J. Microbiol. Biotechnol. (2006), 16(4), –
Production and Characterization of Monoclonal and Recombinant Antibodies
Against Antimicrobial Sulfamethazine
YANG, ZHENG-YOU, WON-BO SHIM, MIN-GON KIM1, KYU-HO LEE2, KEUN-SUNG KIM3,
KWANG-YUP KIM4, CHEOL-HO KIM5, SANG-DO HA3, AND DUCK-HWA CHUNG*
Division of Applied Life Science, Graduate School of Gyeongsang National University, Gyeongnam 660-701, Korea
1Laboratory of Integrative Biotechnology, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea
2Department of Environmental Engineering and Biotechnology, Hankuk University of Foreign Studies, Gyunggi-Do 449-791, Korea
3Department of Food Science and Technology, Chung-Ang University, Gyunggi-do 456-756, Korea
4Department of Food Science and Technology, Chungbuk University, Chungbuk 361-764, Korea
5Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University and NRL-Glycobiology,
Gyungbuk 780-741, Korea
antimicrobial sulfamethazine was prepared and characterized
by an indirect competitive enzyme-linked immunosorbent
assay (IC-ELISA). Sulfamethazine in the range of 0.2 and
45 ng/ml could be determined with the mab by IC-ELISA.
cDNAs encoding a variable heavy chain and variable light chain
of the mab were cloned to produce recombinant antibodies
using phage display technology. Following phage rescue and
three rounds of panning, a single-chain variable fragment
(scFv) antibody with high sulfamethazine-binding affinity
was obtained. ELISA analysis revealed that scFv antibody and
parent mab showed similar, but not identical, characteristics.
The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml,
compared with 1.6 ng/ml with the parent mab. Performances
of the assays in the presence of milk matrix were compared;
the mab-based assay was less affected than the scFv-based
assay. Sixty milk samples were analyzed by mab-based IC-
ELISA, and four samples were sulfamethazine positive; these
results were favorably correlated with those obtained by
A monoclonal antibody (mab) against the
Key words: ELISA, sulfamethazine, antimicrobial, monoclonal
antibody, scFv antibody
Sulfonamides are antimicrobial agents widely used in
veterinary medicine for prophylactic, therapeutic, and growth-
promoting purposes . Improper use or improper time
of withdrawal can lead to the presence of illegal residues in
milk, meat, and other animal byproducts . Sulfamethazine
has been identified as the major agent in 95% of all
sulfonamide tissue violations . The safety of sulfamethazine
for consumers has been questioned because of its potential
toxic effects. Adverse effects known to be associated with
high levels of antibiotic residues include allergenic effects,
carcinogenicity, and antibiotic resistance of microorganisms.
To prevent any problem with consumers, the maximum
residue limit (MRL) for the sum of sulfonamides has been
set to 100 µg/kg in the United States and the European
Union member states, and the Codex Committee of FAO/
WHO has specified a maximum residue limit to 25 µg
sulfamethazine per kg of milk . To comply with these
regulations, it is necessary to use simple, fast, and sensitive
methods to screen for sulfamethazine in food samples.
Generally, high-performance liquid chromatography (HPLC)
and/or HPLC/mass spectrometry (HPLC/MS) have been
used to quantify the amount of sulfamethazine residues in
samples [1, 5, 6, 23]. Although these instrumental analytical
methods are highly accurate and sensitive, they are also
time-consuming and labor-intensive. On the other hand,
immunoassays are analytical methods based on the interaction
of an analyte with an antibody that recognizes it with high
affinity and specificity. They are simple, cost-effective, do
not require sophisticated instrumentation, and are able to
analyze many samples simultaneously . These features
make ELISAs very powerful tools for sulfonamide residues
analysis [4, 13, 16, 18].
Currently, recombinant antibody technology enables one
to manipulate antibody genes for the engineering of single-
chain variable fragment antibodies consisting of the variable
heavy chain (VH) and variable light chain (VL) of an antibody
Phone: 82-55-751-5480; Fax: 82-55-757-5485;
2YANG et al.
[9, 17, 21, 25], both of which are connected by a flexible
peptide linker, and for the large-scale production of
recombinant antibodies in Escherichia coli.
In the present study, we produced and characterized
monoclonal and recombinant antibodies against the
antimicrobial sulfamethazine, and used these antibodies in
the construction of an immunoassay to detect sulfamethazine
MATERIALS AND METHODS
Sulfamethazine and other sulfonamides were purchased
from Sigma Chemical Co. (MO, U.S.A.). Sulfamethazine
haptens S1 and S2 (Fig. 1) were synthesized by Dr. Sergey
A. Eremin (Lomonosov Moscow State University, Russia).
Keyhole limpet hemocyanin (KLH), soybean trypsin inhibitor
type II (STI), and ovalbumin (OVA) were purchased from
Sigma Chemical Co. (MO, U.S.A.). DNA polymerase
and DNA restriction endonuclease were purchased from
TaKaRa Bio, Inc. (Shiga, Japan). HRP-conjugated anti-E
tag antibody, HRP-conjugated anti-M13 antibody, phagemid
pCANTAB5E, E. coli TG1, and E. coli HB2151 were
purchased from Amersham Biosciences (Piscataway, NJ,
U.S.A.). The thio-affinity T-gel was purchased from Pierce
(Rockford, IL, U.S.A.). All other chemicals and organic
solvents were commercially available and were of reagent
grade or better.
Preparation of Hapten-Protein Conjugates
Sulfamethazine haptens S1 and S2 were covalently attached
to carrier proteins KLH, STI, and OVA, according to the
active esters method described by Schneider and Hammock
. The conjugates formed were purified by dialysis in
0.05 M phosphate-buffered saline (pH 7.4) for three days.
The resulting conjugates were used as immunogens for
mice, except for S1-OVA and S2-OVA used as coating
antigens in ELISA.
Production of Monoclonal Antibodies
Seven-week-old BALB/c female mice were immunized
with S1-KLH, S1-STI, S2-KLH, or S2-STI as described
previously . Immunized mouse spleen cells (6×107) were
fused with myeloma cells (P3-X63-Ag8.653) according to
standard procedures . ELISA-positive hybridoma cells
were cloned twice by the limiting dilution method. Then,
the hybridoma cells were injected into BALB/c mice, and
ascites were produced and collected. Mabs were purified
from mice ascites by ammonium sulfate precipitation,
followed by protein G affinity chromatography. The purified
mabs were analyzed by SDS-PAGE and stored at -20oC.
The protein concentration of purified mabs was determined
using a protein assay kit (Bio-Rad). The isotype of a cloned
mab was determined with a mouse monoclonal antibody
isotyping kit (Roche Applied Science, U.S.A.) according
to the instructions of the manufacturer.
Production of ScFv Antibodies
For cloning of monoclonal antibody variable chain fragment
genes, mRNA was extracted from 2×107 hybridoma cells
using mRNA purification kit (Amersham Biosciences,
U.S.A.). About 1 µg of mRNA was reverse transcribed
using an random hexamer primers and the first-strand cDNA
synthesis kit (MBI Fermentas). The scFv DNA fragments
were constructed by assembling the amplified VH and VL
genes with a linker coding for a (Gly4Ser)3 peptide and
ligated into pCANTAB5E vector (Amersham Biosciences,
U.S.A.) as described by Marks et al. . E. coli TG1 cells
were transformed with the recombinant pCANTAB5E
vector and used to prepare phage particles for panning by
rescue using the M13K07 helper phage, as described by
Yuan et al. .
Sulfamethazine-specific recombinant phages were isolated
by three rounds of panning with sulfamethazine-OVA
coated cell culture flasks as described by Choi et al. 
and used to infect E. coli HB2151 to produce soluble scFv
antibodies. Colonies raised from infected E. coli HB2151
cells on the plates were picked into 96-deep, well cell culture
plates (Bioneer, Korea) containing 0.75ml of 2×YT medium
with ampicillin (100 µg/ml) and glucose (0.1%, w/v).
Fig. 1. Chemical structures of sulfamethazine and two haptens
synthesized in this study.
ANTI-SULFAMETHAZINE MONOCLONAL AND RECOMBINANT ANTIBODIES
Following 4 h of incubation at 37oC, 0.25 ml of 2×YT
medium containing IPTG (3 mM) was added to each well,
and incubation was continued for 20 h at 28oC. Finally,
1 ml of PBST was added to each well, and the plates were
centrifuged at 1,000 ×g for 20 min. The supernatant in each
well was tested for the antibody activity to sulfamethazine
by screening ELISA using an HRP-conjugated anti-E tag
antibody (Amersham Biosciences).
One selected clone of E. coli HB2151 that can produce
sulfamethazine-specific scFv antibody was inoculated into
a 2-l flask containing 1.5 l of SB medium supplemented
with 100 µg/ml of ampicilin for large-scale preparation of
scFv antibody. Culture supernatant containing the extracellular
soluble scFv antibody was separated from the cell pellets
by centrifugation at 2,500 ×g for 15 min and filtered
through a 0.45-µm-pore-size filter. The cell pellets were
used to prepare cytoplasmic and periplasmic extracts using
the protocol of McCarthy and Hill . The scFv antibody
in periplasmic extracts was purified by thiophilic adsorption
chromatography, as described by Schulze et al. . The
purified scFv antibody was analyzed by SDS-PAGE. Protein
concentration of the purified scFv was determined using a
protein assay kit (Bio-Rad).
SDS-PAGE and Western Blot Analysis of ScFv Antibody
Proteins in 15 µl of concentrated supernatant (0.3 ml of
supernatant concentrated by 10% trichloroacetic acid),
15 µl of periplasmic extract, or 15 µl of cytoplasmic extract
were separated on 12.5% SDS-polyacrylamide and transferred
to nitrocellulose membranes. The membranes were blocked
with 3% skim milk in PBS for 1 h at room temperature.
HRP-conjugated anti-E tag antibody (diluted 1:2,000 with
PBS) was incubated with the membrane for another 1 h.
The membrane was washed twice with PBST for 10 min at
room temperature. The Western blot was visualized using
the ECL Western blotting analysis system (Pharmacia
Biotech) according to the manufacturer’s instruction.
DNA Sequence Analysis
The clone producing scFv antibody with the highest
affinity for sulfamethazine in E. coli HB2151 was chosen
for DNA sequencing by an automated DNA sequencer
(PerkinElmer Life Sciences).
IC-ELISA Based on Mab
Each well of microtiter plates (Nunc, Denmark) was
coated overnight at 4oC with 100 µl of sulfamethazine-
OVA (1 µg/ml) in 0.05 M carbonate-bicarbonate buffer
(pH 9.6) and blocked with 1% skim milk in PBS at 37oC
for 1 h. After the plates were washed with PBST, 50 µl of
standard solutions or samples and 50 µl (1.5 µg/ml) of
mab were added to each well, followed by incubation for
1 h at 37oC. After washing, 100 µl of goat anti-mouse IgG
conjugated to HRP antibody (diluted 1:5,000 in PBS) was
added and incubated for 1 h at 37oC. Then, 100 µl/well of
ABTS solution containing 0.03% H2O2 was added and
incubated for 30 min in the dark at room temperature.
Absorbance was measured at 405 nm using a microtiter
plate reader (Bio-Rad, Benchmark 550).
IC-ELISA Based on ScFv Antibody
The ELISA assay was performed with an incubation for
1 h at 37oC as described above, except for addition of 50 µl
(4 µg/ml) of a scFv antibody in place of a mab, followed
by washing and adding 100 µl/well of HRP-conjugated
anti-E tag mouse monoclonal antibody (diluted 1:2,000
with PBS) with incubation for another 1 h at 37oC.
To measure the cross-reactivity of the compounds
structurally related to sulfamethazine with mab and scFv
antibody, standard curves of sulfadiazine, sulfapyridine,
sulfamethoxazole, sulfaquinoxaline, sulfisozole, sulfathiazole,
and sulfadimethoxine were constructed in the same manner
as for IC-ELISA. The cross-reaction was calculated as
the ratio of the mass of sulfonamides giving 50% inhibition
of the maximum responses to the concentration of
sulfamethazine as standard.
Milk samples were obtained from local markets. Before
the spike and recovery studies, each test sample was
verified by HPLC to contain <1 ng/ml of sulfamethazine.
For a spiking study, 10 ml of milk samples were spiked
with different levels of sulfamethazine dissolved in methanol,
thoroughly mixed, and then 10 ml of acetone was added
and shaken by hand. After centrifugation for 5 min at
2,500 ×g, the middle clear liquid was diluted 1:20 with
PBST and analyzed by IC-ELISA, based on mab 1H11 or
HPLC Assay for Sulfamethazine
The test sample was verified using DIONEX (Germany)
HPLC equipped with a P580 pump with UVD and ASI-
100 automated sample injector. A C18 column (250 mm×
4.6 mm i.d., 5 µl) was used. The analyses were performed
at 265 nm, and the mobile phase was 0.02 M PBS buffer
(pH 3.1)/ACN (155: 47) at a flow rate of 1.0 ml/ml.
RESULTS AND DISCUSSION
Production and Characterization of Anti-Sulfamethazine
Six female BALB/c mice were immunized with S1-KLH or
S1-STI and six female BALB/c mice were immunized with
S2-KLH or S2-STI. Following a series of boosts, the titers
4YANG et al.
of antisera from mice were examined by a noncompetitive
ELISA. All mice immunized with S2-KLH showed a good
antibody titer and reactivity to sulfamethazine. However,
mice immunized with S1 conjugates or S2-STI exhibited
an insufficient antibody titer, and three of six antisera raised
against S1 conjugates did not react to sulfamethazine. In
theory, immunogens with longer spacers between hapten
and carrier protein are preferable to those with shorter
spacers . In this study, hapten S1 had a two-methylene
chain as the linker arm for the conjugation with carrier
protein, whereas hapten S2 had a three-methylene chain
for the conjugation with carrier protein, and this might be
the reason that mice immunized with S1 conjugates
exhibited an insufficient antibody titer. Considering that
mice immunized with S1-STI and S2-STI conjugates did
not produce anti-sulfamethazine antibodies, STI protein
might not be a good sulfamethazine carrier protein for
Cell fusion between myeloma cells and spleen cells
from the immunized mice was carried out, and fifteen
hybridoma cell lines secreting anti-sulfamethazine monoclonal
antibodies were obtained. The most reactive mab with
sulfamethazine was named 1H11 that was derived from the
mouse immunized with S2-KLH. The hybridoma cell line
1H11 was adapted to an ascites preparation. Monoclonal
antibody 1H11 was purified by ammonium sulfate precipitation,
followed by protein G affinity chromatography. The purified
mab 1H11 was confirmed by SDS-PAGE analysis (Fig. 2).
The protein concentration of purified mab 1H11 was
1.5 mg/ml, as determined by the protein assay kit (Bio-
Rad). The purified mab was used in indirect competitive
ELISA. Isotyping experiment revealed that the isotype of
mab 1H11 was IgG1 with a kappa light chain.
The anti-sulfamethazine mab 1H11 was characterized
by IC-ELISA. The representative standard curve of 1H11
toward sulfamethazine is shown in Fig. 3. The IC50 value
(concentration of analyte giving 50% inhibition) value
for sulfamethazine was 1.6 ng/ml, and the detection
range was from 0.2 to 45 ng/ml. When a hybridoma is
used as an mRNA source for construction of scFv
antibody, it is highly desirable to use a cell line that
produces a high-affinity antibody for target antigen .
The hybridoma cell line 1H11 produced a monoclonal
antibody with high affinity to sulfamethazine, which
made the cloning of high-affinity scFv antibody against
Production and Characterization of Anti-Sulfamethazine
To clone the genes of coding for VH and VL domains of
mab 1H11, cDNAs were synthesized from mRNA isolated
from hybridoma cells. Based on the constructed cDNAs,
we generated the anti-sulfamethazine scFv antibodies by
phage display techniques, as described in Materials and
Methods. The prepared scFv genes were inserted into the
expression vector pCANTAB5E and introduced into E.
coli TG1 cells to produce the corresponding fusion protein,
scFv-g3p, displayed on the outer coat protein of the
The prepared recombinant phages displaying scFv
antibodies, which bound to sulfamethazine-OVA, were
isolated by the panning method. The isolation process was
Fig. 2. SDS-PAGE analysis of purified mab 1H11.
Lane M, standard protein marker; lane 1, mab purified by ammonium
sulfate; lane 2, unbound proteins by Protein G column; lane 3 and lane 4,
eluted fractions of purified mab 1H11.
Fig. 3. Standard curves for mAb 1H11 (●) and SP4scFv (▲).
The reactivity of SP4scFv and mab against sulfamethazine was determined
by IC-ELISA, as described in Materials and Methods.
ANTI-SULFAMETHAZINE MONOCLONAL AND RECOMBINANT ANTIBODIES
repeated through three panning rounds, and the phage titers
were determined for each round. An increasing number of
phage binders were obtained following each panning
cycle. After the first round of panning, 3×103 binders were
obtained, whereas the binders numbers were 6×105 after
the second cycle. The affinity of recombinant phage antibodies
to sulfamethazine-OVA after each round of panning was tested.
Prior to panning, the pooled recombinant phage antibodies
exhibited very low binding activity to sulfamethazine-
OVA (Fig. 4). Increase of binding affinity was observed
after the first and second pannings. No substantial increase
was obtained after the third round of panning.
After three rounds of panning, the pooled phages were
used to infect logarithmic phase E. coli HB2151 for
production of soluble scFv antibodies. Total 480 colonies
were randomly selected and respectively cultured in 2×YT
medium. After induction by IPTG, the culture supernatant
from each clone was used directly for detection of antibody
activity to sulfamethazine-OVA by noncompetitive ELISA.
As a result, the scFv antibodies produced by 15% of the
480 clones showed binding significantly above background,
and eight clones gave high binding. The eight selected
scFv antibodies were further assessed by IC-ELISA for
binding to free sulfamethazine. One of the scFv antibodies,
named SP4scFv produced by clone SP4, showed high
binding affinity to free sulfamethazine. The other seven
scFv antibodies showed less or no recognition of free
Clone SP4, producing SP4scFv with high binding
affinity to free sulfamethazine in E. coli HB2151, was
chosen for DNA sequencing. The nucleotide and deduced
amino acid sequences of the heavy and light chain variable
regions in SP4 are shown in Fig. 5. The DNA coding for
VH domain of SP4scFv comprised 348 bp encoding 116
amino acid residues, and appeared to be a member of the
mouse heavy chain subgroup III according to the
classification of Kabat et al. . The VL gene comprised
330 bp encoding 110 amino acid residues, belonging to the
mouse light chain subgroup IA. The complementarity
determining regions (CDRs) 1, 2, and 3 of VH and VL were
positioned as depicted in Fig. 5.
Fig. 4. Sulfamethazine-OVA binding activities of recombinant
phages, following each round of panning, measured by ELISA.
Panning round “0” represents period of panning.
Fig. 5. Nucleotide and deduced amino acid sequences of the
heavy and light chain variable regions of SP4scFv.
The complementarity determining regions are underlined which were
determined according to Kabat et al. . The nucleotide sequence in this
study was submitted to the GenBank Data Bank with accession number
6YANG et al.
SP4scFv in a large scale was prepared in SB medium,
and its location in the cell fraction was analyzed by Western
blot. As shown in Fig. 6, the location of the expressed
SP4scFv was found to be in the periplasm as well as in the
cytoplasm. The expression of SP4scFv in culture supernatant
was very low, and the event that used 20 times concentrated
supernatant sample, gave only a weak band in Western blot
analysis. The SP4scFv antibody in periplasmic extracts
was purified by thiophilic adsorption chromatography. The
purified SP4scFv antibody was confirmed by SDS-PAGE
analysis (Fig. 7). The protein concentration of purified
SP4scFv antibody was 1.2 mg/ml, as determined by protein
assay kit (Bio-Rad). The purified SP4scFv antibody was
used in the subsequent experiments.
The standard curve of SP4scFv toward sulfamethazine is
shown in Fig. 3. The binding of SP4scFv to the immobilized
antigen sulfamethazine-OVA was inhibited by sulfamethazine
in a concentration-dependent manner. The IC50 value of
SP4scFv was 4.8 ng/ml, and this was comparable to that
obtained with the parent mAb 1H11 in the IC-ELISA
Cross-reactivity of mab 1H11 and SP4scFv was determined
by comparing IC50 values for various sulfonamide compounds
in IC-ELISA. The values were expressed as percent cross-
reactivity (IC50 for sulfamethazine/IC50 for test compound
×100). The result of cross-reactivity tests with the
sulfonamide compounds is shown in Table 1. The high cross-
reactivity of both antibodies observed with sulfamerazine
(54% and 59% for mab and scFv antibody, respectively) is
quite reasonable, because of its similar aromatic structure to
sulfamethazine. Relatively little reaction of sulfadiazine
(8%) and sulfachlorpyridazine (4%) with SP4scFv was
exhibited also; however, these two compounds did not
react with mab 1H11 (Table 1). Both antibodies did not
exhibit significant cross-reactivity with other structurally
related sulfonamide compounds tested in this study.
Analysis of Milk Sample
Milk samples were examined in IC-ELISA to measure the
content of sulfamethazine. To gain basic information on the
matrix effect, conformity of the standard curve generated
in PBS was compared with that of curves obtained using
milk matrices. The inhibition curves generated in PBS were
consistent with those prepared in 10, 20, 50, and 100 times
Fig. 6. SDS-PAGE (A) and Western blot (B) analysis of anti-
sulfamethazine SP4scFv antibody expressions.
Lane 1, SP4scFv in concentrated supernatant (0.3 ml supernatant
concentrated by 10% trichloroacetic acid); lane 2, SP4scFv expression in
the periplasm; lane 3, SP4scFv expression in the cytoplasm; lane M,
Fig. 7. SDS-PAGE analysis of purified SP4scFv antibody.
Lane M, standard protein marker; lane 1, cytoplasmic extracts; lane 2,
periplasmic extracts; lane 3, purified SP4scFv antibody by thiophilic
Table 1. Comparison of cross-reactivities of SP4scFv and
monoclonal antibody 1H11 to sulfamethazine analogs.
ANTI-SULFAMETHAZINE MONOCLONAL AND RECOMBINANT ANTIBODIES
diluted milk matrices, but the OD value curves differed
significantly. After removal of protein with acetone, the
milk samples were diluted 10, 20, 50, and 100 times with
PBST and used to generate standard curves. Twenty times
dilution with PBST was found to be good enough to
reduce the matrix effects on detection of sufamethazine by
mab 1H11-based IC-ELISA (Fig. 8), and the IC50 of mab
1H11 under this condition was 2.2 ng/ml. However, the
effect of milk matrix on SP4scFv-based IC-ELISA was
significant (Fig. 8), and the IC50 of SP4scFv under this
condition was 24 ng/ml. The stronger effect of milk matrix
to recombinant antibody SP4scFv than mab 1H11 might
be due to structural difference between scFv antibody and
parent mab. Therefore, the IC-ELISA-based mab 1H11
was used to detect sulfamethazine in milk samples. The
recoveries of sulfamethazine added to milk at 10, 50, and
100 ng/ml are shown in Table 2. The recoveries from the
matrices were in the range of 94-104%. Concerning the
reproducibility, the average interassay coefficient of variation
(CV) values was 9.8%. Therefore, the mab 1H11-based
indirect competitive ELISA presented herein could be
used to determine sulfamethazine in milk samples.
Real milk samples were obtained from local markets for
sulfamethazine screening and determination. Analysis of
60 milk samples by IC-ELISA showed that 4 samples were
positive (sulfamethazine concentration ranged 5.1 to 8.1 ng/
ml). HPLC analysis also confirmed the same positive
samples, thus demonstrating that the developed assay can
be used for analysis of real samples.
We are very grateful to Dr. Sergey A. Eremin from the
Department of Chemical Enzymology, Chemistry Faculty,
M.V. Lomonosov Moscow State University (Moscow,
Russia) for kindly providing the haptens used in the study.
This research was partially supported by the National
Toxicology program of the National Institute of Toxicological
Research, Brain Korea 21 Program from the Ministry of
Education and the Korea Health 21 R&D Project (03-PJ1-
PG1-CH11-003), Ministry of Health & Welfare, Republic
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