Accelerating antibody pair selection for rapid LFIA development through SPRi

Introduction

Response Biomedical is a company specialized in the development and manufacture of point-of-care diagnostic tests, primarily for emergency and intensive care settings. Leveraging the RAMP® (Rapid Analyte Measurement Platform) technology, their products use fluorescence to detect specific analytes in clinical samples, providing critical diagnostic results in minutes rather than hours or days. Their cardiovascular product line features the Troponin I test, a crucial tool for detecting elevated levels of Troponin I in the bloodstream indicative of recent heart attacks. The company’s R&D pipeline includes new assays and enhancements to current cardiovascular assays on the RAMP® platform.

In Lateral Flow ImmunoAssays (LFIA) in sandwich format, selecting two antibodies that exhibit high affinity for distinct epitopes on the target is a primary consideration. Equally crucial is ensuring that these antibodies also possess appropriate kinetic properties. The antibody used for bioconjugation, immobilized on nanoparticles, and serving as the 'detection antibody,' must form a stable complex with the target due to its prolonged contact during migration along the strip. This requirement underscores the need for a low off-rate, ensuring that once the target is captured, it remains bound. Conversely, the second antibody, immobilized on the test line as the sandwich capture antibody, must rapidly associate with the target-antibody complex. This rapid association is critical as the bioconjugate-target complexes do not linger on the test line, necessitating a swift and effective capture to ensure accurate detection. Surface Plasmon Resonance imaging (SPRi) technology plays a pivotal role here, enabling comprehensive measurements of both affinity and kinetic constants bypassing the lengthy traditional selection process of methods like ELISA, which primarily assess complex stability and often require subsequent LFIA testing for kinetic analysis.

Traditionally, pharmaceutical companies utilize SPR/SPRi technologies in the drug discovery and lead optimization phases for the effective screening and characterization of molecular entities. At Kimialys, our solid expertise in both LFIAs and SPR/SPRi sets us apart, enabling us to bring the benefits of multiplexing offered by SPRi to LFIAs, a true innovation in the field of diagnostics. Multiplexing provides the efficiency of simultaneously screening numerous antibodies and characterizing their kinetic properties under varying experimental conditions; the data generated by SPRi can be directly applied to LFIAs.

Let's elaborate: in conventional practices, test developers are confronted with a labor-intensive process. For example, with ten antibodies, testing all combinations necessitates a matrix of 100 distinct tests. This process includes preparing bioconjugates, striping and cutting membranes, assembling test cassettes, conducting a range of test combinations, optimizing reagent concentrations, and often recalibrating instruments to identify the optimal pairing. Such an exhaustive process not only significantly prolongs the development period but demands extensive resources. Additionally, there is a risk of missing out on the most effective antibodies due to suboptimal immobilization or conjugation.

Advantages of the SPRi-based antibody selection approach

• We drastically reduce the selection duration to just 4 days, compared to the several weeks required by traditional methods;
• We enhance the quality and reliability of the results as we prevent the oversight of potential candidates;
• Our method is very resource-efficient, requiring only minimal amounts of antibodies and antigens.

Our approach is universally compatible

Our antibody selection process employs SPRi, which means the biochips we utilize are made of gold. In contrast, Response Biomedical LFIAs employ fluorescent nanoparticles, rather than gold. However, this distinction is not critical. What truly matters are the kinetic properties of the antibodies. The type of nanoparticle whether gold, fluorescent, magnetic, latex, and so on is of minor importance. Our approach is universally compatible, regardless of the nanoparticle type.

Goal of the study: evaluation of Kimialys’ method

In a comparative study to evaluate our SPRi-based antibody selection method for LFIA applications, we contrasted Kimialys' proprietary techniques with the traditional method. Kimialys conducted multiplex SPRi screening and characterization on five chosen antibodies to identify and select the most effective pair. Simultaneously, Response Biomedical performed standard antibody selection approaches, testing 18 out of the 25 possible combinations by LFIA. As the conclusion of this whitepaper, we will compare the results obtained from both combinatory tables.

Materials and Methods

• For this study, we received five antibodies as part of a blinded study.
• These antibodies were covalently immobilized on SPRi sensor chips using Kimialys proprietary surface chemistry, K-One®.
• Six different medium conditions were tested for each antibody.
• The antibodies were distributed over three biochips, with two antibodies per chip (figure 1).
• Analyte: Troponin I.
• Affinity, association, and dissociation rates were measured by SPRi to select the optimal antibody pair for subsequent LFIAs.

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Description of the figure 1

• K-One® chemistry applied to the biochip surface, appearing as dark gray in the image.
• Spots determined by the presence of immobilized ligands appearing as light grey circles on the surface.
• The specific signal is collected from the spots with immobilized ligands, while the non-specific signal or background noise is collected on the passivated surface surrounding the spots, as indicated on the microarray map.
• The contrast is uniform accross all antibody spots indicating that the density of immobilized antibodies is the same whatever the immobilization conditions and the nature of the antibodies. Also, the passivated surface, treated with K-One® chemistry, also displays a similar contrast and thus an uniform repartition of K-One® on the surface. Therefore, for the subsequent experiment (figure 2), any observed variations in signal intensity result solely from the specific antibody-troponin interactions and not in discrepancies in antibody density.

Information on K-One® surface chemistry

K-One® is Kimialys’ technology platform and the culmination of over 10 years of academic research led at Paris-Saclay University, standing as the most effective surface chemistry for biosensing applications to date. Coating the surface of chips and nanoparticles with K-One® chemistry allows us to control the exposure, density, and distribution of ligands, ensuring that more than 90% of them remain functional while fully protecting the surface from undesired interactions. As this whitepaper is focused on our antibody selection method by SPRi, we won’t develop further on K-One®. For more information about how we utilize K-One® to improve LFIAs performance, check our dedicated page: FirstTimeRight method.

In this study, the surface of the chips has been modified with K-One®. However, as we already said before, our SPRi-based antibody selection approach is universally compatible, suitable for passive adsorption or any other surface treatments.

Why screening for the most optimal immobilization conditions is important?

Screening for the best immobilization conditions is a crucial phase, as it helps mitigate the risk of failing to identify the most effective antibodies during the selection of the optimal pair, as each antibody performs best under its optimal conditions.

It’s even more important in our case since we are using the same surface chemistry, K-One®, in the screening phase on the chips and in the bioconjugation phase on the nanoparticles (even if we don’t perform the bioconjugation in this study). This ensures that the antibodies behave consistently and optimally in both phases and is crucial for maintaining reliable and reproducible results throughout the entire experimental process.

SPRi's multiplexing capabilities can go much further...

In this study, each chip contains 12 spots, a number limited by the manual spotting process. However, it can be optimized using a spotter, potentially allowing up to 400 spots per chip, which would significantly save even more time and resources if we had a large number of antibodies to screen and characterize.

Additionnal information on the study

• Instrument: OpenPlex, Horiba.
• Surface Modification: K-One® surface chemistry. Activation method: 0.4 M EDC / 0.1 M NHS.
• Buffer Conditions: Running buffer PBS, pH 7.4. Regeneration solution: Glycine, pH 2.0.

I- Optimization of the immobilization conditions and selection of the detection antibody

With this initial experiment, we had two objectives (figure 2):

•   Identify the best immobilization conditions for each antibody;
•   Determine the most efficient antibody for complexing troponin, which will be selected as the detection antibody to be immobilized on the nanoparticles for subsequent LFIAs.

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Description of the figure 2 - First objective, identifying the optimal immobilization conditions

In this experiment, we have characterized troponin interactions with all immobilized antibodies (Abs) to determine the optimal immobilization conditions. We have selected 3 different optimal conditions for the immobilization of the 5 antibodies. This variation underscores the importance of screening in diverse conditions to ensure that each antibody is immobilized in its most favorable state. The ability to identify these unique optimal conditions for each antibody highlights a significant advantage of our methodology. // Cond.1: Ab #1 and Ab #5 // Cond.4: Ab #2 and Ab #3 // Cond.5: Ab #4 //

Description of the figure 2 - Second objective, selecting the detection antibody

Distinct differences were observed for association/dissociation rates of tested antibodies allowing to select the best detection antibody. Given its superior efficiency in complexing troponin (highest plateau of all the sensorgrams) and its commendable stability, evidenced by a relatively low dissociation rate (curve declines slowly after the association phase has plateaued), Ab #3 has been selected as the detection antibody to be immobilized on the nanoparticles in the condition 4.

II- Selection of the capture antibody

A binning assay is used to ensure that the two antibodies interact with the same analyte. This technique evaluates if two antibodies can simultaneously bind to their respective epitopes on a single molecule without interacting with each other. This ensures that the antibodies will function effectively in a sandwich assay format, as they won't interfere with each other's binding sites.

To conduct the binning assay using SPRi, we immobilized all tested antibodies on the same biochip and injected troponin into the flow cell, followed by the injection of a second antibody. Each antibody was immobilized on the sensor surface under its optimal immobilization conditions and all five antibodies were injected to form a sandwich one after another, in order to define the most suitable capture antibody. Once all the combinations were formed, we overlayed the results of sandwich injections for the selected detection antibody (Ab #3) for easier estimation of the best sandwich couple (figure 3).

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Description of the figure 3 - Third objective, selecting the capture antibody

• Ab #1 exhibited the highest signal intensity among the tested antibodies. This suggests a strong interaction between this antibody and the troponin complex, potentially marking it as an optimal choice for the capture antibody.
• In response to the troponin injections for each of the five antibodies tested, the signal intensity for each antibody remains stable across all five cycles and does not diminish over time. This underscores the assay's reproducibility and the high stability of the antibody-antigen complexes.
• When Ab #3 was injected, there was no observed signal intensity, confirming that Ab #3 does not exhibit any self-interaction. This ensures that its binding activity in the assay is attributed solely to its interaction with the troponin complex and not due to any cross interaction or non-specific binding.
• The absence of background detection (signal intensity from the passivated surface near 0) enables a direct interpretation of the results. High-specificity assays is a direct result from using K-One® surface chemistry.

Conclusion: Ab#3 has been selected as the detection antibody to be immobilized on nanoparticles while Ab#1 has been selected as the capture antibody to be immobilized on the test line. The Ab#3/Ab#1 pairing demonstrated the highest signal intensity along with the fastest on rate and the slowest off rate.

Results Comparison

The primary objective of this study was to evaluate Kimialys' ability to effectively select the optimal antibody pair using multiplex SPRi technology. As explained in the introduction, Response Biomedical performed standard antibody selection approaches, testing 18 out of the 25 possible combinations by LFIA.

The combinatory tables are presented bellow.

Combinatory tables - Kimialys figure 5 a) - Response Biomedical figure 5 b)

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Description of the figure 5 - Conclusion on the study

When comparing the outcomes from both the traditional (figure 5b) and our SPRi-based methods (figure 5a), we observed a significant concordance. Both strategies concurred that Ab #3 was the optimal choice for the detection antibody and should be immobilized onto nanoparticles, and Ab #1 was identified as the best capture antibody for immobilization on the test line. It’s important to note that while the traditional method provided quality results in this case, it may not be as consistent in other scenarios, as there are instances where screening by LFIAs may fail to identify the best candidates.