Application Note: Measurement of Antibody-Antigen Kinetics with the HexagonFab Bolt
Measurement of Antibody-Antigen kinetics using HexagonFab Bolt - a rapid analysis system for biomolecules
A critical need in many areas of Drug Discovery and Process Monitoring is for low cost, rapid, accurate methods for analysing interactions between molecules. Examples are numerous and include:
Studying binding of molecules that could represent future drugs to their target(s)
Screening for the quantity and quality of a valuable protein molecule (e.g. monoclonal antibody) during various developmental and production steps
Mapping antigen epitopes to characterise and optimise antibody binding
These types of analysis are performed on a variety of platforms, including High Performance Liquid Chromatography (HPLC), Enzyme-Linked Immunosorbent Assays (ELISA) and increasingly various label-free assay technologies.
Well-established technologies such as HPLC and ELISA are limited in terms of the range of assays that can be run on these platforms. They also feature comparatively protracted times to result, offer relatively limited data compared to newer technologies and can only be operated by specially trained personnel in a centralised laboratory environment. Nevertheless, they represent an important benchmark against which newer technology is judged.
The limitations of traditional methods such as HPLC and ELISA have led to a move in recent years to label-free assay platforms. Label-free assays offer various benefits to the user, including real-time data read-out, quantitative data and high sensitivity. There are several label-free platforms available, mostly based on the optical detection principles of Surface Plasmon Resonance (SPR) and BioLayer Interferometry (BLi).
The current optical-based systems suffer from some disadvantages, including loss of sensitivity with decreasing molecular size, complexity and hence cost and the need to use such systems in a centralised laboratory with tests performed by specially trained personnel. This limits their use for activities which are better served by a small, portable instrument platform that can be more widely utilised across both research and manufacturing areas of for example a pharmaceutical company.
The HexagonFab Bolt
To address the limitations of optical-based label-free analysis platforms, HexagonFab has developed a graphene-based sensor system, we call this the HexagonFab Bolt. This consists of the Bolt reader and sensor. The Bolt sensor measures changes in the electronic properties of a sensitive membrane at the sensor-analyte interface.
These changes are induced by the presence of charged molecules, e.g. proteins, small molecules or DNA/RNA. The changes in electrical properties are translated into response units. The magnitude of the response units depends on the number of charges at the analyte/sensor interface, thus different responses can be measured for varying concentration – the higher the concentration, the higher the response change.
The Bolt sensors are supplied with a carboxylic acid-functional chemical linker bound to the sensor surface, which is also highly resistant to non-specific protein adsorption. Like other label-free platforms (e.g. SPR/BLi) our sensor chip can be functionalized with a ligand of choice using standard activation techniques (read more: method description). Once loaded, with an analyte, a sensor may then be used immediately with the Bolt reader to detect binding events in analyte solutions.
Aim of this note
The key aim of this application note is to demonstrate how the HexagonFab Bolt can be used for a widely performed type of assay, often referred to generically as Biomolecular Interaction Analysis (BIA). BIA entails the immobilization of a protein ligand to a sensor substrate (e.g. a capture antibody) followed by the complementary binding of the corresponding antigen or antibody in solution. This event is detected by the sensor and the kinetic curves obtained are then used to calculate the association (ka) and dissociation (kd) rate constants for the interaction and the affinity (KD). The most common methods currently used for BIA are immunoassays (e.g. ELISA), which are limited to affinity only, and SPR/BLI instruments, which provide the full range of high-accuracy kinetic measurements.
In this note, we use of the Bolt system to demonstrate its ease of use and resolution capability via a Streptavidin/Anti-Streptavidin antibody binding experiment utilising various concentrations of antibody.
In this application example, the HexagonFab Bolt is used to measure the binding interaction between a target immobilised on the sensor surface (Streptavidin) and its complementary antibody (Anti-Streptavidin). By applying analyte solutions to the sensor surface and measuring sensor response over a set period of time, an increasing signal is obtained. This signal is identical to a typical association curve recorded by an SPR/BLI instrument. Conversely, when the surface is washed with pure buffer solution, the signal returns to baseline and indicating dissociation of the analyte (Anti-Streptavidin) from the capture molecule (Streptavidin).
In the example shown here, we demonstrate that a single binding measurement on the HexagonFab Bolt sensor chip can be completed in as little as 30 minutes. Also that readings obtained for 5 different sequential antibody concentrations demonstrated the expected relationship between antibody concentration and signal (i.e. the higher the antibody concentration, the higher the signal recorded). The included software for curve fitting will also give a determination of the association (ka) and dissociation (kd) constants of the experiment. The methodology described here may also be used as a general recipe for carrying out these experiments with antigen/antibody combinations of the customer’s choice.
The data from the streptavidin/anti-streptavidin antibody binding experiment yielded 5 curves corresponding to the 5 concentrations of antibody studied. The point of where the antibody was introduced to the sensor for each concentration was defined as time 0. This resulting graph is shown below.
The sensor is able to distinguish the signal response for each concentration. These are within the same order of magnitude (measured in nM). Association and dissociation curves are clearly seen after the baseline correction. The analysis gives a set of ka and kd values as follows in Table 1:
The HexagonFab Bolt offers a quick and time efficient method of analysing protein binding in small-scale bench top experiments. Loading is a simple process, requiring only the sensing area to be exposed to the ligand solution. Once loaded, the experiment can be run immediately with little set up, and an experiment can be completed in as little as 30 minutes. The size of the entire apparatus fits onto the bench of small-scale laboratory, where there may not be room for large optical instruments.
The HexagonFab Bolt is a new small-scale and accessible platform for performing biomolecular interaction analysis, and is applicable to almost all the ligand/antibody systems studied previously with established immunoassay platforms. We believe that this tool will become a valuable means of assisting greater numbers of research groups and commercial R&D laboratories to carry out BIA experiments for a lower cost in space and resources.
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