Multiplex ELISA troubleshooting

Problem Solutions

Low level count

Possible cause Recommended actions

Beads aggregate

Vortex stock and working bead
suspensions well before pipetting.

Beads settle on the well bottom

Shake plates at 700 rpm for 30 seconds
prior to acquisition or re-suspend the
beads in a well by pipetting up and down
6–8 times with a P200 pipette prior to
transferring to a sample tube for acquisition.

Vacuum too strong

Adjust the vacuum pressure so that 100
µL of 1x Wash Buffer in the wells can be
clear in 3-5 second.

Low assay signal or sensitivity

Possible cause Recommended actions

Standard not
reconstituted well

Standard(s) should be incubated on ice
for 5min after the addition of standard

Incubation time too

Follow recommended incubation time in
each step.

Excess exposure to

During incubation, cover the plate with
aluminum foil to minimize exposure of the
beads to light.

High Background

Possible cause Recommended actions


Change pipette tips after every transfer.
Remove plate seal carefully and avoid
contents from one well to mix with another.

Low Precision

Possible cause Recommended actions

Poor pipetting

Use calibrated pipettes.

from adjacent wells

Avoid well-to-well contamination during
pipetting and removal of plate seal.

Clogged filter plate

Possible cause Recommended actions

High lipid content
in the serum,
plasma or bodily
fluid samples

Centrifuge the samples at 10,000 x g for
10 min at 2-8°C. Collect the serum,
plasma or bodily fluid fraction.