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Production and characterization of monoclonal
antibodies specific for 3-amino-2-oxazolidinone
Ratthaphol Charlermroj1, Songchan Puthong2, Kittinan Komolpis2, Tanapat Palaga3
1Biotechnology Program, Faculty of Science, 2Institue of Biotechnology and Genetic Engineering, 3Department of Microbiology,
Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330
Abstract
3-Amino-2-oxazolidinone (AOZ) is a tissue bound toxic metabolite derived from the nitrofuran antibiotic, furazolidone. In this study, monoclonal antibodies (MAbs) were
generated to specifically detect AOZ, without the process of derivatisation with o-nitrobenzaldehyde. Derivatised form of AOZ, 3-{[(3-carboxyphenyl)-methylene]amino-2-
oxazolidinone(CPAOZ) was used as the immunizing hapten for the production of MAbs against AOZ. Hybridomas were generated by fusion of murine myeloma cells (NS1 cells)
and splenocytes from BALB/c mice, immunized with CPAOZ-ethylenediamine-bovine serum albumin (CPAOZ-ed-BSA). The antibody produced from hybridoma clone
CPAOZ#12 MAb was characterized in terms of cross-reactivity by competitive enzyme-linked immunosorbent assay (ELISA). The result suggested that CPAOZ#12 MAb was
highly specific for CPAOZ, 3-{[(2-nitrophenyl)methylene] amino}-2-oxazolidinone (NPAOZ), AOZ and furazolidone. It did not exhibit crossreactivity with various nitrofuran
metabolites and other antibiotics. Isotype of CPAOZ#12 MAb was determined by indirect ELISA to be IgG1 subclass. This is the first report of monoclonal antibodies for a
specific detection of a nitrofuran metabolite(AOZ) without derivatisation. This monoclonal antibody could be developed into an immunoassay-based test kit for AOZ residue in
food.
Introduction
Results and Discussion
Nitrofuran, furazolidone is an antibiotic which has been widely used in the form
Preparation of CPAOZ conjugate
of food additives for treatment and prevention of gastrointestinal infections caused by
Escherichia coli and Salmonella spp. in cattle, pig, poultry and fish. It has also been
used as a growth stimulator. However, the European Union (EU) does not assign a
maximum residue limit for furazolidone because of the potential carcinogenic effects
Fig.1 TLC analysis of CPAOZ synthesis (a)
of its residues on human health[1]. Monitoring of furazolidone has not been effective
visualized by ninhydrin stain,(b) visualized by UV
light; lane 1: 3-carboxybenzaldehyde, 2: (3-amino-
in the past due to the instability and rapid metabolism of the drug. However the
2-oxazolidinone), 3: pyridine, 4: sample before
metabolite, 3-amino-2-oxazolidinone (AOZ), is more stable in vivo and in vitro which is
reflux, 5: sample after reflux, 6: sample after flow of
nitrogen gas. Elution solvent : 5% methanol in
released upon mild acid hydrolysis [2-3]. Various methods have been published for
chloroform
the determination of AOZ by HPLC-UV[2,4-5], LC-MS[6] and LC-MS/MS[7].
Nevertheless, there is now an urgent need for rapid, high capacity screening methods
for the detection of AOZ residue. Currently, there are immunoassay-base screening
tests capable of detecting AOZ. The sample, however, must be derivatized with o-
nitrobenzaldehyde
to form 3[[(2-nitrophenyl)-methylene]-amino]-2-
oxazolidinone(NPAOZ) [8,9]. In this study, monoclonal antibodies for specific
Fig.2 Molecular weight
detection of nitrofuran metabolite(AOZ) were produced. They could be developed into
analysis by MALDI-
TOF/MS (a) cBSA, (b)
an immunoassay-based test kit for detecting AOZ residue in food without sample
CPAOZ-ed-BSA and
derivatization.
(c) CPAOZ-OVA
Materials and Methods
Preparation of CPAOZ conjugate
CPAOZ has Rf approximately 0.03 by TLC analysis. Molar ratio of hapten to
carrier proteins after conjugation was calculated to be 14.5:1 for CPAOZ:cBSA and
1:1 for CPAOZ:OVA.
(3-amino-2-
(3-carboxy
Characterization of MAbs
oxazolidinone)
benz aldehyde)
(ethylenediamine-BSA)
IC50 = 74.530 ng/ml
IC50 = 1,042 ng/ml
Competitiors
LOD = 9.616 ng/ml
reactivity(%)
LOD = 11.324 ng/ml
LOQ = 31.733 ng/ml
LOQ = 37.369 ng/ml
<0.01
<0.01
<0.01
(3-{[3-carboxyphenyl)
<0.01
methylene]amino}-2-
CPAOZ (ug/ml)
<0.01
oxazolidinone))
NPAOZ (ug/ml)
<0.01
CPAOZ-ed-BSA
CPAOZ-OVA
IC50 = 15.39 ug/ml
IC50 = 2.832 ng/ml
<0.01
(used for immunization)
(used for ELISA)
LOD = 0.501 ug/ml
LOD = 0.251 ng/ml
Flumequine
<0.01
LOQ = 1.654 ug/ml
LOQ = 0.827 ng/ml
Norfloxacin
<0.01
MALDI-TOF/MS
MALDI-TOF/MS
Penicilin G
<0.01
Clenbuterol
<0.01
Salbutamol
<0.01
Production of monoclonal antibodies (MAbs)
Furazolidone (ug/ml)
AOZ (ug/ml)
Chloramphenicol
<0.01
Inject CPAOZ-ed-BSA,
Oxytetracycline
<0.01
50 ug/mices,
Fig.3 Inhibition curves of competitive ELISA showing the ability of
CPAOZ#12 MAbs to react with (a) CPAOZ, (b) NPOAZ, (c) Furazolidinone
Boost every 2weeks
and (d) AOZ
Isotype of CPAOZ#12 MAb was determined by indirect ELISA to be IgG1
subclass. Sensitivity (IC50) of CPAOZ#12 MAb to CPAOZ, NPAOZ, furazolidinone
Highest Ab titre
and AOZ are 74.530 1,042 2.832 and 15,390 ng/ml, respectively.
Sacrifice
References
Female BALB/c
Myeloma cells
1. J.E.M. Van Koten-Vermeulen, M.F.A. Wouters, F.X.R. Van Leeuwen, Report of the 40th
mice, 6-8 weeks
(NSI cells)
Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA),
(polyethyleneglycol)
World Health Organisation, Geneva, 1993, p.85
2. A. Conneely. et al., Analyst., 2002, 127, 705-709
3. R.J. McCracken. et al., Food Addit. Contam., 1997, 287
positive
Subclone
Screen by
4. A. Conneely. et al., Anal. Chim. Acta., 2003, 483, 91
until monoclone
indirect ELISA
5. L.A.P. Hoogenboom. et al., Food Addit. Contam., 1992, 623
6. R.J. McCracken. et al., J. Chromatogr. B.,1997,691, 87
Hybridoma cells in
7. A. Leitner. et al., J. Chromatogr. A., 2001,939, 49
HAT medium
8. Kevin M. Cooper. et al., Analytica Chimica Acta.,2004, 520,79–86
Characterization
• isotype by isotyping test kit
9. M. VASS. et al., Vet. Med. , 2005,50(7), 300–310
• sensitivity and cross-reactivity by competitive ELISA
Acknowledgment
This research is partially funded by graduate research fund,
Chulalongkorn University

Source: http://www.ibge.chula.ac.th/antibody/Poster%20Print%20Fluke.pdf