Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 117

The binding affi nity between an antibody (Ab) and an antigen (Ag) can generally
be described by the equilibrium expression:

[Ab g]

K

[ []

(1)

Ab Ag

where K is the equilibrium constant for the interaction and Ab ß Ag is the immuno-
complex formed between Ab and its specifi c Ag. Typical values of K range from 10 6
to 10 12 L mol 1 . In general, only antibodies yielding a high K value ( 10 8 L mol 1 )

will exhibit low cross-reactivity. These features thus make many antibodies ideal bio-
logical recognition components in immunoassays and biosensor design. In addition,
monoclonal antibodies are particularly suitable for use in immunoassays [8]. These are
antibodies produced by a cloned cell line, and thus have the same epitopic specifi city
and affi nity. This homogeneous population can be made in large numbers. Therefore,
they offer superior specifi city and homogeneity compared to polyclonal antibodies,
reducing the need for laborious purifi cation.

5.3 IMMUNOASSAYS AND IMMUNOSENSORS

Immunoassays are the quantitative methods of analysis where antibodies are the
primary binding agents for the antigen (which is often the analyte) of interest. Notably,
the K value in Eq. (1) dictates the equilibrium and the binding between an antibody
and its antigen in a particular system. The net results of an immunoassay are thus often
the investigation of the binding between an antibody and its antigen and the differen-
tiation between bound and unbound antigen. In other words, all immunoassays depend
on measuring the fractional occupancy of the recognition sites. However, such a meas-
urement can rely on either the assessment of occupied sites or, indirectly, on meas-
uring unoccupied sites. This leads to the development of either a “competitive” or a
“non-competitive” immunoassay format, as described below.

5.3.1 Competitive immunoassay systems
In a competitive immunoassay, the sample analyte is mixed with labelled analyte,
which both compete for a limited number of antibody-binding sites. This is schemati-
cally depicted in Fig. 5.2a. In electrochemical immunoassays, an enzyme label or an
electroactive label is commonly used (see section 5.5.2). Quantitative analysis can be
achieved by determining the amount of labeled analyte that interacted at the binding
sites. Therefore, with a fi xed number of antibody sites, a smaller signal is expected
when the ratio between the quantity of sample to labeled analyte is large. In con-
trast, a larger signal is obtained when there is a small quantity ratio. Hence, the signal
produced by the bound labeled analyte is usually inversely proportional to the amount
of sample analyte. An example involving competitive immunoassays is a voltammet-
ric enzyme immunosensor for the determination of rabbit IgG [9]. In this work, the