8
ilic acids, formed via ω-oxidation of the alkyl chains. Subsequent
(different alkyl chain lengths and positional isomers), which makes
antibody production even more challenging. Fujita et al. have
reported the preparation of antibodies using 5-(4-sulfophenyl)-
pentanoic acid, a single substance with a short alkyl chain, as an
immunizing hapten.27 This chemical structure addressed antibody
recognition versus the sulfonic group, which is one of the most
antigenic determinants in this molecule; however, studies per-
formed in our group indicate that the contribution of the alkyl
chain in the stabilization of the antibody-analyte should not be
underestimated.28 In the field of catalytic antibodies, several
research groups have made use of heterologous immunization
strategies to elicit antibody immunoresponse against two different
â-oxidation steps result in congeners with shorter alkyl chains,
some of them extremely polar, that may remain in the environ-
ment, since further degradation of the aromatic ring occurs slowly.
The analytical determination of LAS in environmental samples
has relied on the use of chromatographic and spectrometric
techniques, such as liquid chromatography/mass spectrometry
9
-11
(
LC/MS).
The use of gas chromatography/mass spectrometry
(
GC/MS) has also been reported,12 but it involves a derivatization
step. Cleanup/preconcentration steps consisting of liquid-liquid
extraction with organic solvents followed by solid-phase extraction
SPE) procedures are always required. Their ability to adsorb to
(
29-31
the solid surfaces, through their hydrophobic side, prevents
representative results of real environmental concentrations.13
Moreover, the complexity of the chromatograms obtained may
also make routine screening and multiple analyses difficult. As
an effective alternative, immunochemical techniques could not
only afford the necessary detectability and specificity for the target
analyte but also offer other advantages, such as reliability,
simplicity, low cost, and high sample throughput capabilities.14-19
The preparation of optimum haptens as immunogens and
competitors has been regarded as the most crucial step in the
development of an immunochemical technique for small mol-
ecules. Many literature examples prove that an appropriate hapten
design determines the features of the resulting antibodies, which
mainly govern the specificity and the selectivity of an immuno-
epitopes of the molecules, simplifying hapten synthesis.
Thus,
although an ideal hapten preserving both groups would have a
spacer arm at the ortho or meta positions, the synthetic pathway
leading to these types of chemical structures could be trouble-
some. The heterologous immunization strategy devised by the
group of Prof. Masamune3
1-33
consists of successfully immunizing
the animal with two different but structurally related haptens. It
has mainly been used for zwirterionic transition-state analogues
with positive and negative charges, for which chemical preparation
of the corresponding hapten could be problematic. Instead, the
heterologous immunization using two individual haptens contain-
ing a different charge provided an opportunity to simultaneously
generate an acidic and a basic catalytic residue in the antibody
combining site. Thus, Ersoy et al.,2 designed three haptens to
produce, by heterelogous immunization, nucleophile-mediated
(phenol) amide bond cleaving catalytic antibodies having a binding
pocket with (i) a hydrophobic area, (ii) an acidic residue
complementary to the oxyanionic transition state, and (iii) a basic
residue to aid deprotonation of a phenol nucleophile and pro-
tonation of the departing amine. In all those examples, the catalytic
activity of the antibodies generated through heterologous strate-
gies is higher than those obtained by homologous immunization.
Attending to these precedents, the present paper describes for
the first time the use of this immunization approach to obtain
antibodies against an amphoteric molecule such as LAS with two
well differentiated epitopes in its chemical structure. The results
show that the antibodies obtained by applying this strategy provide
better immunoassays than those obtained through homologous
immunization strategies.
9,34
1
4,20-23
chemical technique.
calculations can be useful tools to assist prediction of which hapten
will be the most appropriate to raise antibodies;
Theoretical molecular models and
24-26
however, LAS
exhibit the particularity of being a complex mixture of substances
(
(
8) Sch o¨ berl, P. Tenside, Surfactants, Deterg. 1989, 26, 2.
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72 Analytical Chemistry, Vol. 78, No. 1, January 1, 2006