High-Sensitivity On-Bead ELISA Assay for Cancer Liquid Biopsy Immuno-Diagnostics

Overview of On-Beads ELISA Assay

Magnetic nanoparticles are an increasingly common feature in diagnostics. With their ability to disperse evenly throughout a medium, the reactive surface area of functionalized nanoparticles exhibit superior kinetics in capturing target analytes with high efficiency. NVIGEN’s nanoparticle-based ELISA assays are thus rapid, highly sensitive tests with shorter incubation times that enable a smoother workflow, provide superior results, and support multiplex assays.

NIVGEN On-Bead-ELISA Workflow

Figure 2. Basic Workflow for On-Bead ELISA Assay.

1. Antigen capture: Mix sample with antibody-conjugated beads, which will rapidly capture analyte such as sPD-L1.
2. Wash: Place plate on an NVIGEN magnetic plate (cat#A20008) and wash plasma and other proteins from the plate. The NVIGEN magnetic plate is designed to magnetize the beads into a pellet so that the supernatant can be easily removed.
3. Incubate with enzyme-bound detection antibody: Mix detection antibody in assay diluent and incubate with the beads.
4. Wash: Wash excess enzyme from the plate, again using the NVIGEN magnetic plate to pellet the beads between wash steps.
5. Add Detection Solution: Add detection solution, pipet up and down to mix, incubate for 15 minutes for the reaction to proceed.
6. Transfer detection solution to new plate, read on plate reader: Transfer the supernatant to a clean plate and read the results using a standard plate reader.

Performance:

Our bead-based PDL1 assay notably exhibits over 6x the sensitivity of a standard ELISA in which capture antibodies are adsorbed onto a plate surface. Additionally, due to the optimal bead kinetics, our assay can be performed in a fraction of the time compared to competing assays, with a lower limit of detection at 0.1 ng/mL.

Figure 3. On-Bead Elisa PD-L1 Assay Performance.

Comparison of standard on-plate versus on-bead ELISAs done in human plasma. On-Bead ELISAs demonstrate superior linearity, signal, and sensitivity. Low-end detection threshold is approximately 100 pg/mL with 200 uL of human plasma sample.

Additional Examples of On-Bead ELISA Assay Performance

Figure 4. EGFR assay performance, on-plate vs. on-bead.

Figure 5. HER2 assay performance, on-plate vs. on-bead.

Figure 6. CEA assay performance, on-plate vs. on-bead. CEA (Cancer Embryonic Antigen), more specifically known as CEACAM5 (CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 5), or CD66e, is a biomarker of a multitude of cancers, particularly colorectal cancers¹. Its role in breast cancer is an active field of research². Progressively elevated levels of CEA have been found to correlate with both the progressive stage as well as the grade of breast cancer³.

Reference

1. Beauchemin N, Arabzadeh A (December 2013). “Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis“. Cancer and Metastasis Reviews. Volume 32 p643–671.
2. Shao Y, Sun X (2015). “Elevated Levels of Serum Tumor Markers CEA and CA15-3 Are Prognostic Parameters for Different Molecular Subtypes of Breast Cancer“. PLoS ONE 10(7).
3. Al-Nafakh Z, Al-Dujaili (2020). “Assessment of Cancer Embryonic Antigen (CEA) Biomarker in Women with Breast Cancer Disease“. AIP Conference Proceedings 2290.

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