Chenglong Li

Development and application of a quantitative fluorescence-based immunochromatographic assay for fumonisin b1 in maize.

A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1 (FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies (FM-mAbs) as detection reporters is described. The ICA is based on the competitive reaction between FB1-bovine serum albumin (BSA; test line) and the target FB1 for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12 ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL (corresponding to 250-2000 μg/kg in maize flour samples, according to the extraction procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from 91.4 to 118.2%. A quantitative comparison of the fluorescence-based ICA and HPLC-MS/MS analysis of naturally contaminated maize samples indicated good agreement between the two methods (r(2) = 0.93). By replacing the target of interest, the FM-based ICA can easily be extended to other chemical contaminants and thus represents a versatile strategy for food safety analysis.

A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 μg kg(-1) for DON, 17.8 μg kg(-1) for T-2 toxin and 331.5 μg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis.

Development of a screening fluorescence polarization immunoassay for the simultaneous detection of fumonisins B₁ and B₂ in maize.

This paper reports the development of a screening fluorescence polarization immunoassay (FPIA) for the simultaneous detection of fumonisins B1 (FB1) and B2 (FB2) in maize. Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC), FB1-5-([4,6-dichlorotriazine-2-yl]amino)-fluorescein (FB1-5-DTAF), and FB1-Texas Red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select appropriate tracer-antibody pairs using seven previously produced monoclonal antibodies (mAbs). An FPIA employing the pair of FB1-FITC and mAb 4B9 showing 98.9% cross-reactivity (CR) toward FB2 was used to simultaneously detect FB1 and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After optimization, the FPIA revealed a limit of detection (LOD) of 157.4 μg/kg for FB1 and an LOD of 290.6 μg/kg for FB2, respectively. Recoveries were measured for spiked samples of FB1 or FB2 separately, ranging from 84.7 to 93.6%, with a coefficient of variation (CV) of <9.9%. Total time needed for FPIA including sample pretreatment was <30 min. The FPIA was used to screen naturally contaminated maize samples. Results detected by FPIA showed good agreement with that of HPLC-MS/MS with a fit of R(2) = 0.99 for the simultaneous detection of FB1 and FB2. The established method offered a rapid, simple, sensitive, and high-throughput screening tool for the detection of fumonisins in maize.

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.

New haptens and antibodies for ractopamine.

In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 β-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples.

Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine

We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.

Development and Application of a Gel-Based Immunoassay for the Rapid Screening of Salbutamol and Ractopamine Residues in Pork.

Salbutamol (SAL) and ractopamine (RAC) have been illegally used to promote protein synthesis and to increase the feed conversion rate in livestock. However, the residues of SAL and RAC could cause potential hazards for human health. The Ministry of Agriculture of China banned the use of SAL and RAC as growth promoters. In this paper, we provide detailed information on developing a rapid and sensitive gel-based immunoassay for on-site screening of SAL and RAC residues in pork. The detection time was shortened to 20 min. The limits of detection were 0.5 μg/kg for both SAL and RAC by visual detection, whereas the quantitative gel-based immunoassay enabled the detection of SAL (0.051 μg/kg) and RAC (0.020 μg/kg) in spiked pork samples. The gel-based immunoassay showed promise as a multiplexed immunoassay for on-site surveilling of SAL and RAC residues in pork.

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 highly sensitive and class-specific fluorescence polarisation assay for sulphonamides based on dihydropteroate synthase

We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS) and a fluorescence probe for multi-sulphonamide detection. Dihydropteridine pyrophosphate (DHPPP) was synthesised and acts as the first substrate for DHPS. Under optimised conditions, the half-maximal inhibitory concentrations (IC50) of the assay were less than 100 ng mL(-1) for at least 29 sulphonamides and the time needed for the detection was less than 20 min. More importantly, the assay revealed quite uniform affinities for all of the individual sulphonamides tested, which has never before been achieved in an antibody-based assay.