General Bioluminescence Resonance Energy Transfer Homogeneous Immunoassay for Small Molecules Based on Quantum Dots

Here, we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and <i>Renilla</i> luciferase (Rluc) as the donor (QD-BRET) for the determination of small molecules. The ratio of the donor–acceptor that could produce energy transfer varied in the presence of different concentrations of free enrofloxacin (ENR), an important small molecule in food safety. The calculated Förster distance (<i>R</i>₀) was 7.86 nm. Under optimized conditions, the half-maximal inhibitory concentration (IC₅₀) for ENR was less than 1 ng/mL and the linear range covered 4 orders of magnitude (0.023 to 25.60 ng/mL). The cross-reactivities (CRs) of seven representative fluoroquinolones (FQs) were similar to the data obtained by an enzyme-linked immunosorbent assay (ELISA). The average intra- and interassay recoveries from spiked milk of were 79.8–118.0%, and the relative standard deviations (RSDs) were less than 10%, meeting the requirement of residue detection, which was a satisfactory result. Furthermore, we compared the influence of different luciferase substrates on the performance of the assay. Considering sensitivity and stability, coelenterazine-h was the most appropriate substrate. The results from this study will enable better-informed decisions on the choice of Rluc substrate for QD-BRET systems. For the future, the QD-BRET immunosensor could easily be extended to other small molecules and thus represents a versatile strategy in food safety, the environment, clinical diagnosis, and other fields

Simultaneous determination of chloramphenicol, florfenicol and florfenicol amine in ham sausage with a hybrid chemiluminescent immunoassay

<div><p>A novel chemiluminescent immunoassay utilising two types of primary antibodies (murine monoclonal antibody and rabbit polyclonal antibody) and two types of horseradish peroxidase–labelled secondary antibodies was established for simultaneously detecting multiple amphenicol residues in ham sausage. After combining the extract procedure of the target amphenicol into one simplified method, this hybrid chemiluminescent immunoassay could screen chloramphenicol (CAP), florfenicol (FF) and its metabolite florfenicol amine (FFA) at the same time by adding the corresponding secondary antibody. Ham sausage samples were analysed by using this hybrid immunoassay, with LODs of CAP being 0.01 μg kg⁻¹, of FF being 2.8 μg kg⁻¹ and of FFA being 3.0 μg kg⁻¹. The applicability of the proposed method has been validated by determining CAP, FF and FFA in ham sausage samples with satisfactory results. Good recoveries and high correlation with traditional enzyme-linked immunosorbent assay and LC-MS/MS results illustrated that the developed hybrid chemiluminescent immunoassay could screen high-throughput ultra-trace amphenicol residues effectively at one time.</p></div

Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon Nanoparticles for Detecting Three Fusarium Mycotoxins in Maize

The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6–109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography–tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals

Antibody Production, Immunoassay Establishment, and Specificity Study for Flunixin and 5‑Hydroxyflunixin

Flunixin (FLU) is a nonsteroidal drug that is widely used in animals, causing severe drug residues in animal-derived foods and environment. The development of antibody-based rapid immunoassay methods is of great significance for the monitoring of FLU and its metabolite 5-hydroxyflunixin (5-FLU). We prepared monoclonal antibodies (mAbs) with different recognition spectra through FLU-keyhole limpet hemocyanin conjugates as immunogen coupled with antibody screening strategies. mAb5E6 and mAb6D7 recognized FLU with high affinity, and mAb2H5 and mAb4A4 recognized FLU and 5-FLU with broad specificity. Through evaluating the recognition of these mAbs against more than 11 structural analogues and employing computational chemistry, molecular docking, and molecular dynamics methodologies, we preliminarily determined the recognition epitope and recognition mechanism of these mAbs. Finally, an indirect competitive enzyme-linked immunosorbent assay for FLU based on mAb6D7 was developed, which exhibited limits of detection as low as 0.016–0.042 μg kg –1 (L–1) in milk and muscle samples

Fluorescence Polarization Immunoassay Based on a New Monoclonal Antibody for the Detection of the Zearalenone Class of Mycotoxins in Maize

To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening the zearalenone class of mycotoxins in maize, two new monoclonal antibodies with uniform affinity to the zearalenone class and four fluorescein-labeled tracers were prepared. After careful selection of appropriate tracer–antibody pairs in terms of sensitivity and specificity, a FPIA that could simultaneously detect the zearalenone class with similar sensitivity was developed. Under optimum conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 1.89, 1.97, 2.43, 1.99, 2.27, and 2.44 μg/L for zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and zearalanone, respectively. The limit of detection of FPIA for the zearalenone class was around 12 μg/kg in maize, and the recoveries ranged from 84.6 to 113.8%, with coefficients of variation below 15.3% in spiked samples. Finally, the FPIA was applied for screening naturally contaminated maize samples, and the results indicated a good correlation with that of high-performance liquid chromatography–tandem mass spectrometry