Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 119

Competitive immunoassays may also be used to determine small chemical sub-
stances [10, 11]. An electrochemical immunosensor based on a competitive immu-
noassay for the small molecule estradiol has recently been reported [11]. A schematic
diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG
was physisorbed onto the surface of an SPCE. This was used to bind monoclonal
mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a stand-
ard solution of estradiol (E 2 ), followed by a solution of AP-labeled estradiol (AP-E 2 ). The
E 2 and AP-E 2 competed for a limited number of antigen binding sites of the immo-
bilized anti-estradiol antibody. Quantitative analysis was based on differential pulse
voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the
enzyme substrate 1-naphthyl phosphate by AP-E 2 . The analytical range of this sensor
was between 25 and 500 pg mL 1 of E 2 .
There are many other examples of competitive electrochemical immunoassays and
immunosensors for detecting clinically important analytes [12-14]. Despite simplicity,
a disadvantage of competitive immunoassays is that labeling the analyte may reduce,
or totally remove, its binding affi nity for antibody. This would occur if the analyte
were labeled at a site that is closely associated with an epitope.

5.3.2 Non-competitive immunoassay systems
In a non-competitive immunoassay (also known as a two-site “sandwich” immu-
noassay), the sample analyte is captured by an excess of a capture antibody, separating
it from the bulk sample. The captured analyte is then exposed to an excess of sec-
ond signal antibody, which will only bind to the existing capture antibody-analyte
complex. As shown schematically in Fig. 5.2b, this structure is now a classic two-site
immunoassay complex in which the analyte is sandwiched between two antibodies.
In this system, the signal antibody is often conjugated to either an enzyme label or an
electroactive label that produces a signal proportional to the amount of bound analyte.
In an ideal non-competitive immunoassay, no signal would be produced in the absence
of any analyte because there are no appropriate sites available for binding to the sig-
nal antibody. However, in practice, this is not the case due to non-specifi c interactions
between the signal antibody and other components of the immunoassay. Therefore, it
is always desirable to use a blocking reagent to reduce these non-specifi c interactions.
Non-specifi c adsorption also needs to be considered when determining the quantity of
signal antibody for use in a system. Although this immunoassay format often offers
superior specifi city, it can only be used for the quantifi cation of analytes with two anti-
genic determinants that can be simultaneously recognized. Aguilar et al. proposed a
model system for a miniaturized two-site sandwich immunoassay using murine IgG
as a model analyte [15]. The assay involved immobilizing the capture antibody (anti-
murine IgG) to the surface of a gold disk microelectrode of 50

m in diameter. The
antibody was used to capture the analyte murine IgG, which was subsequently bound
by a signal antibody conjugated with the enzyme AP. The signal antibody was spe-
cifi c for a different site of the analyte IgG. Upon the catalytic conversion of the sub-
strate, 4-aminophenyl phosphate, by AP to form 4-aminophenol, the magnitude of the

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