Next generation bioluminescence-based homogenous immunoassay platform supporting quantitative, highly sensitive and rapid one-step biomolecule quantification in complex human samples
Heterogeneous immunoassays are assays that rely on immobilisation of an antibody-specific epitope, followed by two or more separate washing and incubation steps to achieve antibody recognition and subsequent signal generation. ELISA has proven to be a highly modular and sensitive technique to detect antibodies, but the multi-step nature of the assay makes it less suitable for incorporation in a point-of-care diagnostic device. Similarly, the need to perform multiple washing and incubation steps is impractical in high-throughput screening applications.
Other heterogeneous approaches, such as those based on surface plasmon resonance, suffer from comparable limitations. Although they can in part be circumvented by applying automated mixing and washing strategies, for example based on the use of magnetic beads or microfluidic circuitry, these limitations have inspired the development of homogeneous assays.
In contrast to heterogeneous antibody detection assays, homogeneous assays are one-step assays, where antibodies can be detected directly in solution without the need for any washing steps. Homogeneous assays typically involve the use of a biomolecular switch in which molecular recognition and signal generation are integrated. The fundamental challenge in designing such switches is to devise efficient strategies to translate antibody binding into a robust and easily observable signal.
Fluorescence has been a very important tool in the development of protein-based antibody sensors. However, the necessity for external illumination means that autofluorescence and scattering will inevitably restrict the sensors limit of the detection to the lowest sensor concentration that can reliably be measured.
An alternative to using fluorescence that is much less sensitive to autofluorescence and scattering is luminescence. Luminescent assays are very sensitive and have a wide dynamic range. It is believed that luminescence is the most sensitive detection method currently in use due to the ability of signal multiplication and amplification. The various forms of luminescence (bioluminescence, chemiluminescence, photoluminescence) differ in the way the excited state is reached. For example, photoluminescence is simply fluorescence; the excitation is initiated by light at a particular wavelength. Bioluminescence is characterized by the use of a bioluminescent compound, such as luciferin and firefly luciferase. Chemiluminescence is light produced by a chemical reaction.
Bioluminescence, where light is generated by the enzymatic conversion of a substrate by a luciferase, eliminates the need for external illumination. Until recently, luciferases were not considered for incorporation in diagnostic technology, mainly because of their modest brightness and stability. Recent developments, notably the development of the very bright and stable NanoLuc® have changed this situation, offering the possibility of developing protein-based antibody sensors with a very low limit of detection.
Bioluminescent-based homogeneous sensors that display a change in color upon analyte binding show great promise for measurements in complex media such as blood plasma, as minimal sample pretreatment is required. Unlike fluorescence- based methods, bioluminescent sensors do not need external excitation, thus eliminating issues associated with autofluorescence or light scattering. LUMABS BV founder has developed RAPPID (Ratiometric Plug-and-Play ImmunoDiagnostics), a mix-and-measure immunoassay platform based on the reconstitution of antibody-conjugated split NanoLuc luciferases.
The platform is highly modular, as it entails monoclonal antibodies and conjugation through a protein adaptor. The straightforward development of a RAPPID assay enables the easy exchange of antibodies and hence screening for the best antibody pair and optimal sensor. Furthermore, the RAPPID platform has a high intrinsic maximal change in emission ratio and a robust ratiometric light output, enabled by the introduction of a green-emitting calibrator luciferase, facilitating the accurate detection of biomarkers in the picomolar to nanomolar range. The ratiometric nature of the RAPPID assay makes this platform an attractive diagnostic development tool to detect challenging biomarkers.
BioAnalysis: Supporting the development of novel therapeutics
Prior to approval of a novel therapeutic, drug developers are required to undertake extensive clinical and pre-clinical trials to ensure the safety and efficacy of their candidate products. LUMABS technology is ideally suited to supporting pharmaceutical and biotechnology companies developing antibody therapeutics including biosimilars.
If you have a therapeutic antibody project and can see the benefits of using LUMABS technology, or would like to explore the potential further, please contact us using the inquiry form here. LUMABS BV are keen to hear from you and partner to support, accelerate, and enhance your project. Initial feasibility assessments are confidential, provided free-of-charge and with no obligation.
We are also interested in the development of companion diagnostics for antibody drugs including those already approved and on the market.
Therapeutic Drug Monitoring (TDM)
Therapeutic antibodies need to be administered by injection. Because of their relatively long serum half-life, the time interval between injections can be several weeks. The time between injections and the amount of antibody injected each time should provide the optimal dosing of the drug for the patient. Ideally, this will be modulated to match each patient to ensure the drug is optimally efficacious and minimal side effects, whilst also ensuring minimal wastage through avoiding excess dosing. Interpatient variabilities in mass, distribution, and clearance mean that a single fixed dosage is not optimal, and frequently not suitable, for all patients. Therapeutic Drug Monitoring (TDM) ensures the optimal dose is administered to each individual patient for each injection and is, therefore, the preferred personalized medicine approach to achieve optimal outcomes, superior drug efficacy, minimizing side effects, and maximal drug administration efficiency.
NanoLuc® is a registered trademark of Promega Corporation