Engineering of a Broad Specificity Antibody for Simultaneous Detection of 13 Sulfonamides at the Maximum Residue Level

Article abstract of DOI:10.1021/jf034951i

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.

Full text of DOI:10.1021/jf034951i

40 J. Agric. Food Chem. 2004, 52, 40−47
Engineering of a Broad Specificity Antibody for Simultaneous
Detection of 13 Sulfonamides at the Maximum Residue Level
TEEMU KORPIMA¨ KI,*,† EEVA-CHRISTINE BROCKMANN,† OUTI KURONEN,‡
†
MAIJA SARASTE,† URPO LAMMINMA¨ KI,† AND MIKA TUOMOLA
Departments of Biotechnology and Bio-Organic Chemistry, University of Turku, Turku, Finland
Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide
moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of
medicated animals is contaminated with residual sulfonamides. Current analytical methods for
sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or
specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single
reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity
of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-
directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient
for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the
functionality of one mutant in some real sample matrices.
KEYWORDS: Food safety; drug residues; group specificity; phage display; sulfonamides; directed
mutagenesis
INTRODUCTION
Antibiotic molecules sharing the p-aminobenzenesulfonamide
moiety (sulfa antibiotics or sulfonamides; Figure 1) are used
in veterinary and human medicine for therapeutic and prophyl-
actic purposes. Sulfonamides are also sometimes used as
additives in animal feed, since prolonged ingestion of sulfas is
thought to have growth-promoting effects. If the proper
withdrawal periods are not observed before slaughtering or
milking of the medicated animals, meat and milk from these
animals may be contaminated with residual sulfonamides. It has
been estimated that approximately 5% of the patients on
sulfonamide medication receive unwanted symptoms from the
drugs (1); thus, the presence of sulfonamide residues in food
can be considered harmful to the consumers. A legal maximum
residue limit (MRL) for sulfonamides has been set to 100 µg/
kg in the United States and the European Union, whereas in
Japan, for example, it has been set to 20 µg/kg (2, 3).
Current conventional sulfonamide detection methods capable
of measuring a wide spectrum of different sulfonamides include
bacteriological growth inhibition (4-6), chromatography (7-
13), and capillary electrophoresis (14, 15). While these methods
may be robust, immunochemical assays such as fluorescence
immunoassay (FIA) have a number of advantages including
speed, simplicity, and low costs over these methods. A number
of immunochemical assays with these advantages have been
developed demonstrating the suitability of immunochemical
Figure 1. Structures of selected sulfonamides.
assays for sulfonamide screening (3, 16-21), but unfortunately,
each of these assays was capable of detecting only a single type
of sulfonamide.
Immunochemical assays able to detect more than one type
of sulfonamide with a sufficient sensitivity for the enforcement
of the MRL have also been developed. Situ et al. (22) used a
multichannel surface plasmon resonance biosensor approach and
managed to build a fast assay for sulfamethazine (SHZ) and
sulfadiazine (SDZ) in porcine bile, which they employed in an
on-site study at an abattoir. Several authors (1, 23-25) managed
to isolate monoclonal antibodies (Mab), which were capable of
binding a few different sulfonamides with adequate affinity.
Haasnoot et al. (2) isolated Mab 27G3, which bound a wide
variety of sulfonamides with measurable affinity. Protein
* To whom correspondence should be addressed. Tel: +358-2-3338685.
Fax: +358-2-3338050. E-mail: teemu.korpimaki@utu.fi.
† Department of Biotechnology, University of Turku.
‡ Department of Bio-Organic Chemistry.
10.1021/jf034951i CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/11/2003

An Antibody for Detection of Sulfonamides
J. Agric. Food Chem., Vol. 52, No. 1, 2004 41
engineering of this antibody (26, 27) produced binders capable
of detecting 10 of the tested 13 sulfonamides in a buffer system
with an adequate sensitivity for screening of sulfonamide
contamination below the EU MRL. Recently, using SHZ Mab
21C7 (19), which can bind a wide variety of sulfonamides,
Haasnoot et al. (28) demonstrated the detection of eight
sulfonamides below the MRL concentration from chicken serum
with a surface plasmon resonance biosensor.
So far, there has not been an immunochemical assay that
would be able to detect all sulfonamides with an adequate
sensitivity so that all sulfas could be screened for in a single
reaction. The main obstacle seems to be the development of an
antibody capable of binding all of the different sulfonamides
with affinities yielding sufficient assay sensitivity. In our
previous studies (26, 27), we used protein engineering to
improve Mab 27G3 (2) to recognize a wider range of structurally
different sulfonamides with similar affinities. Several of the
obtained mutants had significantly altered binding properties
and improved broad specificity.
In this study, we aimed at improving the best mutant of the
previous studies (mutant A.3.5) so that it would recognize all
sulfonamides with an affinity sufficient for screening of
foodstuffs. We used site-directed random mutagenesis to mutate
selected residues of mutant A.3.5. The targeted residues were
chosen based on their proximity to the assumed antigen binding
site in a homology model of Mab 27G3 (27) and their tendency
to mutate in the previous studies. The resulting new libraries
were enriched with hyperphage display (29) using a SHZ
derivative as the collection antigen. The binding properties of
the new mutants were evaluated in a competitive time-resolved
fluoroimmunoassay. The functionality of the best obtained
mutant in different sample matrices was also briefly demon-
strated by using it in the analysis of SHZ from semiskimmed
milk, chicken serum, and minced beef samples.
Figure 2. Plasmid vectors used. Two different plasmids constructed in
our lab were used as the gene-carrying vectors in this work. The E. coli
propagation signal (ColE1), the M13 phage packaging signal (f1-IG) and
the chloramphenicol resistance selection marker [Cam(R)] are needed
for the functioning of the vectors as phagemids. ScFv antibody (V , VH,
MATERIALS AND METHODS
L
and linker-domains are separately marked) expression from both plasmids
is controlled by the lac promoter (lac PO) repressed by the lac inhibitor
(lacI). The signal sequence (pel B) activates transport of scFv to periplasm
for folding. The full length M13 phage protein III (M13 pIII) fused to the
N terminus of the scFv by a short trypsin cut site facilitates the display of
scFv antibodies on the surface of M13 hyperphage. The infectivity function
of protein III is restored by trypsin digestion, which removes the fused
scFv. The antibody constant light domain (Cl) enables scFv immobilization
on rabbit antimouse IgG-coated plates.
Strains, Plasmids, Reagents, and Instruments. The bacterial host
used throughout this work was Escherichia coli K12 strain XL1-Blue
(Stratagene, La Jolla, CA). The vectors used (Figure 2) were
constructed from the pAK series of vectors (30) in our lab. The original
pAK vectors were obtained as gifts from the lab of Andreas Plu¨ckthun
(Biochemisches Institut, Universita¨t Zu¨rich, Switzerland). The helper
phage used in the phage production was hyperphage M13K07∆pIII
(KanR, Progen Biotechnik, Heidelberg, Germany). Recombinant single-
chain antibody (scFv) A.3.5 was previously engineered in our lab (26,
27) from mouse antibody 27G3A9B10 (2), which was a gift from
Willem Haasnoot (RIKILT-DLO, Wageningen, The Netherlands). The
residue numbering of scFv A.3.5 follows the scheme devised by Kabat
et al. (31) throughout this article.
antibiotics (Figure 1), fraction V bovine serum albumin (BSA), and
aseptic trypsin from bovine pancreas were purchased from Sigma-
Aldrich.
All reagents used in the organic synthesis were commercially
available and were of reagent grade or better. Rabbit Anti-mouse IgG-
coated microtiter plates, dissociation-enhanced lanthanide fluoroim-
munoassay (DELFIA) assay buffer, DELFIA wash solution, and
DELFIA enhancement solution were obtained from Perkin-Elmer Life
Sciences (Turku, Finland). The biotinylation reagent Biotin-XX-NHS
(catalog no. 203114) was purchased from Calbiochem (San Diego, CA).
The 9dEu-chelate (({2,2′,2′′,2′′′-{[2-(4-isothiocyanatophenyl)ethylimi-
no]bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl}pyridine-
6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium (III)) for tracer
labeling was synthesized in house according to protocols from Innotrac
Diagnostics (Turku, Finland). The LB agar plates, SB medium, and
SOC medium were prepared as described previously (32). The
antibiotics used in the cultures were obtained from Sigma-Aldrich
(Helsinki, Finland). The concentrations of the antibiotics were as
follows: ampicillin, 100 mg/L; chloramphenicol, 25 mg/L; tetracycline,
5 mg/L; and kanamycin, 50 mg/L. Isopropyl-â-D-thiogalactopyranoside
(IPTG) was purchased from Promega (Madison, WI). The various sulfa
1H NMR spectra were recorded at 400 MHz on a JNM-GX-400
spectrometer (JEOL, Peabody, MA) or at 200 MHz on a AM200
spectrometer (Bruker, Ta¨by, Sweden). The chemical shifts are given
in ppm from internal tetramethylsilane. Time-resolved fluorescence was
measured with a Victor 1420 Multilabel Counter (Perkin-Elmer Life
Sciences).
Synthesis of 6-Methyl-N1-{[4-(5-amino)pentyl]-2-pyrimidyl}-
sulfanilamide Monohydrochloride. (6-N-tert-Butoxycarbonylamino)-
caproic Acid a-N-Methoxy-N-methylamide (A). A was synthesized as
previously described (26).
(9-N-tert-Butyloxycarbonylamino)non-2-yn-4-one (B). Propynylmag-
nesium bromide (12.4 mL, commercially available as 0.5 M in
tetrahydrofuran) was added dropwise to a cooled (-78 °C) solution of
A (0.68 g) in dry ether (60 mL). The reaction mixture was stirred for
2 h, allowed to warm to room temperature, and stirred overnight. The
reaction mixture was poured onto a vigorously stirred mixture of Et2O
(80 mL), 1 M KH2PO4 (160 mL), and ice. The phases were separated,
and the water phase was extracted with Et2O (3 × 60 mL). The

42 J. Agric. Food Chem., Vol. 52, No. 1, 2004
Korpima¨ki et al.
Table 1. Site-Directed Mutagenesis of scFv A.3.5 Amino Acid
Residues
residue
in library
residue
in library
L:D28
L:Y32
T, A, N, or D
any
L:K30
L:N34
any
F, L, I, M, V, S, P, T, A,
Y, H, Q, N, K, D, E
F, S, Y, C, I, T, N, S
G, A, V
I, T, N, S
L, I, M, V, H, Q, N,
Figure 3. SHZ derivative with an amino linker. 6-Methyl-N1-{[4-(5-amino)-
pentyl]-2-pyrimidyl}sulfanilamide was synthesized for biotinylation and for
labeling with an Eu-chelate.
L:R46
L:Q50
L:N53
L:F94
K, R
any
any
F, Y, L, H, I, N
L:Y49
L:G51
L:T92
L:Q96
K, D, E
P, Q, R, A, E, G
T, N, S
I, T, S, V, A, G
K, R
A, G
combined organic phases were washed with aqueous 1 M KH2PO4 (2
× 60 mL) and brine (2 × 60 mL), dried (Na2SO4), and evaporated.
The product was purified with flash chromatography (FC) using silica
gel and 30% ethyl acetate in petroleum ether.
H:Y32
H:M34
H:N52
H:R94
H:G97
H:G99
any
H:G33
H:T35
H:A52a
H:R95
H:G98
L, I, M
T, A, N, K, D, E
K, R
A, G
A, G
{2-Amino-6-methyl-[4-(5-N-tert-butyloxycarbonylamino)pentyl]}-
pyrimidine (C). A mixture of B (0.128 g) and guanidium nitrate (0.074
g) in ethanol (15 mL) was heated in an oil bath at 66-68 °C until the
guanidium nitrate dissolved. A solution of NaOH in water (0.05 g/0.5
mL) was added. The reaction mixture was stirred at this temperature
for 6 h and allowed to cool to room temperature before evaporation.
The product was purified with FC (silica gel, gradient from 2% MeOH/
dichloromethane to 8% MeOH/dichloromethane).
Table 2. Sequences of the PCR Primers
primer
sequencea
5′-GGAATTCGGCCCCCGAGGCC-3′
5′-TTACTCGCGGCCCAGCCGGCCATGGCG-3′
5′-GGAGACTGGCCTGGCCTCTGTAACATCCA-
SDNCAASNNTGTSNNGCCRKYACTATGTAA-
GAGGCTCTGACTTGACTTGC-3′
5′-CAGAGGCCAGGCCAGTCTCCAAAGARRCT-
AATCNWYNNSGBNTCTNNSCTGGACTCTGG-
GGCCCCTGAC-3′
5′-CTTGGAGCCTCCTCCGAACGTNWBGGG-
RWDATGRNTACCTTGCCAGCAATAATAA-
ACCCC-3′
ScRev:
ScFor:
Mut1:
N4-Acetyl-6-methyl-{N1-[4-(5-N-tert-butyloxycarbonylamino)pentyl]-
2-pyrimidyl}sulfanilamide (D). C (0.06 g) was dissolved in dry pyridine
and cooled in an ice bath. A cooled solution of N-acetylsulfanilyl
chloride (0.055 g) in dry pyridine was added dropwise. The reaction
mixture was stirred overnight at room temperature, evaporated, and
dissolved in dichloromethane (10 mL). The solution was washed with
brine (5 mL), dried (Na2SO4), and evaporated. The product was purified
with FC (silica gel, first 3/5/1 petroleum ether/ethyl acetate/triethylamine
and finally 10% MeOH/dichloromethane).
Mut2:
Mut3:
6-Methyl-N1-{[4-(5-N-tert-butyloxycarbonylamino)pentyl]-2-
pyrimidyl}sulfanilamide (E). D (0.085 g) was dissolved in 2 M NaOH
(20 mL) and stirred at room temperature overnight. NaCl was added
to the reaction mixture followed by chloroform (30 mL). The phases
were separated, and the water phase was extracted with chloroform (2
× 35 mL). The combined organic phases were dried (Na2SO4) and
evaporated. The product was purified with FC (silica gel, gradient from
2% MeOH/dichloromethane to 8% MeOH/dichloromethane).
Mut4:
Mut5:
5′-ACGTTCGGAGGAGGCTCCAAG-3′
5′-CTGTAAACCCTTTCCTGGAGCCTGCTTCAC-
CCARBTYAKYBSSNNGTTTGTGAAGGTATA-
CCCAGAGGCC-3′
5′-CAGGCTCCAGGAAAGGGTTTACAGTGGATG-
GGCTGGATARMNRBYTACACTGGGGAGCCA-
ACATATGCAG-3′
5′-GAGTCCCTTGGCCCCAGTAAGCNSCNSCNS-
CATCYYTYYTTGCACAGAAATATGTAGCCAT-
GTCCTC-3′
5′-GCTTACTGGGGCCAAGGGACTC-3′
5′-CAGAGGCCAGGCCAGTCTCC-3′
5′-GGAGACTGGCCTGGCCTCTG-3′
Mut6:
Mut7:
6-Methyl-N1-{[4-(5-amino)pentyl]-2-pyrimidyl}sulfanilamide. E (0.022
g) was dissolved in 1 M HCl and stirred overnight at room temperature.
The reaction mixture was evaporated, and the product (Figure 3) was
Mut8:
Mut9f:
Mut9r:
1
used without further purification. H NMR (200 MHz, D2O): δ 7.87
(d, 2H, J ) 8.6 Hz), 7.37 (d, 2H, J ) 8.6 Hz), 6.59 (s, 1H), 2.75 (t,
2H, J ) 7.5 Hz), 2.43 (t, 2H, J ) 7.5 Hz), 2.18 (s, 3H), 1.50-1.29 (m,
4H), 1.22-1.00 (m, 2H). MS (EI) m/z (relative %): 193 (100), 291
(45), 349 (10) M+.
a Degenerated bases are in bold, and SfiI restriction sites are italicized.
detected at 320 nm, t ) 20.52 min. The product, from hereon referred
to as Eu-labeled SHZ (Eu-SHZ), was dried and dissolved to 1 mL of
DMF.
Biotinylation and Labeling of the Sulfonamide Derivatives.
Biotin-XX-NHS (8.2 mg) was added to the solution of 6-methyl-N1-
{[4-(5-amino)pentyl]-2-pyrimidyl}sulfanilamide (4.3 mg) in a mixture
of pyridine, water, and triethylamine (90:15:1, 0.9 mL). The reaction
mixture was stirred overnight at room temperature. The product was
purified with reversed phase high-performance liquid chromatography
(RP-HPLC) using a 150 mm × 4.6 mm, 5 µm HyPurity Elite C18
column (Thermo Hypersil, Runcorn, U.K.) with a gradient from aqueous
0.1% TFA to acetonitrile in 30 min and a flow rate of 1.0 mL/min.
The peak was detected at 265 nm, t ) 23.55 min. The product, from
hereon referred to as biotinylated SHZ (Bio-SHZ), was dried and
dissolved to 1 mL of dimethyl formamide (DMF). Because the final
product mass was too low to be measured with our equipment, dilution
of the stock solution used is given in the text instead.
Site-Directed Mutagenesis Libraries. On the basis of the model
of antibody 27G3 (27) and the mutation data of previous studies (26,
27), several amino acid residues of the CDR loops of scFv A.3.5 were
selected for site-directed mutagenesis (Table 1). A selection of
degenerated oligonucleotides (Table 2) was synthesized in order to
construct libraries with the desired mutations in the gene of scFv A.3.5.
A selection of scFv A.3.5 gene fragments was polymerase chain reaction
(PCR) amplified with appropriate primer pairs, the resulting fragments
were purified with agarose gel electrophoresis, and then, four different
libraries were assembled with strand overlap extension PCR (SOE-
PCR, 33). Lib 1 was assembled from PCR fragments given by primer
pairs ScFor-Mut9r, Mut2-Mut7, and Mut8-ScRev; Lib 2 was built from
PCR fragments amplified by primer pairs ScFor-Mut1, Mut9f-Mut3,
and Mut4-ScRev; Lib 3 was put together from PCR fragments generated
by primer pairs ScFor-Mut1 and Mut2-ScRev; and finally, Lib 4 was
constructed from PCR fragments ScFor-Mut5 and Mut6-ScRev. A
typical PCR reaction had 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2-
SO4, 10 mM KCl, 0.1% Triton X-100, 0.1 mg/mL BSA, 2 mM MgSO4,
200 µM each dNTP, 40 ng each of appropriate template fragment(s),
6-Methyl-N1-{[4-(5-amino)pentyl]-2-pyrimidyl}sulfanilamide (5.0
2-
mg) was dissolved in aqueous 50 mM CO3 buffer and stirred for 5
min before an equimolar amount of 9dEu-chelate was added. The
reaction mixture was stirred overnight at room temperature. The product
was purified with RP-HPLC using a 150 mm × 4.6 mm, 5 µm Hypersil
ODS column (Thermo Hypersil), a gradient from 95% A and 5% B to
100% B in 30 min, and a flow rate of 1.0 mL/min (A ) aqueous 20
mM TEAA and B ) 20 mM TEAA/50% acetonitrile). The peak was

An Antibody for Detection of Sulfonamides
J. Agric. Food Chem., Vol. 52, No. 1, 2004 43
1.25 U Pfu-polymerase, and 0.4 µM of each primer in a total reaction
volume of 50 µL. The PCR program used was 95 °C for 2 min, 14
cycles of 95 °C for 1 min, 54-56 °C (depending on the primers) for
1 min and 72 °C for 35 s, and the final extension of 72 °C for 5 min.
Characterization of the Active Clones. Active antibody mutants
were sequenced in order to ascertain that each contained different
mutations and that they were therefore different mutants. These mutants
were also produced in 100 mL cultures as previously described (26),
and their cross-reaction profiles for different sulfonamide antibiotics
were determined. Rabbit anti-mouse IgG-coated microtiter plates were
coated with the mutant antibodies as above. To each well, 100 µL of
assay buffer containing 6 ng/mL of Eu-SHZ was applied, followed by
a 100 µL aliquot of assay buffer containing varying concentrations of
different sulfonamides (Figure 1). The plates were incubated for 1 h
at room temperature with gentle shaking and washed four times. Time-
resolved fluorescence was measured as above. Six different concentra-
tions were measured for each sulfonamide in order to obtain data points
for the whole range of inhibition, and sigmoidal curves were fitted to
these data. The R2 values obtained from the comparison of the fitted
curves to the raw data varied between 1 and 0.97, being almost always
better than 0.99.
Mutant Functionality in Real Sample Matrices. To demonstrate
that the new mutants of this study also work in real sample matrices,
M.3.4 was applied to the measurement of SHZ from semiskimmed milk,
minced beef, and chicken serum. Semiskimmed milk (pasteurized,
unhomogenized, approximately 1.5% fat) was spiked with varying
concentrations of SHZ and incubated for 24 h in 4 °C. The milk samples
were then briefly vortexed and diluted 10-fold in assay buffer before
the assay.
Minced beef was spiked with varying concentrations of SHZ,
thoroughly mixed, and incubated for 24 h at 4 °C. To each sample of
beef, 10 mL/g of assay buffer was then added and the sample was
thoroughly vortexed. After the sample was centrifuged (4000g, 20 min),
the supernatant was used in the assay.
Chicken serum was spiked with varying concentrations of SHZ,
thoroughly mixed, and incubated for 1 h in 25 °C. The serum samples
were then briefly vortexed and diluted 10-fold in assay buffer before
the analysis.
Rabbit anti-mouse IgG-coated microtiter plates were coated with
scFv M.3.4 as above. After they were washed, 100 µL of assay buffer
containing 12 ng/mL of Eu-SHZ was applied to each well, followed
by a 100 µL aliquot of the sample. The plates were incubated for 30
min at room temperature with gentle shaking and washed four times.
A 200 µL aliquot of enhancement solution was added to each well,
and after 30 min of incubation with gentle shaking, time-resolved
fluorescence was measured. Six different concentrations were measured
for each sulfonamide, and sigmoidal curves were fitted to these data.
The R2 values obtained from the comparison of the fitted curves to the
raw data varied between 1 and 0.98.
The PCR products (about 2.5 µg per library) were cloned to pAKp3fl
phagemid vector (Figure 2) using the SfiI sites. The ligation reactions
were precipitated with ethanol and transformed to electrocompetent E.
coli XL1-Blue cells with electroporation in six separate aliquots each,
yielding approximately 2 × 107 transformants per library (determined
with platings on LB/chloramphenicol agar). Electroporated cells were
diluted to 20 mL total volume in SOC medium, and they were incubated
in shake flasks at 37 °C with 200 rpm for 1 h. Cultures were then
diluted to 100 mL with SB medium containing tetracycline, chloram-
phenicol, and 0.2% glucose. Growth was continued at 37 °C with 240
rpm, until the optical density (OD600nm) of the cultures reached 0.5.
The cultures were infected with 2.5 × 1011 pfu of helper phage
M13K07∆pIII each for 15 min at 37 °C without shaking. Cells were
pelleted with centrifugation (3000g, 10 min, 4 °C) and resuspended in
20 mL of SB medium containing tetracycline, chloramphenicol, and
0.2% glucose. After 2 h of growth at 30 °C with 240 rpm, IPTG and
kanamycin were added to the cultures to final concentrations of 500
µM and 70 mg/L, respectively. Phage production was continued
overnight (26 °C, 220 rpm). After the bacteria were removed by
centrifugation, the phages were precipitated twice by adding poly-
(ethylene glycol) 8000 (Sigma-Aldrich) to 5% and NaCl to 4%. Phages
were finally dissolved in 200 µL of TBS/BSA buffer (50 mM Tris-
HCl, pH 7.5, 150 mM NaCl, 1% BSA) and stored at 4 °C.
Enrichment of the Libraries. All four libraries were enriched with
three rounds of hyperphage display (29). The wells of a streptavidin-
coated microtiter plate (Innotrac Diagnostics) were coated by incubating
an appropriate dilution of Bio-SHZ (panning round 1, dilution 1:3000;
2, 1:3000; and 3, 1:20 000) in 200 µL of assay buffer each well for 1
h in slow shaking after which the wells were washed four times with
200 µL aliquots of wash solution. Phage libraries were diluted in TBT-
0.05 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Sigma-
Aldrich fraction V BSA, and 0.05% Tween20) to final concentration
of 3 × 1013 (for first round) or 5 × 1011 (for rounds 2 and 3) pfu/mL.
Aliquots of 200 µL of each diluted library were applied to one Bio-
SHZ-coated microtiter well and one uncoated well. The microtiter plate
was incubated in slow shaking for 1 h at 25 °C and washed 10 times
as above. Phages were eluted by adding 200 µL of TBS buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl) with 14 µg/mL of trypsin to
each well and incubating the plate in 37 °C for 1 h without shaking.
One milliliter of fresh XL1-Blue cells was mixed with each batch
of eluate. Cells were incubated for 30 min at 37 °C without agitation,
and serial dilutions of each cell batch were plated on LB/chloram-
phenicol agar. Shake flasks containing 20 mL of SB medium with
tetracycline, chloramphenicol, and 0.2% glucose were inoculated with
1 mL of infected cell suspension from each of the replicas that had
been eluted from the Bio-SHZ-coated wells. The flasks were shaken
at 37 °C with 300 rpm, until the OD600nm of the cultures reached 0.4.
The cells were then infected with 5 × 1010 pfu of helper phage
M13K07∆pIII each, and the new recombinant phage preparations were
produced as described above and used as the starting material of the
next round of enrichment.
RESULTS
Construction and Enrichment of the Site-Directed Mu-
tagenesis Libraries. On the basis of the model of antibody 27G3
(27) and the mutation data of previous studies (26, 27), a
selection of amino acid residues in the CDR loops of scFv A.3.5
was targeted for site-directed mutagenesis (Table 1). Residues
L:D28, L:K30, L:Y32, L:Q50, L:G51, L:N53, H:T35, H:N52,
and H:A52a were picked for mutagenesis, because in the model
the side chains of these residues were located in close proximity
to the assumed antigen binding site and because several mutants
with mutations in these residues were found in the previous
studies (26, 27). The rest of the residues in Table 1 were selected
for mutagenesis, regardless of the fact that no mutations in these
residues had been found in the previous studies (26, 27), because
in the model these residues were located close by the assumed
antigen binding site of the antibody. When deciding the variety
of amino acid changes applied to each residue, the environment
of each residue in the model was considered and only mutations
that we believed each particular location could accommodate
were chosen. The variety of mutations selected for each residue
is shown in Table 1. A set of primers with degenerated bases
for the introduction of the planned mutations to the scFv A.3.5
Isolation of Active Clones. The genes of 20 random clones from
each enriched library were transferred to the pAK100Cl vector (Figure
2) using the SfiI restriction sites, and cell lysates were prepared for
antibody activity measurements as previously described (26). SHZ
binding activity was determined using a time-resolved DELFIA (34).
Rabbit anti-mouse IgG-coated microtiter plates were prewashed once
with wash solution. A 100 µL aliquot of assay buffer was applied to
each well followed by 100 µL of the cell lysate sample. The plates
were incubated for 1 h at room temperature with gentle shaking and
washed four times as above. To each well, 200 µL of assay buffer
containing 10 ng/mL of Eu-SHZ was applied. The plates were incubated
for 1 h at room temperature with gentle shaking and washed four times
as above. A 200 µL aliquot of enhancement solution was added to
each well. After 30 min of incubation in gentle shaking, time-resolved
fluorescence was measured.

44 J. Agric. Food Chem., Vol. 52, No. 1, 2004
Korpima¨ki et al.
Table 3. Performance of Selected Clones in a Competitive
Sulfonamide Assay for 13 Different Sulfonamides
IC50 concentration (µgL-1
)
sulfaa
27G3
A.3.5
M.1.12
M.1.17
M.3.1
M.3.4
SMT
STZ
SCP
SMP
SDO
SPY
SMX
SDZ
SMZ
SFX
SQX
SDM
SHZ
1.2
8.7
9
19
22
0.24
2.5
1.3
3.1
0.83
9.6
6.8
5.8
11
19
0.11
0.12
0.41
0.63
0.44
1.3
0.94
0.48
0.33
3.8
0.029
0.33
0.24
0.38
0.23
1
0.58
0.47
0.83
3.4
0.019
0.085
0.066
0.12
0.11
0.27
0.28
0.26
0.26
2.21
16
0.05
0.15
0.19
0.25
0.11
0.42
0.34
0.24
0.68
2.75
14
35
69
160
310
420
760
1100
4200
230
360
480
18
35
29
31
60
64
23
47
48
19
Figure 4. Number of phages collected during each panning round. Shown
for each of the four different libraries is the number of phages collected
from the input pool (3 × 1013 phages for first round and 5 × 1011 phages
for rounds 2 and 3) with the panning antigen (the bars) and by unspecific
binding (the shaded area) during each round of panning.
a Sulfonamides not previously defined are abbreviated as follows: sulfathiazole
(STZ), sulfachlororpyridazine (SCP), sulfamethoxypyridazine (SMP), and sulfam-
erazine (SMZ).
gene were designed (Table 2). Four different mutant libraries
were assembled with these primers in SOE-PCR reactions. In
each of these four libraries, only two structurally adjacent CDR
loops had mutations (Table 1) in them (Lib 1, CDR-H3 + CDR-
L2; Lib 2, CDR-L1 + CDR-L3; Lib 3, CDR-L1 + CDR-L2;
and Lib 4, CDR-H1 + CDR-H2).
All four PCR products were cloned to a pAKp3fl phagemid
vector (Figure 2). The scFv mutants were produced as fusions
to full-length filamentous phage coat protein III from this vector.
The coat protein III had an artificial trypsin cut site engineered
to the N terminus of the protein. The usage of vector pAKp3fl
enabled the enrichment of the mutant libraries with hyperphage
display (29). After transformation to E. coli XL1-Blue cells,
the libraries contained approximately 2 × 107 independent
clones each.
The libraries were biopanned with three rounds of hyperphage
display (29), where sulfa binders were repeatedly enriched by
collecting phages with Bio-SHZ attached to streptavidin-coated
microtiter wells followed by phage amplification in vivo. The
amount of collection antigen used was reduced with each passing
round (panning round 1, Bio-SHZ dilution 1:3000; 2, 1:3000;
and 3, 1:20 000). Figure 4 shows how many phages were
collected from each library with the SHZ antigen during each
round of panning and how many of those were collected by
unspecific background binding. The increasing number of
collected phages as the rounds progress, even when the amount
of collection antigen decreases, and the fact that the unspecific
background stays in very low levels indicates that phages
displaying antigen binders were indeed enriched.
Isolation of Active Clones. For the purpose of isolating the
active clones from the libraries, phagemid DNA was purified
from cultures of cells infected with each enriched library and
scFv genes were transferred to the pAK100CL vector (Figure
2) with SfiI digestion and ligation. Expressed from this vector,
scFv antibodies were produced as mouse IgG constant light
domain fusions, which enabled immobilization on anti-mouse
IgG-coated surfaces. Twenty individual clones from each of the
four libraries were grown in minicultures and Eu-SHZ binding
activity was measured. Of the selection of 20 clones picked
from library 1, seven clones showed significant (S/N > 7) SHZ
binding activity, whereas five of the library 2 clones and six of
the library 3 clones also did so. Not a single clone with
significant Eu-SHZ binding activity was found from library 4.
Several clones from all enriched libraries displayed measurable
Table 4. Performance of Mutant M.3.4 in a Competitive Assay of SHZ
from Different Sample Matrices
2
sample
IC50 (µgL-1
)
R
buffer
20
34
27
40
0.98
0.98
1.00
1.00
1:10 semiskimmed milk
1:10 chicken serum
1:10 minced beef
binding activity (S/N > 2), but these were judged to be binders
of low affinity and were not chosen for further study. It is likely
that site-directed mutagenesis of antibody CDR loops produces
libraries of mostly inactive mutants. The high frequency
presence of significantly and weakly active SHZ binding clones
in the enriched libraries clearly tells us that the panning process
with hyperphage display had resulted in enrichment of SHZ
binders.
Characterization of Active Clones. Each isolated antibody
mutant displaying significant Eu-SHZ binding activity (S/N >
7) was sequenced in order to ascertain that each of the mutants
had a unique sequence (results not shown). The mutants were
also produced in 100 mL cultures and tested with a time-
resolved competitive sulfa fluoroimmunoassay for their ability
to bind SHZ. In this assay, sulfonamides compete with the Eu-
SHZ tracer for binding to the selected antibody. The concentra-
tion of SHZ needed to inhibit 50% of the tracer binding (IC50)
was calculated for each mutant antibody (data not shown). Four
clones with the lowest IC50 values for SHZ (ranging from 30
to 60 µg/L) were picked for further study. Two of these clones
were from library 1 and two from library 3. The performance
of these mutants in a competitive sulfa assay for 13 different
sulfonamides is shown in Table 3. The results show that not
only are nine of the sulfonamides recognized with the IC50
values varying within a range of little over a decade but also
the IC50 values for all sulfas are well below the 100 µg/L MRL,
in the case of all four of the characterized mutants.
Mutant Functionality in Sample Matrices. To briefly
demonstrate that the mutant with the lowest IC50 for SHZ, M.3.4,
also works in some real sample matrices, the measurement of
SHZ from semiskimmed milk, minced beef, and chicken serum
was done. Table 4 shows the IC50 values of SHZ and the R2
values of the sigmoidal fits in each of these sample matrices
diluted 10-fold in assay buffer before the assay. The data refer
to the actual SHZ concentration in the assay reaction. The results

An Antibody for Detection of Sulfonamides
J. Agric. Food Chem., Vol. 52, No. 1, 2004 45
show that the IC50 value of SHZ is only slightly affected by
the sample matrices. The worst effect is observed in the minced
beef matrix, which gives a 2-fold increase in the IC50 value.
Nevertheless, a MRL contamination of each sample matrix (100
µg/g of SHZ in sample), diluted 10-fold for the assay, reduced
the assay signal about 25%, demonstrating that an illegal sample
contamination should be detected with an immunoassay using
mutant M.3.4.
the recognition of this sulfa more than any other. By enriching
the four mutant libraries with hyperphage display with Bio-
SHZ as the collection antigen, we managed to get rapid
enrichment of binders in only three rounds; furthermore, the
unspecific background of each panning round was very small
(Figure 4). The panning reactions were performed in microtiter
wells coated with the collection antigen prior to addition of
phage libraries so that the hyperphage could bind multivalently
on the randomly distributed antigen on the well surface.
DISCUSSION
Mutant Cross-Reaction Profiles. There have been a few
studies (3, 24), which have modeled the minimum energy
conformations of different sulfonamides and then calculated the
electrostatic and steric properties of these conformations. These
properties seem to vary considerably between different sulfona-
mides in the minimum energy conformation, which seems to
suggest that any generic antibody must bind and thereby stabilize
some higher energy conformation of each sulfonamide, which
is supported by the fact that sulfonamides have rather flat
potential energy maps of rotation around the dihedral angles
(3); therefore, a range of conformations only slightly more
energetic than the minimum are available.
Libraries and Enrichment. A lot of research (1, 2, 23-27)
has revolved around the subject of developing antibodies capable
of binding all different sulfonamide antibiotics with an affinity
strong enough for detection of sulfas below the established MRL
(100 µg/L). However, despite the effort, such antibodies have
so far not been isolated. In our previous studies (26, 27), we
set out to transform a monoclonal sulfa antibody 27G3 to the
direction of generic binding. We first used random mutagenesis
of the whole scFv antibody gene combined to enrichment with
phage display followed by, in the later study, recombination of
the enriched mutants to produce the much improved antibody
A.3.5. This antibody, although significantly improved over
27G3, still failed to detect three of the 13 sulfonamides below
the MRL concentration.
As a result of the previous studies, we had an abundancy of
mutational data concerning antibody 27G3. This enabled us to
undertake the current study with the goal of improving mutant
A.3.5 using site-directed saturation mutagenesis: A thorough
mutagenesis was targeted to the areas of the CDR loops deemed
important for sulfa binding on the basis of the mutational data
and the antibody homology model constructed previously (27).
The advantages of site-directed mutagenesis are tremendous,
since it enables the complete screening of the whole mutational
space of a few residues of a protein. Before such an approach
can be used, however, enough data on the protein is needed so
that correct residues can be targeted. Four libraries in total were
constructed, each having mutations targeted to the residues of
two adjacent CDR loops. Together, the libraries covered all of
the CDR loops of antibody A.3.5.
Generic antibodies probably often have only low or medium
affinities for their antigens (since specificity and affinity are
somewhat dependent on each other), so for the enrichment of
the libraries we decided to use the new hyperphage display (29).
The essence of this technique is the use of helper phages, which
do not produce any wild-type pIII, thus all ∼5 pIII proteins on
the surface of each recombinant phage have a scFv fragment
fused to them, the phagemid vector being the only source of
pIII protein. These recombinant phages can bind multivalently
to antigen-coated surfaces of sufficient antigen density, thus
giving better signal-to-noise ratios in panning reactions than
phages with only a lone scFv displayed on their surface. This
is important when panning low or medium affinity binders so
that specific binding is not lost in the background. However, it
has to be remembered that since each hyperphage has the same
number of scFv on their surface, high-affinity antibody mutants
have a competitive edge over low-affinity mutants in enrichment,
even if these low-affinity antibody mutants have better expres-
sion properties in E. coli. This is because the mutants with better
expression yields do not end up producing phages with higher
valency than the low expression yield mutants, which is the
case in ordinary phage display.
All of the new mutants isolated in this study seemed to behave
in quite a similar manner. Sulfamethizole (SMT) was the
sulfonamide most readily recognized by the new mutants
followed by nine other sulfonamides, which had their IC50 values
spread within a range of 2 orders of magnitude from the IC50
of SMT, mutant M.1.12 having the narrowest range of them all
(a little over 1 order of magnitude). The case of M.1.12 was a
clear improvement over A.3.5, which had a cross-reaction profile
spread across 2 orders of magnitude for the same 10 sulfona-
mides. However, all of the new mutants had on the average a
10-fold improvement of the IC50 value of each tested sulfona-
mide over A.3.5, bringing all of the mutant IC50 values below
the MRL of 100 µg/L.
It seems that the sulfonamide binding of the new mutants is
not affected whether the N1 ring of the sulfonamide in question
is of the five or six carbon variety, since, for example,
sulfamethoxazole (SMX) and sulfapyridine (SPY) are recog-
nized with almost the same affinity by all of the new mutants.
However, it seems that there are a few details in the structure
of sulfonamides, which are responsible for the poorer binding
of sulfaquinoxaline (SQX), sulfadimethoxine (SDM), and SHZ.
First of all, it seems to be beneficial for recognition that there
is a nitrogen atom (as opposed to a carbon or an oxygen for
example) in the N1 ring in an ortho position of the ring as shown,
for example, by the 5-fold better affinity of the new mutants
for SMX over sulfisoxazole (SFX). Second, it seems to be not
favorable affinitywise, if both meta positions or both ortho
positions of the N1 ring have methyl or larger groups attached,
as demonstrated, for example, by the 100-200-fold better
affinity of the new mutants for sulfadoxine (SDO) over SDM,
which seems to implicate that upon binding the N1 ring of the
sulfonamides turns so that any methyl or larger groups face away
from the binding site surface if possible (for example, SMT
and SDO can accomplish this). Thus, the worse binding of SHZ
and SDM is probably caused by the fact that both of these sulfas
have both meta positions of the N1 ring occupied, whereas the
worse binding of SQX is maybe caused by the N1 ring turning
so upon binding that the attached secondary ring is facing away
from the antibody binding site surface, which causes a lack of
a nitrogen atom in the ortho position of the N1 ring facing the
binding site surface.
We synthesized a Bio-SHZ derivative for use as the collection
antigen, since SHZ was the sulfonamide that antibody A.3.5
recognized with the worst affinity and we wanted to improve
Sometimes, the performances of antibodies that function well
in simple buffer systems are adversely affected by the introduc-

46 J. Agric. Food Chem., Vol. 52, No. 1, 2004
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CONCLUSIONS
In our earlier studies (26, 27), we demonstrated, by improving
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Received for review August 22, 2003. Revised manuscript received
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supported by a grant from the Finnish National Technology Agency
(TEKES).
JF034951I