A standard sandwich ELISA in a commercial pre-coated kit takes 3.5 to 4.5 hours, from equilibration to reading the plate. Optimised rapid kits bring that down to about 2.5 hours. If you coat the plate yourself, add an overnight step: 12–16 hours at 4 °C plus 1–2 hours of blocking, for a total of 14–18 hours.
The useful part is not the total — it is which stage you can compress without losing sensitivity.
Total time by format
| Format | Total time | What changes |
|---|---|---|
| Standard pre-coated kit | 3.5 – 4.5 h | The reference protocol. Best stability and reproducibility. |
| Rapid kit | 2.5 – 3 h | Higher antibody concentration and shorter incubations. Suitable for screening; sensitivity drops slightly. |
| In-house coated plate | 14 – 18 h | Overnight coating at 4 °C (12–16 h) plus blocking (1–2 h), then the normal sequence. |
Stage-by-stage, for a standard kit
| Stage | Time | Condition |
|---|---|---|
| Reagent and sample preparation | 30 – 60 min | Bring kit to room temperature, centrifuge sample, prepare dilution series |
| Sample / antigen incubation | 1 – 1.5 h | 37 °C — antigen binds the capture antibody |
| Wash | ~10 min | 5 washes and pat dry |
| Enzyme-labelled detection antibody | 30 – 60 min | 37 °C — completes the capture–antigen–detector sandwich |
| Second wash | ~10 min | 5 washes — removes unbound conjugate and keeps background down |
| TMB development | 15 – 30 min | 37 °C, in the dark, until a clear blue gradient appears |
| Stop and read | within 15 min | Read OD at 450 nm immediately after adding stop solution |
Where the time can and cannot be cut
Washing is not the place to save time. Five short washes remove interfering material better than one long soak, and they already take only about ten minutes. Shortening them costs background.
Incubations are where the savings are — but they are a trade. Reducing antigen and conjugate incubation is exactly how a rapid kit gets to 2.5 hours, and the price is a little sensitivity. For a screen that is a fair trade; for a low-abundance analyte it usually is not.
Automation trims 15–20 % of the total, mostly by removing the manual gaps between steps: an automated washer and temperature-controlled incubation cut the waiting, not the chemistry.
The read is not negotiable. Add stop solution and read at 450 nm within 15 minutes; the colour keeps developing and the numbers drift.
Frequently asked questions
Can I leave the plate overnight between steps?
Not in the middle of the sequence. The sandwich is held together by non-covalent binding, and an overnight pause between steps lets it come apart and lets background rise. Plan the run so the incubations happen in one sitting.
Why is my run taking longer than 4.5 hours?
Usually sample preparation and dilution series, which are outside the kit protocol and can take an hour on their own. Plate washing by hand also stretches the two wash steps.
Does a longer incubation give a better result?
Beyond the specified time, no. Once binding has equilibrated, extra time adds background rather than signal.
Related reading
Note: The information on the scientific research products above is for reference only!
Leave a Reply