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.

Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay.

KRAS is one of the most heavily mutated oncogenes in cancer and targeting mutant KRAS with drugs has proven difficult. However, recent FDA approval of the KRAS G12C selective inhibitor sotorasib (AMG-510), has breathed new life into the drive to develop mutant KRAS inhibitors. In an effort to study RAS inhibitors in cells and identify new compounds that inhibit Ras signaling, western blotting and ELISA assays are commonly used. These traditional immunoassays are tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we applied Lumit immunoassay technology to analyze RAS signaling pathway activation and inhibition through the detection of phosphorylated ERK. The assay we developed was used to rank order potencies of allele specific inhibitors within cell lines harboring various activating KRAS mutations. An inhibition profile was obtained indicating various potencies and selectivity of the inhibitors, including MRTX-1133, which was shown to be highly potent against KRAS G12D signaling. MRTX-1133 had approximately 40 and 400 times less inhibitory potency against G12C and G12V mutant KRAS, respectively, while no inhibition of WT KRAS was observed. The potency of PROTAC compound LC-2 targeting selective degradation of KRAS G12C was also tested using the Lumit pERK immunoassay, and a maximal decrease in RAS signaling was achieved. Lumit immunoassays provide a rapid, homogeneous platform for detecting signaling pathway activation and inhibition. Our results demonstrate that this bioluminescent technology can streamline the analysis of signaling pathways of interest, such as RAS-dependent pathways, and be used to identify much needed inhibitors. The results further imply that similar assay designs could be applied to other signaling pathway nodes.

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors.

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.

Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation. This immunoassay takes less than two hours to complete in a homogeneous "Add and Read" format and was successfully used to monitor multiple signaling pathways' activation through specific nodes of phosphorylation (e.g pIκBα, pAKT, and pSTAT3). We also tested deactivation of these pathways with small and large molecule inhibitors and obtained the expected pharmacology. This approach does not require cell engineering. Therefore, the phosphorylation of an endogenous substrate is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific inhibitors.

Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of action for many therapeutic antibodies. Classic ADCC assays measure antibody-dependent target cell cytotoxicity induced by primary effector cells that are isolated from human blood. They suffer from high assay variability due to the genetic and immune-status-mediated variation from blood donors. Here we report the development of a robust reporter-based ADCC assay that uses an engineered Jurkat stable cell line as the source of effector cells. These engineered effector cells were further developed as frozen, thaw-and-use format that can be plated for assay immediately after thaw. We demonstrate that frozen, thaw-and-use Jurkat effector cells showed appropriate Fc effector function similar to fresh cells from continuous culture, with added benefits of convenience and consistency. This robust assay is able to measure antibody potency for several therapeutic antibodies targeted to hematopoietic or solid tumors. The assay can distinguish effector functions for different antibody IgG isotypes in two antibody model systems: anti-CD20 and anti-EGFR. It is able to detect changes in ADCC biological activity for heat-stressed rituximab and trastuzumab, demonstrating that it possesses proper stability-indicting property. When compared with a classic PBMC-based ADCC assay, the ADCC reporter assay showed better assay precision and similar correlation of antibody glycosylation with ADCC biological activity for a panel of glyco-modified trastuzumab mixtures. Together these data demonstrate that this robust ADCC reporter assay meets the requirement of a potency bioassay that can quantify antibody Fc effector function in ADCC mechanism of action during drug discovery and development.

Methods for the purification of HQ-tagged proteins.

The HQ (H = histidine, Q = glutamine) tag is a small fusion tag that can be isolated using immobilized metal affinity columns. HQ-tagged proteins can be expressed and purified from bacterial cells under native and denaturing conditions, mammalian cells, insect cells, wheat germ and rabbit reticulocyte. Furthermore, HQ-tagged proteins can be purified using magnetic or non-magnetic resins in multiple formats from small to large-scale and manual or automated. In this chapter, we have described various protocols for the purification of HQ-tagged proteins.