Common Problems about ELISA

Problem

Possible causes

Solutions

Poor precision

Incomplete washing

Ensure that the washing apparatus is working correctly;

Ensure adequate time of soaking.

Inadequate aspiration of wells

Wells should be fully dry after aspiration.

Incomplete mixing of reagents

Ensure adequate mixing.

Buffers contaminated

Prepare fresh buffer.

Improper dilution of standard

Use diluent in the kit as blank value;

Ensure accurate dilution of highest standard;

Ensure accurate completion of 2-fold dilution series.

Poor standard curve

Incomplete washing

Ensure that the washing apparatus is working correctly.

Inadequate aspiration of wells

Wells should be fully dry after aspiration.

Unequal volumes added to wells

Check pipettes;

Recalibrate the pipette if necessary.

Edge effect

Uneven temperatures among wells

Adhere to recommended incubation time and temperatures.

Evaporation caused by inadequate covering of plate sealer

Ensure correct use of plate sealer.

The assay was interrupted

The assay should be continuous.

All standards/samples should be prepared appropriately before the assay.

High background

Reagents are not at room temperature

Allow all reagents to reach room temperature (18-25℃) before use.

Incomplete washing

Ensure that the washing apparatus is working correctly.

Ensure adequate time of soaking.

Inadequate aspiration of wells

Wells should be fully dry after aspiration.

Standard curve achieved but poor discrimination between points.

Improper calculation of standard curve dilutions

Check the calculation results and make new standard curve

Insufficient washing

Ensure sufficient washing

Plate sealer reused

Use a fresh plate sealer for each step

Not used plate sealers

Use plate sealers

Buffer contaminated

Prepare fresh buffer

Insufficient washing

Ensure sufficient washing

If using an automatic plate washer, check that all ports are clean and unobstructed.

Variations in incubation temperature

Adhere to recommended incubation temperature;

Avoid putting plates in variable environmental conditions

Poor reproducibility

Plate sealer reused, thus resulting in presence of residual HRP

Use fresh plate sealer for each step.

Pipetting error

Check pipettes;

Low signal or no signal

Buffer contaminated

Make fresh buffer.

Determinant level contained in samples is above the kit’s detection range

Dilute samples and run again

Incorrect wavelength

Check the filters/reader.

Stop solution was not added

Add stop solution to each well.

Insufficient incubation time

Increase the incubation time.

Incubation temperature vary wildly

Check the incubator.

Incorrect dilutions of detection Ab/HRP-conjugate

Adhere to the manual.

The kit has lost its activity

Get a new kit.

Plate not incubated long enough

Increase Substrate Solution incubation time.