Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2 h to complete in a homogeneous "Add and Read" format. In this chapter, we describe step-by-step protocols to create Lumit immunoassays for the detection of (1) secreted cytokines from cells, (2) phosphorylation levels of a specific signaling pathway node protein, and (3) a biochemical protein-protein interaction between a viral surface protein and its human receptor.

A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors.

Fc engineering efforts are increasingly being employed to modulate interaction of antibodies with variety of Fc receptors in an effort to improve the efficacy and safety of the therapeutic antibodies. Among the various Fc receptors, Fc gamma receptors (FcγRs) present on variety of immune cells are especially relevant since they can activate multiple effector functions including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the desired mechanism of action (MOA) of the antibody, interactions between Fc domain of the antibody and FcγR (denoted as Fc/FcγR) may need to be enhanced or abolished. Therefore, during the antibody discovery process, biochemical methods are routinely used to measure the affinities of Fc/FcγR interactions. To enable such screening, we developed a plate based, simple to use, homogeneous immunoassays for six FcγRs by leveraging a luminescent protein complementation technology (NanoBiT). An added advantage of the NanoBiT immunoassays is their solution-based format, which minimizes well known surface related artifacts associated with traditional biosensor platforms (e.g., surface plasmon resonance and biolayer interferometry). With NanoBiT FcγRs assays, we demonstrate that assays are specific, report IgG subclass specific affinities and detect modulation in Fc/FcγR interactions in response to the changes in the Fc domain. We subsequently screen a panel of therapeutic antibodies including seven monoclonal antibodies (mAbs) and four polyclonal intravenous immunoglobulin (IVIg) products and highlight the advantages of parallel screening method for developing new antibody therapies.

Bioluminescent Bridging Immunoassay for Anti-Drug Antibody (ADA) Detection.

Any immune reaction to therapeutic antibodies will impact the drug efficacy and can have serious consequences for patient safety. Therefore, detection and reporting of anti-drug antibodies (ADA) during clinical trials is required by regulatory agencies during drug approval process. We have developed a bioluminescent bridging immunoassay for ADA detection, which uses an extremely bright NanoLuc enzyme as a reporter. The assay is sensitive with a wide dynamic range and meets the FDA drug tolerance guideline of detecting 100 ng/ml of ADA in the presence of 500-fold excess of free drug. We describe detailed protocols for development of ADA assays using therapeutic Trastuzumab as a model drug and an anti-Trastuzumab antibody as an example of immune response.

Deciphering the Interaction between Neonatal Fc Receptor and Antibodies Using a Homogeneous Bioluminescent Immunoassay.

Long half-life of therapeutic Abs and Fc fusion proteins is crucial to their efficacy and is, in part, regulated by their interaction with neonatal Fc receptor (FcRn). However, the current methods (e.g., surface plasmon resonance and biolayer interferometry) for measurement of interaction between IgG and FcRn (IgG/FcRn) require either FcRn or IgG to be immobilized on the surface, which is known to introduce experimental artifacts and have led to conflicting data. To study IgG/FcRn interactions in solution, without a need for surface immobilization, we developed a novel (to our knowledge), solution-based homogeneous binding immunoassay based on NanoBiT luminescent protein complementation technology. We optimized the assay (NanoBiT FcRn assay) for human FcRn, mouse FcRn, rat FcRn, and cynomolgus FcRn and used them to determine the binding affinities of a panel of eight Abs. Assays could successfully capture the modulation in IgG/FcRn binding based on changes in Fc fragment of the Abs. We also looked at the individual contribution of Fc and F(ab)2 on the IgG/FcRn interaction and found that Fc is the main driver for the interaction at pH 6. Our work highlights the importance of using orthogonal methods to validate affinity data generated using biosensor platforms. Moreover, the simple add-and-read format of the NanoBiT FcRn assay is amenable for high-throughput screening during early Ab discovery phase.

Development of a novel homogeneous immunoassay using the engineered luminescent enzyme NanoLuc for the quantification of the mycotoxin fumonisin B1.

Compared to the traditional heterogeneous assays, a homogeneous immunoassay is a preferred format for its simplicity. By cloning and isolating luminescent proteins from bioluminescent organisms, bioluminescence has been widely used for various biological applications. In this study, we present the development of a homogeneous luminescence immunoassay (FNanoBiT assay) for detection of fumonisin B1 (FB1), based on the binding of two subunits of an engineered luminescent protein (NanoLuc). For the detection of the mycotoxin FB1 in foods, the anti-fumonisin antibody was conjugated to the large subunit of NanoLuc (FLgBiT), and the FB1 was conjugated to the small subunit (FSmBiT). The conjugates were used for the detection of FB1 in a competitive immunoassay format without the need of a secondary antibody, or washing steps. The developed FNanoBiT assay revealed high specificity toward FB1 with no cross-reactivity with other mycotoxins, and it demonstrated acceptable recovery (higher than 94%) and relative standard deviation from spiked maize samples. Further, the assay was successfully applied for the detection of FB1 in naturally contaminated maize, with a dynamic range of 0.533-6.81 ng mL-1 and a detection limit of 0.079 ng mL-1. The results derived with FNanoBiT assay of all spiked samples showed a strong correlation to those obtained by the High-performance liquid chromatography method. Thus, the FNanoBiT based homogeneous immunoassay could be used as a rapid, and simple tool for the analysis of mycotoxin-contaminated foods.

Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

Anti-drug antibodies (ADAs) are generated in-vivo as an immune response to therapeutic antibody drugs and can significantly affect the efficacy and safety of the drugs. Hence, detection of ADAs is recommended by regulatory agencies during drug development process. A widely accepted method for measuring ADAs is "bridging" immunoassay and is frequently performed using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence (ECL) platform developed by Meso Scale Discovery (MSD). ELISA is preferable due to widely available reagents and instruments and broad familiarity with the technology; however, MSD platform has gained wide acceptability due to a simpler workflow, higher sensitivity, and a broad dynamic range but requires proprietary reagents and instruments. We describe the development of a new bridging immunoassay where a small (19kDa) but ultra-bright NanoLuc luciferase enzyme is used as an antibody label and signal is luminescence. The method combines the convenience of ELISA format with assay performance similar to that of the MSD platform. Advantages of the NanoLuc bridging immunoassay are highlighted by using Trastuzumab and Cetuximab as model drugs and developing assays for detection of anti-Trastuzumab antibodies (ATA) and anti-Cetuximab antibodies (ACA). During development of the assay several aspects of the method were optimized including: (a) two different approaches for labeling drugs with NanoLuc; (b) sensitivity and dynamic range; and (c) compatibility with the acid dissociation step for improved drug tolerance. Assays showed high sensitivity of at least 1.0ng/mL, dynamic range of greater than four log orders, and drug tolerance of >500.

Identification and characterization of small molecule inhibitors of a plant homeodomain finger.

A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modeling and site-directed mutagenesis of JARID1A PHD3 provided insights into the biochemical basis of competitive inhibition.