RayBiotech: Next generation of ultra-sensitive ELISA
ELISA (enzyme-linked immunosorbent assay) is the gold standard for protein detection, with three key platforms: standard ELISA (sELISA), immuno-PCR (IQELISA®/BIQ-ELISA™), and single molecule array (Simoa).
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Find products for your researchEach platform offers distinct advantages in sensitivity, sample requirements, and cost.
Standard ELISA
sELISA uses a 96-well microplate with a capture antibody that binds the target protein, while a detection antibody linked to an enzyme catalyzes a colorimetric reaction. Data can be obtained using a plate reader within ~5.5 hours. Its sensitivity ranges typically from 1 pg/mL to 100 pg/mL. sELISA is widely used due to its simplicity, compatibility with a broad range of protein targets, and doesn’t require costly equipment. However, it has the lowest sensitivity compared to more recently advanced platforms.
Immuno-PCR and Bead-based Immuno-PCR
Immuno-PCR, including IQELISA and BIQ-ELISA, merges ELISA with PCR amplification. These two platforms differ in that IQELISA adheres capture antibodies onto a 96-well PCR plate, while on the BIQ-ELISA platform, detection antibodies are coupled to microspheres. In both cases, the antibodies are tagged with DNA barcodes, producing signals that are measured via real-time PCR. BIQ-ELISA achieves very low background noise, enabling 100- to 1000-fold greater sensitivity than sELISA, with limits of detection (LOD) in the femtomolar range (3–25 fg/mL). Additionally, BIQ-ELISA requires only 5 µL of sample per replicate and offers a streamlined workflow (~4 hours). While highly sensitive, both of these methods demand precise pipetting and are technically more challenging than sELISA.
Simoa
Simoa achieves ultrasensitivity using antibody-coated beads and fluorescent detection within an array of over 235,000 microwells. Its LOD can range from 10 fg/mL to 1 pg/mL—up to 5000-fold more sensitive than sELISA. Data collection is fast (~3 hours), but Simoa’s costly, specialized instrumentation and significant sample volume requirements limit accessibility.
Key Takeaways
- Sensitivity: Simoa > BIQ-ELISA > IQELISA > sELISA.
- Sample Volume: BIQ-ELISA is ideal for limited samples.
- Cost: sELISA is the most affordable; Simoa and BIQ-ELISA incur higher costs.
- Multiplexing: Both immuno-PCR and Simoa can detect multiple proteins simultaneously, enhancing efficiency.
| Target | (pg/mL) | sELISA | BIQ-ELISA | IQELISA | Simoa |
|---|---|---|---|---|---|
| IL-6 | Range | 1.4 – 1000 | 0.27 – 200 | 0.24 – 1000 | 0.16 – 120 |
| LOD | 3 | 0.03 | 0.23 | 0.05 | |
| IFNγ | Range | 21 – 15000 | 0.27 – 200 | 0.7 – 3000 | 0.55 – 400 |
| LOD | 15 | 0.015 | 0.73 | 0.03 | |
| TNFα | Range | 24 – 6000 | 0.55 – 400 | 11 – 8000 | 0.55 – 400 |
| LOD | 30 | 0.025 | 7.8 | 0.01 | |
| IL-1β | Range | 0.4 – 100 | 0.14 – 100 | 0.006 – 100 | 0.08 – 60 |
| LOD | 0.3 | 0.006 | 0.006 | 0.01 | |
| IL-12p70 | Range | 0.8 – 600 | 0.27 – 200 | 0.05 – 40 | |
| LOD | 1 | 0.022 | 0.005 |
The choice of ELISA depends on the study’s sensitivity requirements, sample availability, and budget. BIQ-ELISA stands out for its combination of affordability, low sample consumption, and exceptional sensitivity, offering a practical alternative to Simoa for many applications.
