples might require some purification steps for certain types of ELISA because the non-analyte components in the sample may increase the overall ELISA signal through its non-specific binding to enzyme-conjugated antibodies.
3. Screening of Antibody-Antigen Pairs
One of the key aspects of LFA development that requires optimization is the antibody-antigen pair. SPR allows simultaneous and real-time screening of antibody-antigen pairs. For example, antibodies can be immobilized using one of the strategies described in section (1) above and each channel of the SPR sensor chip can be dedicated for a different antibody-antigen pair (Fig. 1). Screening is a vital part of LFA development to test any steric hindrance issues and determine the dissociation constant (KD) of the biomolecular interaction, which affects the sensitivity of the assay [1]. Using SPR such as one of Affinité's SPR platforms, the specificity of each antigen-antibody pair and their KD can be calculated by using the system’s software. Doing so will easily lead to the best pair to use for the LFA.
Although the determination of KD can be achieved with ELISA, it is an endpoint detection method and its procedures are more tedious and long. ELISA also tends to miss low affinity interactions as they can be removed easily during the washing steps. SPR, on the other hand, is a real-time technique that can detect low KD interactions.
4. Time Window of Binding Kinetics
SPR is a better analytical tool to evaluate the kinetics of the binding interaction of an antibody-antigen complex. In LFA, the typical reaction time window is between 5-30 min. Therefore, the association rate (kon) is a very critical parameter to determine [2]. SPR has an experimental time frame of 30 min, which is similar to that of LFA [3]. Furthermore, the key feature in LFA is a very fast kon [3]. In contrast, ELISA is dictated by a slow dissociation rate and the hours required by ELISA does not represent the fast binding time that occurs in LFA. Although one can theoretically perform kinetic ELISA (k-ELISA), in which the optical density is monitored with time, it still does not come close to having a constant flow condition. In contrast, kinetic conditions in an SPR experiment can be simply supplied by a pump. Therefore, SPR would be a more useful tool to assess the binding kinetics of a biomolecular interaction.
Conclusions
SPR provides a more suitable testing platform than ELISA to develop LFA rapidly. Capture molecules can be immobilized via different strategies onto an SPR sensor surface rather than being passively adsorbed in ELISA, which would create a more realistic environment for the biomolecular interaction in question. Furthermore, SPR allows the antibody immobilization steps and protein interactions to be monitored closely in real-time. Complex biological samples can be tested on SPR sensor chips without much sample preparation, but it is generally not ideal to use them in most types of ELISA. Quantitative affinity information can be obtained very easily and quickly from an SPR instrument versus using an ELISA. In addition, ELISA may also miss low-affinity interactions. Finally, SPR is a better tool to gather kinetics information on antibody-antigen pairs due to the availability of compatible pumps to provide controlled flow conditions.
References
Katarzyna M. Koczula and Andrea Gallotta. Lateral Flow Assays. Essays in Biochemistry, 2016, 60, 111–120.
Emilie Ernst, Patricia Wolfe, Corrine Stahura, Katie A. Edwards. Technical considerations to development of serological tests for SARS-CoV-2. Talanta. 2021, 224, 121883.
Helen V. Hsieh, Jeffrey L. Dantzler and Bernhard H. Weigl. Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays. MDPI Diagnostics: Diagnostics | Free Full-Text | Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays (mdpi.com)
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