Last reviewed: 18 October 2026.
This guide gives a step-by-step sandwich ELISA protocol for quantifying a protein antigen, explains what each step does, and covers the standard curve, controls and the most common causes of weak signal or high background. Timings follow a typical RayBiotech sandwich ELISA kit; always use the manual for your specific kit.
2-minute animation: sandwich ELISA, step by step · ▶ Watch on YouTube
Key points
- A sandwich ELISA captures the antigen between an immobilised capture antibody and a labelled detection antibody, giving high specificity for complex samples such as serum and culture supernatant.
- Run standards, blanks and samples in duplicate or triplicate on the same plate.
- A typical RayBiotech sandwich ELISA: 100 µL standard or sample for 2.5 hours at room temperature (or overnight at 4 °C), 100 µL biotinylated detection antibody for 1 hour, 100 µL HRP–streptavidin for 45 minutes, 100 µL TMB substrate for 30 minutes, then 50 µL stop solution and read at 450 nm immediately.
- Thorough, consistent washing between steps is the single biggest factor in background.
- TMB turns blue with HRP; acid stop solution turns it yellow, which is read at 450 nm.
- Fit the standard curve with a four-parameter logistic (4PL) model and only report samples that fall inside the standard range.
- Dilute samples that read above the top standard and re-assay; do not extrapolate.
Which ELISA format should I use?
| Format | How it works | Best for |
|---|---|---|
| Direct | Antigen on plate, labelled primary antibody | Quick checks of immobilised antigen |
| Indirect | Antigen on plate, primary antibody, labelled secondary | Measuring antibodies in serum (e.g. titres) |
| Sandwich | Capture antibody, antigen, labelled detection antibody | Quantifying proteins in complex samples |
| Competitive | Sample antigen competes with labelled antigen for limited antibody | Small molecules and antigens with a single epitope |
Sandwich ELISA protocol, step by step
- Bring reagents to room temperature and prepare wash buffer, standards and samples. Prepare a serial dilution of the standard as described in the kit manual.
- Add standards and samples: 100 µL per well in the pre-coated plate. Incubate 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.
- Wash the wells several times with wash buffer; remove all liquid after the last wash by inverting and blotting the plate.
- Add detection antibody: 100 µL biotinylated antibody per well, 1 hour at room temperature. Wash.
- Add enzyme conjugate: 100 µL HRP–streptavidin per well, 45 minutes at room temperature. Wash.
- Add substrate: 100 µL TMB per well, 30 minutes at room temperature in the dark.
- Stop the reaction: 50 µL stop solution per well; the colour changes from blue to yellow.
- Read absorbance at 450 nm immediately.
If you coat your own plates, coat the capture antibody overnight in carbonate or PBS buffer, then block (for example with BSA or casein) before step 2. Pre-coated kits skip both steps.
How do I calculate concentrations?
- Average replicate readings and subtract the blank (zero standard).
- Plot standard concentration against absorbance and fit a four-parameter logistic (4PL) curve; linear fits are only valid over a narrow range.
- Read sample concentrations from the curve and multiply by the dilution factor.
- Check replicate agreement; repeat samples whose replicates disagree.
Troubleshooting
| Problem | Common causes | Fix |
|---|---|---|
| High background | Insufficient washing, too much detection antibody, plate dried out, substrate exposed to light | Wash more thoroughly, follow reagent dilutions, never let wells dry, keep TMB in the dark |
| Weak or no signal | Reagents cold or expired, wrong order of reagents, short incubations, sample below detection limit | Equilibrate reagents, follow the step order, use full incubation times, concentrate or reduce sample dilution |
| Poor standard curve | Standard dilution errors, pipetting inaccuracy | Prepare fresh serial dilutions, change tips each step, calibrate pipettes |
| Poor replicates | Uneven washing, bubbles, edge effects | Use a multichannel or plate washer, remove bubbles, seal plates during incubation |
Accurate pipetting matters at every step; see how to choose pipette tips and calibrate pipettes.
ELISA kits
RayBiotech offers sandwich ELISA kits for human, mouse and rat cytokines, growth factors and other targets, plus antibody arrays for measuring many proteins at once. See the RayBiotech ELISA kits and antibody arrays overview or request a quote.
Sources
- Crowther JR. The ELISA Guidebook. 2nd ed. Methods in Molecular Biology, vol. 516. Humana Press; 2009. doi:10.1007/978-1-60327-254-4
- RayBiotech. Rabbit IFN-gamma ELISA Kit (ELL-IFNg) manual: incubation volumes, times and 450 nm read-out. raybiotech.com
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