sulfonamide moiety was situated at the immunodominant position
by linkage through the N1 group, antibodies with a broad cross-
reactivity pattern were not formed. Haasnoot et al.3,23 and Pastor-
Navaro et al.24 used various sulfonamide derivatives with the
common moiety remote to the protein surface for immunization
of animals in an attempt to raise generic antibodies. Their
polyclonal and monoclonal antibodies were surprisingly specific,
and only a limited number of sulfonamide species could be
detected in ELISA. Spinks et al.25 proposed a strategy based on
the heterologous combination of antibodies against sulfachloro-
pyridazine and coating conjugates being modified at the N1
position. Although this reagent combination proved to have
specificity, recognizing 15 out of the 21 drugs tested, the assay
response to most of the sulfonamide species was weak and
therefore not applicable in real screening analysis.
Recently, recombinant antibodies with a broad-specificity
pattern for the class of sulfonamide compounds have been
prepared by Korpim¨aki et al.26-29 Mutagenesis and phage display
selection26 were employed by these authors to manipulate analyti-
cal properties of the monoclonal antibody produced by Haasnoot
et al.23 The use of protein engineering in the following develop-
ment phases resulted in antibody mutants with broad selectivity,
capable of simultaneous detection of 10-18 sulfonamides at the
regulatory levels.27-29
From our previous study, it appeared that the cross-reactivity
pattern for sulfonylurea herbicides was considerably enlarged
when the s-triazine moiety, common to these herbicides, was
linked to a carrier protein. In this case, a one-ring s-triazine moiety
representing a part of the sulfonylurea molecule was employed
as a fragment-derived hapten evoking a generic immune re-
sponse.30 The produced monoclonal antibody exhibited a sensitive
response toward eight related sulfonylurea species having a cross-
reactivity pattern in the range of 21-167%. The results prompted
us to apply this approach to a broad family of sulfonamide drugs,
which have an aromatic one-ring moiety common to them all. The
unique chemical pattern of the sulfonamide species offered us a
suitable model system to study the general validity of the above
concept based on the use of the fragment-derived hapten ap-
proach. The aim of this study was to particularly explore the
capability of the p-aminobenzenesulfonamide moiety attached to
a carrier protein through the N1 position to evoke a generic
response of interest. The evaluation of the peroxidase tracers and
coating conjugates based on sulfonamide-derived hapten frag-
ments in heterologous assay systems was an additional objective
of this work.
EXPERIMENTAL SECTION
Reagents and Chemicals. 2-(4-Aminobenzensulfonylamino)-
ethanoic acid, 4-(4-aminobenzensulfonylamino)butanoic acid, 6-(4-
aminobenzensulfonylamino)hexanoic acid, 4-(4-aminobenzensul-
fonylamino)benzoic acid, and 6-(4-aminobenzensulfonylamino)py-
ridine-3-carboxylic acid were synthesized at Biosfor (J. Socha; Na
Labisti 533 53 009, Pardubice, Czech Republic). Bovine serum
albumin (BSA), thyroglobulin (TG), ovalbumin (OV), dimethyl-
formamide (DMF), dimethyl sulfoxide (DMSO), N-hydroxysuc-
cinimide (NHS), dicyclohexylcarbodiimide (DCC), horseradish
peroxidase (HRP), swine immunoglobulin against rabbit immu-
noglobulin-HRP (SwAR), Freund’s Complete Adjuvant (FCA),
Freund’s Incomplete Adjuvant, Sephadex G-25, p-aminobenzoic
acid, sulfamethazine, sulfamerazine, sulfadimethoxine, sulfaceta-
mide, sulfamethoxazole, sulfachloropyridazine, sulfamethoxypy-
ridazine, sulfapyridine, sulfathiazole, sulfamethizole, sulfaguani-
dine, and sulfisoxazole were obtained from Sigma Aldrich Chemie
(Steinheim, Germany). 3,3′,5,5′-Tetramethylbenzidine was pur-
chased from Serva (Heidelberg, Germany). Sulfadiazine, sul-
famethoxydiazine, sulfanilamide, sulfachloropyrazine, sulfaqui-
noxaline, sulfadoxine, and sulfaphenazole were donated by K.
Frgalova (Institute for State Control of Veterinary Biologicals and
Medicaments, Brno, Czech Republic); N4-acetyl sulfadiazine and
N4 acetyl sulfamethazine were received from CH. T. Elliott
(Veterinary Sciences Division, Department of Agriculture and
Rural Development, Belfast, Nothern Ireland, U.K.).
Instrumentation. Maxisorp microtiter plates were purchased
from Nunc (Roskilde, Denmark) and Vivapore 5 mL concentrators
from Vivascience (Hannover, Germany). Autostrip washer EL 50
(Bio-Tec Instruments, Inc.) was used for washing the microtiter
plates. Ultramicroplate reader EL 808 with software KC4 v3.1 (Bio-
Tec Instruments, Inc.) and Origin v7.5 (OriginLab Corp.) were
used for the absorbance measurement in plates and the processing
of ELISA results.
Buffers and Solutions. The following solutions were used:
(1) coating buffer, prepared as 50 mmol‚L-1 carbonate buffer (pH
9.6); (2) assay buffer (phosphate-buffered saline, PBS), 10 mmol‚L-1
phosphate buffer with 145 mmol‚L-1 NaCl (pH 7.2); (3) assay
buffer-BSA, 10 mmol‚L-1 phosphate buffer with 145 mmol‚L-1
NaCl (pH 7.2), 5 g‚L-1 BSA (blocking agent); (4) wash buffer:
PBS buffer with 0.1% (v/v) Tween 20 (PBST20), (5) acetate
(substrate) buffer, 0.1 mol‚L-1 sodium acetate adjusted to pH 5.5
by addition of 1 mol‚L-1citric acid; (6) dialysis solution, 0.1 mol‚L-1
(NH4)2CO3 in water; (7) 0.13 mol‚L-1 NaHCO3 in water; (8) TMB
solution, 10 mg‚mL-1 TMB in DMSO; (9) substrate solution,
prepared by addition of 1 mL of acetate buffer, 200 µL of TMB
solution, and 20 µL of 6% H2O2 to 20 mL water; (10) 2 mol‚L-1
H2SO4 as the stopping reagent.
(19) Thomson, C. A.; Sporns, P. J. Food Sci. 1995, 60, 872-879.
(20) Thomson, C. A.; Sporns, P. J. Food Sci. 1995, 60, 409-415.
(21) Cliquet, P.; Cox, E.; Haasnoot, W.; Schachet, E.; Goddeeris, B. A. Anal. Chim.
Acta 2003, 494, 21-28.
(22) Kohen, F.; Gayer, B.; Zaltsman, Y. A.; O’Keeffe, M. Food Agric. Immunol.
2000, 12, 193-201.
(23) Haasnoot, W.; Du Pre, J.; Cazemier, G.; KemmersVoncken, A.; Verheijen,
R.; Jansen, B. J. M. Food Agric. Immunol. 2000, 12, 127-138.
(24) Pastor-Navarro, N.; Garcia-Bover, C.; Maquieira, A.; Puchades, R. Anal.
Bioanal. Chem. 2004, 379, 1088-1099.
(25) Spinks, C. A.; Wyatt, G. M.; Everest, S.; Jackman, R.; Morgan, M. R. A. J.
Sci. Food Agric. 2002, 82, 428-434.
(26) Korpima¨ki, T.; Rosenberg, J.; Virtanen, P.; Karskela, T.; Lamminma¨ki, U.;
Tuomola, M.; Vehnia¨inen, M.; Saviranta, P. J. Agric. Food Chem. 2002, 50,
4194-4201.
(27) Korpima¨ki, T.; Rosenberg, J.; Virtanen, P.; Lamminma¨ki, U.; Tuomola, M.;
Saviranta, P. Protein Eng. 2003, 16, 1, 37-46.
(28) Korpim¨aki, T.; Brockmann, E. Ch.; Kuronen, O.; Saraste, M.; Lamminm¨aki,
U.; Tuomola, M. J. Agric. Food Chem. 2004, 52, 40-47.
(29) Korpima¨ki, T.; Hagren, V.; Brockmann, E. Ch.; Tuomola, M. Anal. Chem.
2004, 76, 3091-3098.
Standards. Stock solutions were prepared by adding 1 mg of
sulfonamide to 10 mL of methanol (100 mg‚L-1). For the
calibration series and for testing inhibition reactions, series of
concentrations were prepared in the range of 0.5-10 000 µg‚L-1
,
using individual sulfonamides. An equimolar mixture consisting
of 19 sulfonamides was prepared from aliquots of each stock
(30) Kolar, V.; Deng, A.; Franek, M. Food Agric. Immunol. 2002, 14, 41-105.
Analytical Chemistry, Vol. 78, No. 5, March 1, 2006 1561