Preparation of Polyclonal Antibodies to a Derivative of 1-Aminohydantoin (AHD) and Development of an Indirect Competitive ELISA for the Detection of Nitrofurantoin Residue in Water

Abstract

Nitrofurans are used widely to treat animal diseases and were identified as the major compounds in many worldwide drug residue violations. To develop a rapid and convenient detection method to measure the residue of nitrofurantoin, we designed an immunogen and prepared a polyclonal antibody to develop an immunoassay in this study. The antibodies obtained were characterized by an indirect cELISA method and showed excellent specificity and sensitivity with IC50 of 3.2 ppb and no cross-reaction with most related species and compounds. Considering that nitrofurans often are used illegally to feed animals through drinking water, we measured the residue of nitrofurantoin in water spiked by the drug. The recovery rates are in the ranges of 88-103% for interassay and 90-103% for intra-assay. The CVs are in the ranges of 3.1-11.4% for interassay and 2.7-6.2% for intra-assay. The detection limit was determined to be 0.2 ppb. The immunoassay developed in this study is suitable to be used as a screening method to detect residues of nitrofurantoin in drinking water for animals.

Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

“…For nitrofuran metabolites, haptens were always designed by derivatising the metabolites with 3-CBA or 4-CBA [6,7,[9][10][11][12][13][14][15]. Although this strategy was able to generate antibodies with high specificity and sensitivity, it's believed that there are some other strategies to further improve the antibody characteristics.…”

“…The first enzyme-linked immunosorbent assay (ELISA) capable of detecting the furazolidone metabolite (AOZ) was developed by Cooper et al [6] under the support of a multi-national EU research project "FoodBRAND" [2]. Since then, immunoassays for the determination of AOZ [7][8][9], SEM [10][11][12], AHD [13,14] and AMOZ [15] were reported in succession.…”

“…NPAOZ, NASEM, NPAHD and NPAMOZ) [6][7][8][9][10][11][12][13][14][15]. In most studies, nitrofuran metabolites were derivatised with 3-carboxybenzaldehyle (3-CBA) or 4-carboxybenzaldehyle (4-CBA) to form immunizing haptens [6,7,[9][10][11][12][13][14][15].…”

Separation and Determination of Nitrofuran Antibiotics in Turbot Fish by Microemulsion Electrokinetic Chromatography

“…Several methods have been established to determine some of the NFs and metabolites contained in the feeds, water or food derived from animals, using thin-layer chromatography (TLC), 6 enzyme immunoassay (EIA), [7][8][9] liquid chromatography (LC), [10][11][12][13][14][15][16] liquid chromatography-mass spectrometry (LC-MS) [17][18][19] and liquid chromatography-tandem mass spectrometry (LC-MS/ MS). [20][21][22][23][24][25][26][27][28][29] However, these methods suffer from materials and are time consuming, since large amounts of organic reagents and many operation steps are often required.…”

Development of a Competitive ELISA for the Detection of a Furaltadone Marker Residue, 3-Amino-5-Morpholinomethyl-2-Oxazolidinone (AMOZ), in Cultured Fish Samples

“…ELISAs for the furazolidone metabolite AOZ were developed using the carboxy phenyl AOZ (CPAOZ) hapten for antibody production [4,9,10,14], and were used in assays specific for porcine, beef, chicken, shrimp and fish tissue analysis. Liu et al produced polyclonal antibodies against CPAHD that were specific to nitrofurantoin and used for in drinking water [20]. Similar approaches were undertaken for the development of the ELISA formats based on monoclonal and polyclonal antibodies against CPSEM as a metabolic target of nitrofurazone by Cooper et al [13], Gao et al [15] and Vass et al [24], which were considered to be effective screening assays for porcine muscle, chicken tissue and egg samples.…”