Agilent EL406 User Manual

Application Note

Molecular Biology and

Biochemical

Using the Agilent BioTek EL406
Combination Washer Dispenser to
Semi-Automate a Competitive ELISA

for Melamine

Semi-automated melamine quantitation

Author

Paul Held, PhD
Agilent Technologies, Inc.

Abstract

Melamine is a nitrogen rich compound normally used as either a flame retardant
or in conjunction with formaldehyde to produce melamine resin, a durable
thermosetting plastic used in the manufacture of countertops, fabrics, and
glues. However, in addition to the normal use of melamine, several illicit uses
for the material have been reported. This application note describes the use of

the AgilentBioTek EL406 combination washer dispenser in conjunction with
AgilentBioTek BioStack and AgilentBioTek liquid handling control (LHC) software to
semi-automate a melamine ELISA.

Introduction

Melamine is a nitrogen rich compound that has gained
notoriety as a result of its illicit use as an additive that mimics
protein in foods. The practice of using melamine scrap as
an additive to animal feed and food products to give the
appearance of increased protein content is widespread in
many countries. The basis for this are the presence of three

primary amine groups, which are known to react with biuret

reagent used in many of the commonly used protein assays

(Figure 1). Recently, a scandal in China has implicated over

two dozen companies and numerous individuals of adding

melamine to milk and infant formula, leading to kidney stones

and renal failure, resulting in the deaths of several infants and

the sickening over 53,000 others. Trace amounts have been
reported by the FDA to be in some infant formula products in
the US as well.

Figure 1. Chemical structure of melamine.

The melamine assay is a competitive ELISA, where
melamine-HRP conjugate competes for binding to the

melamine antibody attached to the wells of the microplate.

Following the completion of the binding reaction, unbound
sample and conjugate is removed by washing. Substrate

reagent is immediately added and the color allowed to
develop. The color-development reaction is terminated by the
addition of stop solution and the absorbance of each well is

determined. Unknown concentrations are then determined

by interpolation from a standard curve generated by running

standards of known melamine concentrations. To easily

compare multiple experiments, the data are expressed as
a ratio to the zero-standard. This ratio is often expressed

asB/Bo.

Materials and methods

The melamine assay used was an ELISA kit from Abraxis
(Warminster, PA) and was performed as described by the
kit instructions. Samples and standards were prepared
offline and 100 µL of each was pipetted manually into the
assay plate. Plates were loaded into the AgilentBioTek
BioStack storage stacker and the assay initiated. Plates were
automatically transferred sequentially to an AgilentBioTek
EL406, where 50 µL of assay conjugate was added using the

peristaltic pump dispenser. The plates were then returned

to the BioStack and restacked to restore their original
order and allowed to complete the 30-minute incubation at

room temperature. After incubation, the plates were again

transferred automatically to the EL406 and washed four
times with 300 µL of washer buffer followed by the addition
of 100 µL of substrate solution using one of the syringe

pump dispensers. Plates were automatically returned to the

BioStack and restacked. The color development continued
for 20 minutes. After color development, the plates were
automatically returned to the EL406. A 100 µL amount of stop

solution was added using the second syringe-pump dispenser

and the plates returned to the BioStack. After restacking, the
absorbance of each well at 450 nm was determined using an
AgilentBioTek Synergy 4 multimode microplate reader.

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