Technical Overview

Benchtop ELISA Automation with anAgilent BenchCel Microplate Handler
Introduction
The enzyme-linked immunosorbent assay (ELISA) is a common technique usedin labs to detect a variety of biomolecules. The process of running ELISA plates
requires several liquid handling steps between incubations and a final detection step.
When performed manually, this is a laborious process that limits throughput and canintroduce human error.
Automated systems increase throughput and assay precision, but some limit
users to proprietary reagents and consumables, or are too large for benchtop
implementation. The Agilent BenchCel microplate handler is a versatile benchtop
system designed to move microplates between two instruments that are commonlyused for ELISA – a microplate washer dispenser and a microplate reader.
Liquid handlers such as the Agilent BioTek EL406 washer dispenser automate thetedious steps required in a typical ELISA process. The EL406 is used for washingand ELISA reagent dispensing in a single platform. Multimode readers, suchas the Agilent BioTek Synergy Neo2 hybrid multimode reader, are used for theendpoint detection step of an ELISA, which is commonly done through absorbance(colorimetric) measurement. However, the Synergy Neo2 can also be used for
fluorescence- and luminescence-based ELISA, offering assay flexibility overabsorbance-only readers.
Systems such as these are well established in labs running ELISA, but a common
bottleneck in this type of workflow is the manual intervention needed to move plates
between the two instruments, limiting throughput and efficiency. The BenchCel
microplate handler bridges the two instruments, enabling fully automated batchprocessing of ELISA plates post sample addition.

Automating a typical ELISA workflow
When automating any multistep laboratory protocol such as
ELISA, throughput requirements must be balanced with assay
requirements. Correct scheduling and batch size ensuresthat each sample is handled in the same throughout fashion,
regardless of whether it is on the first plate or the last.
Important considerations for designing an automatedworkflow are:–Identifying the rate limiting step–

Understanding what time constraints exist in the assay

Automated liquid handlers are typically faster than hand
pipetting, but different operations within an ELISA workflow
will have varying times. For instance, the EL406 can add
100 µL of reagent to a 96-well microplate in 20 seconds, buta 5x wash cycle can take nearly a minute. An important time
constraint in the ELISA workflow is the incubation periods
following the additions of the secondary antibody, detection
reagent, and stop reagent. The key is to ensure that intervals
for processing the first plate at each sequential step is thesame for processing the last plate.
Consider the example ELISA protocol described in Table 1,
run on the BenchCel microplate handler with the EL406
and Synergy Neo2. Each run begins after the sample has
been added to the coated plate and the initial incubation
has occurred. The subsequent steps and estimated timings
are listed. The timings of liquid handling and read steps
are representative of those achievable on an EL406 andSynergy Neo2.
Following this protocol, it would be possible to handle batch
sizes of nine plates while adhering to the incubation times
set in the protocol, with the protocol requiring a total run
time of 81 minutes. In this case, it is assumed that eachplate contains 80 samples while allotting the remaining wells
for controls. In a 12-hour period, it would be feasible to run
eight batches of this protocol, translating to a throughput
of 72 plates or 5,760 samples (Table 2). The throughput
is determined by the timing constraints of the assay
protocol, and not the processing capability of the automatedsystem itself.
The Agilent VWorks dynamic scheduling software that
underpins the operation of the system can be used todetermine procedure run times through the use of embedded
simulation modeling. The model can be updated to include
timings for the individual protocol steps, to provide moreaccurate predictions. Before finalizing the automation
procedure, it is best practice to perform a physical dry-run on
the system to obtain real-world timings.

2Figure 1. An Agilent BenchCel microplate handler with an integrated
Agilent BioTek EL406 washer dispenser and Agilent BioTek Synergy Neo2
hybrid multimode reader fits easily on a standard lab benchtop.
Table 1. Representative ELISA protocol schedule using an
Agilent BenchCel microplate handler, Agilent BioTek EL406
washer dispenser, and Agilent BioTek Synergy Neo2 hybrid
multimode reader.
Assay Step

VolumeDuration

3x Wash350 µL

42 s

Dispense Secondary Antibody

100 µL

19 s

Incubate

NA60 min

3x Wash350 µL

42 s

Dispense Detection Reagent

100 µL

19 s

Incubate

100 µL10 minDispense Stop Reagent

100 µL

19 s

Read Absorbance

NA

27 s

Table 2. Summary of representative ELISA throughput.
Parameter

ValueProtocol Run Time81 minNumber of 96-Well Plates per Run9Average Number of Samples per Plate

80

Number of Samples per Run

720

Operation Time per Day

12 hrsNumber of 96-Well Plates per 12-Hour Shift72

Total Number of Samples per 12-Hour Shift

5,760

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This information is subject to change without notice.
© Agilent Technologies, Inc. 2021, 2022
Printed in the USA, June 20, 2022
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