New Immunoassay Tools: From Samples to Results Faster

Immunoassay data help us understand how a drug behaves in the body and whether it is working as intended. Newer assay formats aim at simpler workflows and adaptability with New Approach Methodologies (NAMs).

Many biologically important molecules, such as hormones, cytokines, or antibodies, naturally occur at very low levels, sometimes picograms per milliliter (pg/mL). A sensitive assay can still reliably detect and quantify these molecules, even if only present in low abundance in body fluids. This capability is essential in preclinical drug development, where early and precise data determine a drug’s potential. One method that can capture trace amounts of these biomolecules is immunoassays. These antibody-based tests inform about a drug’s behavior, safety, and effect in the body, especially when sample volume or drug concentration is limited.

Moreover, preclinical animal studies often require highly sensitive readouts that give accurate results from microsampling to minimize animal use and stress. Immunoassays can detect how much of a drug is present over time to understand how it is absorbed, distributed, broken down, and removed from the body.
Some disease or drug-response biomarkers change subtly or exist at baseline in healthy organisms at minimal concentrations. High assay sensitivity allows these small changes to be tracked, providing insights into drug efficacy, toxicity, or mechanism of action.

Immunoassays are also used to detect immune responses to the drug, such as unwanted antibodies that could cause side effects or reduce the drug’s effectiveness. Less sensitive measurements may lead to false negative results, thus incorrectly concluding that a drug is not working or that a biomarker is absent. In short, immunoassays help answer key questions about a drug’s safety, effectiveness, and behavior in the body before it ever reaches clinical trials. 

These assay types remain widely used due to:

a.    Their familiarity and ease of use: ELISAs, the classic form of immunoassay, are affordable, relatively simple to perform, and compatible with standard equipment like plate readers and washers¹.
b.    their versatility: options include enzyme-based (ELISA), chemiluminescence (CLIA), fluorescence (FIA), lateral flow (LFIA), among others. Enzyme immunoassays dominated ~64.7% of the immunoassay market in 2023. However, CLIA and FIA have been growing quickly due to their high sensitivity, automation, and compatibility with multiplexing.

New immunoassay formats can now measure femto or even attomolar concentrations of unknown analytes in biofluids. Their advantages in speed and simplicity over traditional ELISAs align with the re-ignited quest to reduce and replace animals in preclinical research and testing. Among these innovations are Lumit™ immunoassays developed by Promega. These luminescence-based formats are designed to provide a faster, simpler, and more sensitive alternative to traditional ELISAs.

They appear particularly useful for decision-making in discovery phases, for high-throughput screenings, and for applications where rapid, no-wash workflows are crucial^2,3. They can also be used to measure real-time secretion of cytokines or other proteins directly in cell culture supernatants. Lumit assays use Promega’s NanoBiT® luciferase system⁴. It requires two fragments of luciferase (LgBiT and SmBiT) being attached to antibodies or binding proteins. When a target analyte is present, the sysyem brings the fragments together, and this reconstitution creates a bright luminescent signal proportionate to analyte levels. 

Another alternative format that uses bioluminescence and nucleic acid-based proximity detection to measure biomolecules rapidly and with high sensitivity, without requiring wash steps, is the Exazym® BOLD technology introduced by Cavidi. BOLD stands for Binding Oligo Ladder Detection, an ELISA-enhancing technology. It confirms the presence of a target analyte by using two antibodies (or binding proteins), each conjugated to a short oligonucleotide (DNA/RNA strand). When both antibodies bind to the same target molecule, their oligonucleotides come into close proximity, enabling a templated enzymatic or hybridization reaction that generates an amplified bioluminescent signal for immunoassays. Light intensity is measured using a luminometer, correlating directly with analyte concentration. There are several design variants, such as:

•    Split-luciferase complementation, which resembles Lumit immunoassays, but uses oligonucleotide guidance.
•    DNA-ligation-based systems where a ligase joins oligonucleotiodes only when close enough.
•    Hybridization-induced polymerase extension, enabling signal amplification.

The BOLD format embodies key NAM principles of replacement, reduction, and refinement by:

(a) enabling robust biomarker analysis in vitro, thereby reducing animal experiments

(b) detecting low-abundance biomarker signals in minimal sample volume applications (e.g. as run on the Gyrolab platform)

(c) allowing detailed insights from cell-based non-animal models. BOLD makes NAM assays more reliable and accessible by unlocking attomole-level sensitivity without added complexity. 

It can strengthen the bridge between sophisticated in vitro systems and meaningful translational insights, advancing the 3Rs framework. NAMs rely on alternatives like cell-based assays, organoids, microfluidics (organs on chips), or human-derived specimens. BOLD assays easily integrate into these setups due to development flexibility and lack of proprietary instrumentation. 

Essentially, BOLD and Lumit formats can be considered for fast, high-sensitivity, and automation-ready workflows, whereas ELISAs are suited best for cost-sensitive, flexible, and well-established applications with existing infrastructure. Lumit and BOLD immunoassays can be implemented without major capital investments. Both outperform traditional ELISA methods due to the following key advantages:

In preclinical drug development, immunoassays remain widely used due to their proven reliability, sensitivity, and integration into existing laboratory workflows. They are flexible ranging from classic ELISAs to multiplex and miniaturized platforms like DMF and biochip arrays. Newer technologies like immuno mass spectrometry (immune-MS), HTS/HCS, SDR assays, and organ-on-a-chip systems are complementary, offering enhanced sensitivity (MS), higher throughput/comprehensive phenotyping (HTS/HCS), rapid small volume screens (SDR), and better human relevance (organ chips). However, these often require more specialized equipment, validation, or are still gaining regulatory acceptance. As the industry evolves driven by trends like personalized medicine and NAMs, immunoassays continue to adapt and stay central, while newer formats grow into their own roles.

References:

1.    Song JG, Baral KC, Kim GL, et al. Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques. Drug Deliv., 2023, 30:2183816. doi: 10.1080/10717544.2023.2183816.
2.    Meng S, Meng Y, Yang X, et al. Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing fluorescence protein and luciferase. BMC Biol., 2025, 23:51. doi: 10.1186/s12915-025-02153-7.
3.    Brian Hwang B, Alves J, Lazar D, et al. Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets. Methods Mol Biol., 2023, 2612:195-224. doi: 10.1007/978-1-0716-2903-1_15.
4.    Dixon AS, Schwinn MK, Hall MP, et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol.,2016, 11:400-8. doi: 10.1021/acschembio.5b00753.