Design and Synthesis of Immunoconjugates and Development of an Indirect ELISA for Rapid Detection of 3, 5-Dinitrosalicyclic Acid Hydrazide

Abstract

In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acid-ovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a “substructural coating antigen” to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the “substructural coating antigen” concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC50 of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue.

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)

“…The result of this study demonstrated a previous conclusion that using only a partial structure of the target molecule as the coating hapten may be a good strategy to improve assay sensitivity [22,23].…”

“…In this work, four benzaldehyde derivatives (including 4-CBA (hapten 1, containing 1 carbon spacer), 3-CBA (hapten 2, containing 1 carbon spacer), 2-(4-formylphenoxy)acetic acid (hapten 3, containing 2 carbon and 1 oxygen spacer), and 2-(3-formylphenoxy)acetic acid (hapten 4, containing 2 carbon and 1 oxygen spacer)) were attached to AMOZ for immunizing haptens to study the effect of spacer group on production of specific antibodies against the NPAMOZ. Moreover, another hapten consisting of AMOZ moiety and a simple carboxyl spacer was synthesized as a coating hapten, because previous studies had revealed that using only a partial structure of the target molecule as the coating hapten may be a good strategy to improve assay sensitivity [22,23].…”

Development of Antibodies and Immunoassays for Monitoring of Nitrofuran Antibiotics in the Food Chain

“…Antibodies have been produced to bind the metabolites (Fig. 1), or derivatives of the metabolites, of the five major nitrofuran drugs: AOZ [5,[7][8][9][10], SEM [11][12][13][14][15], AMOZ [16][17][18][19][20], AHD [21][22][23][24][25] and DNSH [26]. The majority of publications focus on analysis of food products of animal origin in which these metabolites are the target analytes.…”

“…The marker metabolite of NFS is 3,5-dinitrosalicyclic acid hydrazide (DNSH). Shen et al [26] prepared a glyoxylic acid derivative of DNSH by reaction in methanol at room temperature for 3 h (Fig. 8).…”

“…The extraction steps required generally depend upon the specificity and sensitivity of the antibody for the target in question and the matrix under analysis. Some published methods [18,19,23,26,45,55,56], especially for immunochromatographic and biosensor applications, use no sample derivatisation. In these cases the authors claim that antibody sensitivity and specificity for the underivatised nitrofuran metabolites is sufficient such that, when methods are calibrated with underivatised standards, detection can be achieved to meet the MRPL.…”