Xuezhi Yu

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 Renilla 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 (R0) 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.

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.

Development and validation of a chemiluminescent ELISA for simultaneous determination of florfenicol and its metabolite florfenicol amine in chicken muscle

A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC50 value of the method was 0.153 μg kg−1 for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA–F–BSA (FFA–formaldehyde–BSA) and FF–G–OVA (FF–glutaric anhydride–OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40:1 ethyl acetate–ammonia mixture, obtaining recoveries of 70.3–100% (FFA) and 71.8–102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCβ) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 μg kg−1 for FFA and 0.526 μg kg−1 for FF (10-fold dilution) and 0.453 μg kg−1 for FFA and 0.657 μg kg−1 for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCβ values of 1.0 μg kg−1 for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.

An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis.

Development of a rapid chemiluminescent ciELISA for simultaneous determination of florfenicol and its metabolite florfenicol amine in animal meat products

BACKGROUND A rapid one-step chemiluminescent competitive indirect enzyme-linked immunosorbent assay (CL-ciELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in animal meat products has been developed. RESULTS The 50% binding inhibition (IC₅₀) values of the method were 0.195 µg kg⁻¹ for FFA and 0.24 µg kg⁻¹ for FF under optimum conditions. The cross-reactive rates for FF and FFA were 100.0% and 81.2%, respectively. FF and FFA were easily extracted from animal meat product with an FF/FFA extraction buffer, obtaining recoveries of 81.8-92.0% (FF) and 77.2-100% (FFA). The whole one-step CL-ciELISA test can be accomplished within 40 min in theory. The detection limits (LODs) of the assay were 0.98 µg kg⁻¹ for FF and 0.80 µg kg⁻¹ for FFA in animal meat samples. Finally, field animal meat samples were analyzed with the CL-ciELISA method, and the results correlated well with those obtained using traditional ELISA and a previously reported liquid chromatographic-tandem mass spectrometric method. CONCLUSION The combined results confirmed the utility of this faster one-step CL-ciELISA for simultaneous trace analysis of FF and FFA. To date, this is the most rapid developed ELISA and CL-ELISA method for detection of FF and FFA.

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

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.

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.

A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein

A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)–alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv–AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv–AP fusion protein in combination with a norfloxacin–ovalbumin conjugate (NOR–OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg−1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv–AP), and the linear response range of the assay was extended from 0.04 to 1.08 μg kg−1. The limits of detection (LODs) of the assay for NOR were 0.017 μg kg−1 in shrimp and 0.018 μg kg−1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg−1 for ciprofloxacin (CIP) to 4.19 μg kg−1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv–AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp.

A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk

Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarisation immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labelled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR)) with different excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits in milk samples. Recoveries for spiked milk samples were from 76.4% to 128.4%, with a relative standard deviation lower than 13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC-MS/MS. All three instances in which FQs and SAs were present at concentrations near or above the assay limit of detection were identified as positive by the developed DB-FPIA, demonstrating that the method is suitable for rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for fast multi-analyte screening of low-molecular weight chemical residues in food samples. Graphical Abstract

Generic Hapten Synthesis, Broad-Specificity Monoclonal Antibodies Preparation, and Ultrasensitive ELISA for Five Antibacterial Synergists in Chicken and Milk

An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immunoassay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using trimethoprim as the starting molecule. These haptens contained carboxyl groups on the meta position of trimethoxybenzene for conjugating to protein, while, the common moiety of ASGs, i.e., diaminopyrimidine, was intentionally and maximally exposed to the immune system in animals in order to induce antibodies with broad specificity against ASGs. Five monoclonal antibodies (mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm, named Hapten A, showed not only uniform broad specificity but also high affinity to all five ASGs. We further determined the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. An indirect competitive ELISA (icELISA)-based 5C4 was established and exhibited IC50 values of 0.067-0.139 μg L-1 with cross-reactivity of 48.2%-418.7% for the five ASGs in buffer under optimal conditions. The calculated limits of detection of the icELISA for chicken and milk were 0.06-0.8 μg kg-1 and 0.05-0.6 μg L-1, respectively. The recoveries in spiked chicken and milk samples were 75.2%-101.4% with a coefficient of variation less than 14.3%. In summary, we have developed, for the first time, a rapid and reliable icELISA for ASGs with significantly improved sensitivity and class selectivity.