Enzyme-linked immunosorbent assay (ELISA) is a powerful and widely used technique for detecting and quantifying proteins, antibodies, and other biomolecules. However, even experienced researchers can encounter challenges that affect accuracy and reproducibility.
To help you achieve reliable results, we’ve compiled answers to the most frequently asked questions about ELISA experiments. Whether you’re troubleshooting unexpected results or optimizing your protocol, this FAQ covers the essentials—from sample handling to kit components.
1. What causes false negative results in ELISA?
False negatives—where a sample truly containing the target appears negative—can stem from several factors:
- Improper sample storage and handling: Repeated freeze-thaw cycles degrade proteins. Always aliquot samples and avoid multiple freeze-thaws.
- Insufficient kit sensitivity: If your target concentration is near the detection limit, a kit with inadequate sensitivity may miss it.
- Operational errors: Common mistakes include insufficient sample volume, too short incubation times, or excessive washing that removes bound antigen.
Yanda Bio’s advantage: Our ELISA kits are validated for robust sensitivity across a wide range of targets. Need higher sensitivity? We support custom sensitivity adjustments. For example, a researcher required a 50‑fold sensitivity increase for our Parathyroid Hormone 1‑34 kit—from 3000 pg/mL to 60 pg/mL. We optimized the assay to meet their exact needs. Contact us to discuss your requirements.
2. How can I optimize ELISA sensitivity?
To push your assay to its maximum sensitivity:
- Verify sample quality: Use fresh aliquots stored appropriately (short‑term at 4°C for ≤3 days; long‑term at –20°C or –80°C). Avoid repeated freeze‑thaw.
- Choose a sensitivity‑matched kit: Select a kit whose detection range aligns with your expected analyte concentration.
- Strictly follow incubation and wash protocols: Deviations in time or wash steps directly impact signal intensity.
3. How many samples can a 96‑well ELISA kit measure?
A standard 96‑well plate includes 8 wells for the standard curve (7 standards + 1 blank). The remaining 88 wells are available for samples—ideal for moderate‑throughput screening.
4. What is the recommended sample volume per well?
Always refer to the specific kit manual. Most Yanda Bio ELISA kits require 50 µL per well. Using the correct volume ensures accurate results and avoids cross‑contamination.
5. Can I mix common components from different ELISA kits?
For Yanda Bio kits: Our common components—TMB substrate, stop solution, and wash buffer concentrate—are formulated for consistency and can be used interchangeably across our kits.
However, do not mix components from different manufacturers. Formulations vary, and cross‑using them can lead to poor performance or invalid results.
6. Should I store samples at –20°C or –80°C?
For most ELISA applications, –80°C is preferable for long‑term preservation. It minimizes degradation and maintains analyte integrity.
- At –20°C: Use samples within one month.
- At –80°C: Samples remain stable for up to six months (or longer, depending on the target).
7. Can hemolyzed samples be used for ELISA?
Severe hemolysis interferes with antibody binding and can produce false results. If hemolysis is significant, we recommend collecting fresh samples before proceeding.
Mild hemolysis may sometimes be acceptable, but it’s best to evaluate on a case‑by‑case basis. Contact our technical support for guidance.
8. What types of serum samples are unsuitable for ELISA?
The following sample conditions are generally not acceptable for reliable ELISA:
- Severe hemolysis (red discoloration)
- Icteric (jaundiced) samples (yellowish from high bilirubin)
- Lipemic samples (cloudy from high lipids)
These can interfere with optical density readings and antigen‑antibody interactions. Always visually inspect samples before use.
9. Are the ELISA plates removable (strip wells)?
Yes. Our 96‑well plates are designed as break‑apart strips. You can use only the number of strips you need and reseal the remaining wells.
Each Yanda Bio kit includes a resealable zipper bag for storing unused strips at 4°C—keeping them dry and protected.
10. How long can I store an opened ELISA kit?
Once opened, store all kit components at 4°C. For best results, we recommend using the kit within one month to maintain antibody activity and reagent stability.
11. How much tissue sample is needed for an ELISA?
For tissue lysates, we recommend starting with 0.1 g of tissue. The minimum acceptable amount is 0.05 g, but using less may compromise detection if the target is low‑abundance.
12. Can you provide quality control (QC) samples?
Yes! If you need QC samples for validation, please contact our sales team. We are happy to provide them free of charge—you only pay the shipping cost.
13. What if my sample concentration is too high?
If you suspect your samples contain high analyte levels, perform a preliminary dilution test:
- Take 1–2 representative samples from each experimental group.
- Test them at 1:1 (neat), 1:10, and 1:100 dilutions.
- Identify the dilution that falls within the linear range of your standard curve, then use that dilution for your full experiment.
This simple pilot saves reagents and ensures accurate quantification.
Need More ELISA Support?
Yanda Bio is committed to empowering your research with high‑quality, affordable ELISA kits and expert technical assistance.
Why choose Yanda Bio?
- 6,000+ targets covering immunology, oncology, neuroscience, metabolism, and infectious diseases
- Custom sensitivity adjustments available
- Free ELISA testing service with kit purchase
- Rapid delivery: same‑day dispatch for orders before 3:30 PM; next‑day for others
- Bulk pricing: Standard kits from just $120—contact us for unbeatable quotes on large orders
Ready to place an order or have a technical question?
