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NexaTag® qIPCR ELISA
The Next Generation of Ultrasensitive Protein Quantification
RayBiotech NexaTag® technology is a bead-based quantitative immuno-PCR (qIPCR) platform that delivers ultrasensitive protein quantification by combining the specificity of sandwich-based ELISA, the amplification power of real-time PCR, and the versatility of magnetic bead handling. With detection limits in the femtogram per milliliter (fg/mL) range from as little as 5 µL of diluted sample, NexaTag kits achieve a level of sensitivity rivaling digital immunoassays like Simoa—without the need for dedicated capital equipment.
Because each detection antibody carries its own DNA barcode paired with its own sequence-specific probe, multiplex panels enable simultaneous quantification of up to 5 proteins in a single reaction without sacrificing sensitivity.
How NexaTag Works
NexaTag leverages the principles of quantitative immuno-PCR (qIPCR) in a bead-based sandwich assay format:
Assay Procedure
- Mix – Magnetic beads pre-coated with capture antibodies are mixed with detection antibodies that are conjugated to a unique DNA oligonucleotide (barcode).
- Incubate – The samples and standards are added and incubated for 2.5 hours with shaking. If the target antigen is present, it binds to both the capture and detection antibodies to form a sandwich immunocomplex.
- Wash – Assay diluent is added and the beads are washed using a magnetic plate separator (automated plate washer compatible but not required).
- Amplify – The DNA barcodes are eluted and amplified via real-time PCR.
- Quantify – Fluorescent signals from dsDNA-binding dyes are measured, with signal intensity proportional to the target protein concentration.
Choosing your format: Singleplex or Multiplex
Both formats use the same technology, the same 4-hour workflow, and similar instrumentation. The choice comes down to how many targets you need and how much sample you have.
Singleplex | Multiplex | |
|---|---|---|
Targets per well | 1 | Up to 5 |
Sample volume per well | 5 μL (diluted) | 5 μL (diluted) |
Sample volume per target | 5 μL | 1 μL |
qPCR channels required | 1 | 5 |
Sensitivity | As low as fg/mL (target-dependent) | As low as fg/mL (target-dependent) |
Assay time | ~4 hours | ~4 hours |
Best for | Single validated biomarker; deep dose-response; instruments with limited channel capacity | Panel-level profiling; precious or volume-limited samples; multi-analyte screening with minimal sample draw |
NexaTag brings together the best of ELISA and qPCR — no trade-offs, just results you can trust.
Key Advantages
Ultrasensitive Detection | Limits of detection down to femtogram/mL — up to 1000 times more sensitive than standard ELISA |
|---|---|
Low Sample Volume | Requires as little as 5 μL per replicate of diluted sample per well (1 µL per target in a 5-plex panel) — ideal for precious samples (e.g., CSF) |
High Specificity | Sandwich-based format ensures dual recognition of the target antigen, reducing false positives |
Fast Protocol | Complete processing in ~4 hours, with antibodies mixed prior to sample incubation for streamlined workflow |
Multiplex Advantage | Five targets quantified simultaneously in a single well using sequence-specific probes across five discrete fluorescence channels — with sensitivity matching singleplex |
Applications
- Inflammation and immune profiling — resolve cytokine networks rather than single mediators, from one small-volume sample
- Biomarker discovery and validation — screen panels of low-abundance candidates in plasma, serum, and complex matrices
- Neuroscience and neurodegeneration — multi-analyte CSF profiling where sample volume is the binding constraint
- Oncology and immuno-oncology — coordinated readouts across immune signaling axes
- Longitudinal and preclinical studies — repeated sampling from small animals without exhausting the specimen
- Translational and clinical research — panel-level data with ELISA-grade specificity
Considerations
While NexaTag delivers unmatched sensitivity, it requires:
- Magnetic plate separator (Catalog #BIQ-MPS or similar)
- Exceptional pipetting precision
- Triplicate sample measurements to account for small-volume variability
With good lab technique, NexaTag enables highly precise quantification with just 7.5 µL of sample using a 2X dilution.
See What Ultrasensitivity Can Do For Your Research
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