Mouse ELISA Kits: Handling Precautions and Plate Washing

A mouse ELISA kit fails for unglamorous reasons: haemolysed serum, a wash step shortened under time pressure, or a plate left to dry between steps. The assay itself is robust — most of the variability comes from handling before and after the incubation.

Before you start: samples and reagents

Serum and plasma should be separated cleanly and spun down before freezing. Haemolysis, lipaemia and repeated freeze-thaw all interfere with the readout, and none of them is recoverable once the sample is in the well. Aliquot on first thaw and never refreeze a working aliquot.

Bring every reagent out of 2–8 °C at least 30 minutes before use so it reaches room temperature. Cold reagent added to a room-temperature plate binds unevenly across the plate. If crystals have formed in the concentrated wash buffer, dissolve them completely in a 37 °C water bath before diluting — crystals left in the buffer mean a wash concentration that varies from well to well.

During the assay

• Change tips between every reagent and every sample. Cross-contamination between adjacent wells is the most common cause of a single odd value on an otherwise clean plate.
• Hold to the incubation temperature and time in the protocol. Extending the incubation “to be safe” increases non-specific binding and raises background directly.
• Seal the plate properly. An imperfect seal lets liquid evaporate from the perimeter wells and concentrates the reaction there — the classic edge effect.
• Put a humidification tray in the incubator. It costs nothing and removes most of the evaporation problem.

Colour development and reading

Keep the TMB substrate away from light throughout, and discard it if it has already turned pale blue before use. Add the stop solution in the same order and at the same rate as the substrate, then read within 30 minutes. The coloured product fades, and an OD read later than that is simply lower than the true value.

Storage between runs

Store unopened kits at 2–8 °C, protected from light, and do not freeze them. Return unused strips to the foil pouch with the desiccant, reseal it, and use them within a month. A strip that has been sitting in an open pouch for two months is the usual explanation for a plate that worked in September and does not work in November.

Standard plate-washing procedure

Washing is where backgrounds are made or lost. The protocol is short, and every step matters:

1. Dilute the concentrate with fresh deionised water at the ratio stated in the instructions — 20-fold for most BUNSEN kits — and use it the same day.
2. Fill each well with about 300 µL, right to the brim without overflowing.
3. Soak 30–60 seconds so the buffer contacts the whole well wall, not just the bottom.
4. Drain and blot: invert the plate over a sink, then tap it firmly face-down on a thick pad of absorbent paper. Do not wipe the wells with tissue — paper fibres left behind read as signal.
5. Repeat five times. On the last cycle make sure no residual liquid remains in any well, because leftover conjugate raises background across the whole plate.
6. Move straight on to the next reagent. A plate left to dry out blocks unevenly.

Washing mistakes that raise background

• Fewer than three cycles when the protocol says five.
• Wash buffer diluted the day before, or made with tap water.
• Blotting gently instead of firmly, leaving a film of unbound protein in the wells.
• Letting the plate stand dry for more than a couple of minutes between washing and the next step.

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Note: The information on the scientific research products above is for reference only!