Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay Based on Monoclonal Antibodies for the Detection of 2-Dodecylcyclobutanone in Irradiated Beef

Abstract

A highly sensitive and specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on monoclonal antibodies was developed for the detection of 2-dodecylcyclobutanone (2-DCB), a chemical marker for irradiated lipid-containing foods. 2-Oxocyclobutane undecanoic acid was used as an alternative to 2-DCB and conjugated to BSA and OVA via a conventional carbodiimide condensation reaction to prepare the immunogen and the coating antigen for 2-DCB. The monoclonal antibody against 2-DCB was obtained using the hybridoma technique, with a high specificity and low cross-reactivity for 2-tetradecylcyclobutanone (2-TCB; <8%) and other structurally related compounds (<0.1%). The ciELISA method was applicable at optimal experimental conditions of 0.001-100 μg/mL 2-DCB in a buffer solution, with an IC50 value of 0.25 μg/mL and a limit of detection (defined as the IC20) of approximately 0.004 μg/mL. The recovery efficiency of 2-DCB from ground beef patties ranged from 84.4 to 109.8%. The intra-assay and interassay coefficients of variation were <10.0 and <12.0%, respectively. The proposed method was validated by gas chromatography-mass spectrometry with high correlation. The same method was used to detect 2-DCB in ground beef patties irradiated at 0.5, 1.0, 3.0, 5.0, and 7.0 kGy; the 2-DCB concentration linearly increased with the radiation dose.

Potential allergenicity response to structural modification of irradiated bovine α-lactalbumin

“…Given that the interior buried linear and conformational epitopes of the native molecule are mainly hydrophobic amino acid residues, more IgG and IgE epitopes were exposed on the molecule surface as the dose was increased. 36 The exposed epitopes were destroyed severely by irradiation, resulting in a decrease in IgG-and IgE-binding ability of the irradiated α-La. Previous studies have confirmed that irradiation treatment could destroy linear and conformational epitopes by denaturation, aggregation, fragmentation, and amino acid modification of proteins.…”

Monoclonal Antibody Production and the Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Screening Spiramycin in Milk

“…During the last two decades, high-performance liquid chromatography , and liquid chromatography-tandem mass spectrometry − (LC-MS/MS) have been widely used to measure the macrolide antibiotic residues in various foods of animal origin. , These methods are typically labor-intensive, time-consuming, and require sophisticated instruments; thus, they are unsuitable for screening large numbers of samples . However, immunoassays, especially enzyme-linked immunosorbent assays (ELISA), have been confirmed as alternative screening methods for rapid analysis , without some of the limitations of chromatographic methods. , The ELISA method could simply and reliably provide high sample throughput, sensitivity, and selectivity. , To the best of our knowledge, no other reports have yet been published concerning the development of an immunoassay to detect SP residues in food. In this paper, we detail the production of highly specific mAbs and the development of an icELISA to screen for SP residues in milk without requiring complicated sample preparation.…”

“…10 However, immunoassays, especially enzyme-linked immunosorbent assays (ELISA), have been confirmed as alternative screening methods for rapid analysis 15,16 without some of the limitations of chromatographic methods. 17,18 The ELISA method could simply and reliably provide high sample throughput, sensitivity, and selectivity. 19,20 To the best of our knowledge, no other reports have yet been published concerning the development of an immunoassay to detect SP residues in food.…”

Development of a novel biosensor based on F0F1-ATPase for the detection of 2-dodecylcyclobutanone in irradiated beef

“…The microplate luminometer was a Centro XS3 LB 960 (Germany). Chromatophores containing F 0 F 1 -ATPase, biotinylated e-subunit monoclonal antibodies, ATP synthesis buffer, and biotinylated 2-DCB monoclonal antibodies were prepared according to previous study (Zhao, Wang, Li, & Ha, 2013;. All other analytically purified reagents were purchased domestically.…”