Antigens synthesis and antibodies preparation for furazolidone and its metabolite 3-amino-2-oxazolidinone

Article abstract of DOI:10.1016/j.cclet.2010.04.001

Two haptens of 3-[(5-amino-furan-2-ylmethylene)amino]oxazolidin-5-one(FZ-NH₂) and 3-{[(4-carboxyphenyl)methylene]-amino}-2-oxazolidinone (CPAOZ) were synthesized. For FZ-NH₂, immunogens were prepared by glutaraldehyde and diazo salt methods. For CPAOZ, immunogens were connected by the methods of the active ester and mixed acid anhydride. Compared with the combination, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with coating antigen of FZ-NH₂-OVA via the glutaraldehyde method and immunogen of CPAOZ-KLH via active ester method. For furazolidone and its metabolite AOZ (NPAOZ as derivative), the sensitivities (IC₅₀) were 2.0μg/L and 2.5μg/L, limits of detection (IC₁₅) were 0.09μg/L and 0.25μg/L, respectively. A sensitive method was developed for the simultaneous determination of furazolidone in feed and its metabolite AOZ in tissue.

Full text of DOI:10.1016/j.cclet.2010.04.001

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 1049–1052
www.elsevier.com/locate/cclet
Antigens synthesis and antibodies preparation for furazolidone
and its metabolite 3-amino-2-oxazolidinone
Hua Ping Zhu, Ting Ting Liu, Bing Liu, Hui Ling Yin, Xiao Long Li,
*
Li Wang, Shuo Wang
Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China
Received 5 January 2010
Abstract
Two haptens of 3-[(5-amino-furan-2-ylmethylene)amino]oxazolidin-5-one(FZ-NH2) and 3-{[(4-carboxyphenyl)methylene]-
amino}-2-oxazolidinone (CPAOZ) were synthesized. For FZ-NH2, immunogens were prepared by glutaraldehyde and diazo salt
methods. For CPAOZ, immunogens were connected by the methods of the active ester and mixed acid anhydride. Compared with
the combination, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with coating antigen of FZ-
NH2-OVA via the glutaraldehyde method and immunogen of CPAOZ-KLH via active ester method. For furazolidone and its
metabolite AOZ (NPAOZ as derivative), the sensitivities (IC50) were 2.0 mg/L and 2.5 mg/L, limits of detection (IC15) were
0.09 mg/L and 0.25 mg/L, respectively. A sensitive method was developed for the simultaneous determination of furazolidone in
feed and its metabolite AOZ in tissue.
# 2010 Shuo Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Furzolidone; AOZ; Antigen; Antibody; ELISA
Furazolidone [N-(5-nitro-2-furfurylidene-3-amino)-2-oxazolidinone] belongs to the nitrofuran antibiotic drugs,
which have been employed for the prevention and treatment of gastrointestinal infections. Because the parent drug and
its metabolite 3-amino-2-oxazolidinone (AOZ) showed carcinogenic and mutagenic characteristics [1], since
furazolidone was prohibited for food animals in the European Union in 1995, it has also been banned in the China,
United States, and other countries. However, furazolidone was still used due to its low cost and high benefit till now.
The detection for furazolidone residue was mainly based on the AOZ derivative 3-[(2-nitrophenyl)methylene-
amino]-2-oxazolidinone (NPAOZ) [2–4]. Various instrumental methods were used to detect the residues of
furazolidone and AOZ by HPLC, LC–MS, LC–MS/MS. But these instrumental analytical procedures were not fit for
the well-accepted convenient, so ELISA is accepted and performed in the worldwide due to the low cost, high sensitive
and rapid screening.
To obtain a specific antibody for furazolidone and its metabolite AOZ, the design of the corresponding immunogen
is important. Furazolidone molecule has no active group and is hard to connect with carrier proteins, directly.
Therefore, we utilized its NO2 group to obtain active NH2 group by Zn/HCl systems as Fig. 1. Then the immunogens of
* Corresponding author.
E-mail address: elisasw2002@yahoo.com.cn (S. Wang).
1001-8417/$ – see front matter # 2010 Shuo Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.04.001

[(Fig._1)TD$FIG]
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H.P. Zhu et al. / Chinese Chemical Letters 21 (2010) 1049–1052
Fig. 1. Synthetic route for haptens.
FZ-NH2-1 (I, II) and FZ-NH2-2 (III, IV) were respectively connected by the diazotization and glutaraldehyde method
[5,6], but the immune result for the furazolidone or AOZ are unsatisfactory, it is concerned that the furazolidone of low
chemical stability might were damaged in rabbit body, and the released AOZ of light molecular is difficult to arouse
the immune response. Subsequently, the hapten 3-[(4-carboxyphenyl)methylene]amino-2-oxazolidinone (CPAOZ)
was synthesized as Fig. 1. A carboxylic acid moiety in the hapten was a convenient functional group for conjugation to
carrier protein, and this similar structural element could increase the chance of high cross-reaction with furazolidone
drug. Then the antibodies were achieved from different immunogen haptens of CPAOZ-KLH-1 (V, VI) and CPAOZ-
KLH-2 (VII, VIII) via the methods of the active ester and the mixed acid anhydride [7,8], respectively. The
identification of conjugates was determined by UV spectrophotometer [9]. The UV absorbance spectra of FZ-NH2-
KLH (lmax, 345 nm) and CPAOZ-KLH (lmax, 290 nm) were different from the spectra of KLH (lmax, 275 nm), FZ-
NH2 (lmax, 365 nm) and CPAOZ (lmax, 295 nm). It showed that haptens conjugate to the protein successfully.
The polyclonal antibodies were produced by immunizing eight rabbits [10], and the antiserums were purified by a
Protein A-Sepharose 4B affinity column. Using the indirect ELISA [11], antibodies titer and sensitivity (IC50) were
showed in Table 1. Comparing the four conjugated methods, the antibodies raised from the CPAOZ-KLH-1 (V, VI)
were better than others. It was due to the higher conjugate ratios by the active ester methods.
The specificity of the antibody was evaluated by the cross-reactivity with nitrofuran drugs (furazolidone,
nitrofurantoin, furaltadone, nitrofurazone) and their metabolites [3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin
(AHD), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) and semicarbazide (SEM)] in Table 2. The
furazolidone (125%) showed a high cross-reactivity, because the CPAOZ structure is similar to furazolidone. It is well
known that antibodies show a preferential recognition to the moiety of the hapten that is farthest to the carrier protein.
Table 1
Antibody titer and sensitivity of each immunization.
I
II
III
IV
V
VI
VII
VIII
Titer
2000
600
2400
700
3000
400
2500
450
6000
5000
3000
16
3500
18
IC50(mg/L)
2.5
3.2
Antibodies of I–VIII were obtained respectively via the diazotization, glutaraldehyde, active ester and a mixed acid anhydride reaction.
Table 2
Cross-reactivity of antibody with various drugs and marker residues.
Competitor
IC50 (mg/L)
Cross-reactivity (%)
NPAOZ
2.5
2.0
100
Furazolidone
AOZ
125
6500
0.038
0.045
0.003
0.050
0.063
AMOZ,
5500
AHD, SEM
Nitrofurantoin
Furaltadone, nitrofurazone
100000
5000
4000

[(Fig._2)TD$FIG]
H.P. Zhu et al. / Chinese Chemical Letters 21 (2010) 1049–1052
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Fig. 2. Competitive indirect ELISA standard curve of NPAOZ and furazolidone.
But the cross-reactivity of AOZ was only 0.038%, so the C N group played an important role in antibody recognition,
despite it is closer to the carrier protein.
In indirect ELISA, the different coating antigens were studied. The OVA conjugates of FZ-NH2 were achieved by
the glutaraldehyde method, and the CPAOZ-OVA were obtained via the active ester and the mixed acid anhydride
methods. Compared the three coating antigens, the effect of the heterologous conjugates were better than the
homologous conjugates. The standard curves were shown in Fig. 2, IC50 (the concentration of 50% inhibition in
inhibition curve) values for NPAOZ and furazolidone were 2.5 and 2.0 mg/L, IC15 (the concentration of 15% inhibition
in inhibition curve) were 0.25 and 0.09 mg/L, respectively.
In conclusion, this paper provided a highly sensitive ELISA method for rapidly analyzing furazolidone and AOZ
residues. It established the foundation to determine furazolidone in animal feed and its metabolite AOZ in animal
tissues in the future.
1. Experimental
Furazolidone (200 mg) in 10 mL solutions of methanol/acetonitrile (30:70, v/v) were heated to 50 8C, then 30 mL
1 mol/L HCl and zinc powder (200 mg) were added slowly and heated to 80 8C for 40 min. The dark red solution was
cooled down to 4 8C. After filtration, the solution was extracted with ethyl acetate three times. The combined organic
solutionsweredried over Na2SO4, andsolventwas removed byreducedpressure. Theresidueswere chromatographedon
silica[ethylacetate/petroleum ether/aceticacid, 5:2:0.05, v/v/v] togive FZ-NH2 in46%yields. Thetarget compoundwas
identified by TLC Rf = 0.67 and HPLC–MS/MS [ESI-MS: 2M++H: 319.4 (C8H9N3O3, MW = 195.16)].
4-Carboxybenzaldehyde (42.3 mg) in 200 mL DMF were slowly dropped into the solution of AOZ (20.4 mg) in
1 mL of 0.1 mol/L HCl, the mixture solution was stirred overnight at 60 8C. The mixture was extracted with ethyl
acetate. The organic layer were dried over Na2SO4 and concentrated by reduced pressure. The residues were purifies
on silica [ethyl acetate/petroleum ether/acetic acid, 5:1:0.05, v/v/v] to give 4-CPAOZ in 62% yields. The obtained
CPAOZ was characterized by TLC Rf = 0.45, HPLC–MS/MS [ESI-MS: MÀ: 233.76 (C11H10N2O4, MW = 234.21)],
1H NMR (300 MHz, DMSO-d6): d 3.94–3.99 (t, 2H, J = 8.0 Hz), 4.49–4.54 (t, 2H, J = 8.0 Hz), 7.82 (s, 1H), 7.84 (s,
1H), 7.89 (s, 1H), 7.99 (s, 1H), 8.02 (s, 1H), 13.08 (br, 1H).
Acknowledgments
The authors are grateful for financial supports from the Ministry of Science and Technology of the People’s
Republic of China (No. 2009ZX09502-027 and No. 2009BADB9B03).
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