Homogeneous Immunoassay Research Articles

Homogeneous Electrochemical Immunoassay Using an Aggregation–Collision Strategy for Alpha-Fetoprotein Detection

Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity and high efficiency. Homogeneous electrochemical assays, however, are not commonly accessed due to the requirement of electrode immobilization of the recognition elements. Herein, we demonstrate a new homogeneous electrochemical immunoassay based on the aggregation-collision strategy for the quantification of tumor protein biomarker alpha-fetoprotein (AFP). The detection principle relies on the aggregation of AgNPs induced by the molecular biorecognition between AFP and AgNPs-anti-AFP probes, which leads to an increased AgNP size and decreased AgNP concentration, allowing an accurate self-validated dual-mode immunoassay by performing nanoimpact electrochemistry (NIE) of the oxidation of AgNPs. The intrinsic one-by-one analytical capability of NIE as well as the participation of all of the atoms of the AgNPs in signal transduction greatly elevates the detection sensitivity. Accordingly, the current sensor enables a limit of detection (LOD) of 5 pg/mL for AFP analysis with high specificity and efficiency. More importantly, reliable detection of AFP in diluted human sera of hepatocellular carcinoma (HCC) patients is successfully achieved, indicating that the NIE-based homogeneous immunoassay shows great potential in HCC liquid biopsy.

Processing Validation Metrics of Syva Enzyme-Multiplied Immunoassay Technique (EMIT) Methotrexate Assay for Beckman Coulter System.

High-dose methotrexate (HDMTX), defined as a dose greater than 500 mg/m2, is used to treat a variety of cancers; and though safe, it can cause major toxicity. Syva enzyme-multiplied immunoassay technique (EMIT) methotrexate (MTX)assay(Gurgaon, India: Siemens Healthcare Diagnostics Ltd.) uses a homogeneous enzyme immunoassay method. Low-end precision performances are very important for laboratory methods, especially when their results have clinical significance at these levels. A total of 25 replicates (five replicates per run, for five runs) were analyzed for profiling. Precision, accuracy, linearity, limit of blank, limit of detection, and limit of quantification were determined using existing guidelines. Imprecision profile and limit of quantitation (LoQ) at 10% were determined by fitting data with hyperbolic regression. The coefficient of variation percentage (CV%) for low, mid, and high-level internal quality control (IQC) was 1.25%, 3.45%,and 1.55%, respectively. Similarly, estimated bias was -4.58%, -3.54%, -7.21% for each level. The assay linearity was maintained from a range of 0.041-1.993 mmol/L with an R2 of 0.959. The limit of detection was estimated to be 0.07 mmol/L. Syva EMIT MTX assay can be precisely and accurately used to measure low levels of serum methotrexate at levels lower than claimed by the manufacturer, aiding in the monitoring of toxicity in patients.

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes.

There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.

Preparation and characterization of a homogeneous immunoassay for point-of-care testing (POCT) of procalcitonin (PCT).

Procalcitonin (PCT) has been recognized as a specific and early marker for microbial infection and sepsis. Sensitive measuring interaction-triggered luminescence experiment (SMILE), a homogeneous immunoassay method, was established for point-of-care testing (POCT) of PCT. SMILE is achieved through the principle of double antibody sandwich, where two antibodies immobilized on the surface of polystyrene microspheres (donor and acceptor beads) bind to the PCT antigen. The donor bead contains phthalocyanine dye (luminol chemiluminescent substance) and the acceptor bead contains dimethylthiophene derivatives and Eu chelates. Therefore, singlet oxygen can be transferred when the distance between donor and acceptor beads is within 200 nm, generating detectable luminescent signals. Scanning electron microscopy (SEM) was used to detect the diameter and polymer dispersity index (PDI) of microspheres before and after binding with antibodies to characterize the immobilization of antibodies. The reaction conditions for antibody immobilization including pH, mass ratio and reaction time have also been optimized. The limit of quantitation (LOQ) of the SMILE method (0.01 ng mL-1) was lower than that of the LFI method (0.1 ng mL-1), the working range (0.01-500 ng mL-1) was wider than that of the LFI method (0.1-50 ng mL-1), and the assay time (10 min) was shorter than that of the LFI method (15 min). So, SMILE is more suitable for POCT of PCT compared with lateral flow immunochromatography (LFI), which is the most used measuring method, due to its advantages of simple operation, saving time, convenience, wide detection range, and high sensitivity and accuracy.

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 2h 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.

Dual-Wavelength Fluorescence Polarization Immunoassay for Simultaneous Detection of Sulfonamides and Antibacterial Synergists in Milk

Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC50 values for 23 SAs below 100 μg L-1 and 5 ASGs below 50 μg L-1. The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.

Effects of genetic polymorphisms on methotrexate levels and toxicity in Chinese patients with acute lymphoblastic leukemia.

Methotrexate (MTX) has an antitumor effect when used for the treatment of acute lymphoblastic leukemia (ALL). This study aims at evaluating the associations between 14 polymorphisms of six genes involved in MTX metabolism with serum MTX concentration and toxicity accompanying high-dose MTX. Polymorphisms in 183 Chinese patients with ALL were analyzed using TaqMan single nucleotide polymorphism genotyping assay. The serum MTX concentration was determined using homogeneous enzyme immunoassay. MTX-related toxicities were also evaluated. Renal toxicity was significantly associated with higher serum MTX concentrations at 24, 48, and 72 hours, and MTX elimination delay (P = 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively), whereas SLCO1B1 rs4149056 was associated with serum MTX concentrations at 48 and 72 hours, and MTX elimination delay in candidate polymorphisms (P = 0.014, P = 0.019, and P = 0.007, respectively). SLC19A1 rs2838958 and rs3788200 were associated with serum MTX concentrations at 24 hours (P = 0.016, P = 0.043, respectively). MTRR rs1801394 was associated with serum MTX concentrations at 72 hours (P = 0.045). Neutropenia was related to SLC19A1 rs4149056 (odds ratio [OR]: 3.172, 95% confidence interval [CI]: 1.310-7.681, P = 0.011). Hepatotoxicity was associated with ABCC2 rs2273697 (OR: 3.494, 95% CI: 1.236-9.873, P = 0.018) and MTRR rs1801394 (OR: 0.231, 95% CI: 0.084-0.632, P = 0.004). Polymorphisms of SLCO1B1, SLC19A1, ABCC2, and MTRR genes help predict higher risk of increased MTX levels or MTX-related toxicities in adult ALL patients.

Development of a multiplexing potency assay using upconverting nanoparticles-based luminescence resonance energy transfer

A homogeneous particle-based immunoassay using upconverting nanoparticles (UCNPs) has been developed for multiplexing potency analysis of two different therapeutic monoclonal antibodies (mAbs) in a fixed-combination formulation.The UCNP, considered as the best donor lumiphore for luminescence resonance energy transfer (LRET), offers long lasting excitation state and increased signal-to-noise (S/N) ratio due to low autofluorescence effect and light scattering from near infrared (NIR) excitation. In this study, the dose-response curves for each therapeutic mAb were generated using two distinct UCNPs. This proof-of-concept LRET-based immunoassay demonstrated a novel approach for increasing testing throughput and analyzing the potency of mixed therapeutic mAbs in co-formulated products.

Development and application of amplified luminescent proximity homogeneous assay for quantitation of heparin-binding protein

A fast and highly sensitive amplified luminescent proximity homogeneous assay (AlphaLISA) method was developed for quantitation of plasma heparin-binding protein levels. In this study, a method directly coupling donor and acceptor beads modified with aldehyde groups to anti-HBP antibodies was proposed, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the developed method required only 15 min of reaction time to generate results. Compared with the approved commercial kit, the developed method had a wider linear range (2.78–500 ng/mL). The excellent linear range means that the method can better exploit the value of HBP in clinical applications. Meanwhile, results of amplified luminescent proximity homogeneous assay and fluorescence dry quantitative immunoassay had good correlation and consistency (ρ = 0.9181). Moreover, the plasma HBP concentrations of patients with bacterial infection were significantly higher than those of healthy individuals (P < 0.0001), indicating the potential applicability of the proposed method for predicting the incidence of bacterial infections. Importantly, the newly developed method is expected to serve as an alternative to the traditional assay method and provides a completely new platform for other biomarkers that require rapid detection.

A homogeneous enzyme-free ratiometric immunoassay for the determination of C-peptide

In this study, a homogeneous enzyme-free ratiometric (HOMO- EF-RA) immunoassay was developed for the sensitive detection of C-peptide. In the immunoassay, there have been a miscible detection system by mixing with the fluorescent quantum dots conjugated antigen (QD-Ag conjugates) and the dylight dye conjugated antibody (DL-Ab conjugates). When connecting between Ag-QD conjugate and Ab-DL conjugate by specific recognition, the system emitted fluorescence resonant energy transfer (FRET). The target C-peptide can inhibit the connection and FRET formation between QD-Ag conjugates and DL-Ab conjugates, thus changing the dual fluorescence. By measuring the ratio dual fluorescence changes of the system, the content of C-peptide was evaluated without any enzyme used and multiple incubation and washing steps. This immunoassay realized the highly sensitive (as low as 0.12 ng mL−1), selective and rapid (as less as 6 min) detection of C-peptide. Furthermore, the simple and convenient immunoassay was applied successfully to the determination of C-peptide in real serum samples.

Measurement of α-Synuclein Dynamics In Vivo Using Microdialysis with a Novel Homogeneous Immunoassay.

Understanding the regulation of α-synuclein release could be important in better understanding Parkinson's disease development, progression, and treatment. Advances in such studies are hindered by technical challenges that limit the ability to monitor α-synuclein concentration in vivo. We developed a novel α-synuclein microdialysis method coupled with a specific and sensitive immunoassay that requires a small sample volume (1 μL). Using this method, basal α-synuclein level was estimated at 254 ± 78 pM in the striatum of freely moving mice. Additionally, we observed that potassium (75 mM) and nicotine (0.5 mg/kg) administration significantly increased α-synuclein in dialysates. These results provide evidence that the methods we report here can be useful to investigate the physiological roles of α-synuclein and support the idea that α-synuclein is secreted to the extracellular space in a neuronal activity-dependent manner.

Development of a rapid homogeneous immunoassay for detection of rotavirus in stool samples.

Rotavirus is the main pathogen causing acute viral gastroenteritis. Accurate and rapid diagnosis of rotavirus infection is important to determine appropriate treatment, prevention of unnecessary antibiotics use and control of infection spread. In this study, we established a rapid, accurate, and sensitive amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) for detecting rotavirus and evaluated its efficacy in human stool samples. Our results demonstrated that the sensitivity of AlphaLISA (5−8) significantly exceeded that of the immunochromatographic assay (ICA, 5−4) for rotavirus antigen detection. The intra-assay and inter-assay coefficients of variation were 2.99–3.85% and 5.27–6.51%, respectively. Furthermore, AlphaLISA was specific for rotavirus and did not cross-react with other common diarrhea viruses. AlphaLISA and real-time reverse transcription polymerase chain reaction (RT-qPCR, which is considered a gold standard for detecting diarrhea viruses) tests showed consistent results on 235 stool samples, with an overall consistency rate of 97.87% and a kappa value of 0.894 (P < 0.001). The overall consistency rate of ICA compared with RT-qPCR was 95.74%. AlphaLISA showed better consistency with RT-qPCR than the routinely used ICA for rotavirus detection in stool samples. The AlphaLISA method can be used in clinical practice for the rapid, accurate, and sensitive detection of rotavirus infection.

One-Step Homogeneous Immunoassay for the Detection of Influenza Virus Using Switching Peptide and Graphene Quencher.

One-step homogeneous immunoassay was developed for detecting influenza viruses A and B (Inf-A and Inf-B) using the switching peptide H2. As the fluorescence-labeled switching peptide dissociated from the binding pocket of detection antibodies, the fluorescence signal could be directly generated by the binding of Inf-A and Inf-B without washing (i.e., one-step immunoassay). For the one-step homogeneous immunoassay with detection antibodies in solution, graphene was labeled with the antibodies as a fluorescence quencher. To test the feasibility of the homogeneous one-step immunoassay, the stability of the antibody complex with the switching peptide was evaluated under different pH and salt conditions. The one-step homogeneous immunoassay with switching peptide was conducted using influenza virus antigens in phosphate-buffered saline and real samples with inactivated Inf-A and Inf-B spiked in serum. Finally, the one-step homogeneous immunoassay results were compared with those of commercially available lateral flow immunoassays.

Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides.

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Creating a Thermostable β-Glucuronidase Switch for Homogeneous Immunoassay by Disruption of Conserved Salt Bridges at Diagonal Interfaces.

Escherichia coli β-glucuronidase (GUS) has been used as a reporter enzyme in molecular biology and engineered as an enzyme switch for the development of homogeneous biosensors. In this study, we developed a thermostable GUS enzyme switch based on the thermostable GUS mutant TR3337 by disrupting a conserved salt bridge (H514-E523) between the diagonal subunits of its homotetramer. A combinatorial library (240 variants) was screened using a novel high-throughput strategy, which led to the identification of mutant DLW (H514D/M516L/Y517W) as a functional enzyme switch in a caffeine-recognizing immunosensor. Molecular dynamics simulations were performed to predict the topology change around position 514, and a side-chain flip of D514 (repulsion with E523) was observed in the DLW mutant. Up to 1.8-fold of signal-to-background ratio was confirmed when measured at up to 45 °C, thereby highlighting the DLW mutant as a versatile tool for developing thermostable immunosensors for in vitro and in cellulo applications.

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.

A simple and rapid homogeneous fluorescence polarization immunoassay for rapid identification of gutter cooking oil by detecting capsaicinoids.

In order to address the widespread concerns with food safety such as adulteration and forgery in the edible oil field, this study developed a fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody in a homogeneous solution system for determination of capsaicinoids in gutter cooking oil by using chemically stable capsaicinoids as an adulteration marker. The prepared fluoresceinthiocarbamyl ethylenediamine (EDF) was coupled with capsaicinoid hapten C, and the synthesized tracer was purified by thin-layer chromatography (TLC) and showed good binding to the monoclonal antibody CPC Ab-D8. The effects of concentration of tracer and recognition components, type and pH of buffer and incubation time on the performance of FPIA were studied. The linear range (IC20 to IC80) was 3.97-97.99 ng/mL, and the half maximal inhibitory concentration (IC50) was 19.73 ng/mL, and the limit of detection (LOD) was 1.56 ng/mL. The recovery rates of corn germ oil, soybean oil and peanut blend oil were in the range of 94.7-132.3%. The experimental results showed that the fluorescence polarization detection system could realize the rapid detection of capsaicinoids, and had the potential to realize on-site identification of gutter cooking oil. As a universal monoclonal antibody, CPC Ab-D8 can also specifically identify capsaicin and dihydrocapsaicin, so the proposed method can be used to quickly monitor for the presence of gutter cooking oil in normal cooking oil.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

Abstract 2977: Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays

Abstract Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG-510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays 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. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants. Citation Format: Hicham Zegzouti, Laurie Engel, Matthew Swiatnicki, Juliano Alves, Said A. Goueli. Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2977.

Abstract 2750: Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy

Abstract Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new NanoBiT-based assay platform-based RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of CD3 bispecific antibodies or BiTEs and similar biologics that induce T Cell-dependent target cell killing and cytokine production. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN gamma) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRab-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and b chains into TCRab-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRab-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Gopal Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2750.