Simultaneous Determination of 13 Fluoroquinolone and 22 Sulfonamide Residues in Milk by a Dual-Colorimetric Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor two classes of antimicrobial residues in different food matrixes. In this paper, we describe a dual-colorimetric ELISA for the simultaneous detection of 13 fluoroquinolone and 22 sulfonamide residues. The limit of detection for fluoroquinolones and sulfonamides was 2.4 and 5.8 ng/mL, respectively. The developed immunoassay is suitable for high-throughput screening of these low-molecular weight contaminants. This is the first report where two different enzymes (alkaline phosphatase and horseradish peroxidase) were used in one immunoassay and together in a single well for simultaneous detection of multiple low-molecular weight chemical residues

Simultaneous Detection of Forbidden Chemical Residues in Milk Using Dual-Label Time-Resolved Reverse Competitive Chemiluminescent Immunoassay Based on Amine Group Functionalized Surface

<div><p>In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) in chemiluminesecence (CL) systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP) for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP) and CLE (ALP-CLE). The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 10⁵) of CLE as background for HRP CL signal value (about 10⁷) of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC₅₀) values of CAP and CLE in milk samples were 0.00501 µg L⁻¹ and 0.0128 µg L⁻¹, with the ranges from 0.0003 µg L⁻¹ to 0.0912 µg L⁻¹ and from 0.00385 µg L⁻¹ to 0.125 µg L⁻¹, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE.</p></div

A novel variable antibody fragment dimerized by the dHLX peptide with enhanced affinity against amantadine compared to its corresponding scFv antibody

<p>Amantadine (AMA) is an illegally used antiviral drug in the poultry industry, it is necessary to establish a fast, accurate and time-saving detection method for poultry food. The antibody-based immunoassay can achieve fast and accurate requirements. We developed a recombinant antibody-based specificity immunoassay for AMA. In the recombinant antibody, the heavy chain variable region (VH) is connected covalently with the light chain variable region (VL) by the artificial linker. Here, two recombinant antibodies’ single-chain variable fragment (scFv) and scFv-dHLX were constructed and functionally expressed in the periplasm of <i>Escherichia coli</i>. The helix-turn-helix peptide was utilized to dimerize VH and VL similar to the IgG counterpart. The ScFv-dHLX protein showed a higher binding ability and affinity resulting in improvement of <i>in vitro</i> affinity activity over its corresponding scFv. Our results not only indicated scFv-dHLX as an alternative for scFv in analytical application, but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced affinity.</p

Inhibition curves of CAP and CLE in buffer and milk extract.

<p>Inhibition curves of CAP and CLE in buffer and milk extract.</p

Recovery of spiked CAP and CLE in milk.

a<p>Each value was repeated five times.</p>b<p>Not detectable, </p><p>Recovery of spiked CAP and CLE in milk.</p

Schematic representation of DLTRRC-CIA for quantitative determination of CAP and CLE.

<p>Schematic representation of DLTRRC-CIA for quantitative determination of CAP and CLE.</p

Determination of milk samples collected from retail outlets in Beijing by the DLTRRC-CIA and traditional ELISA kit.

a<p>Each was determined with 3 repeats.</p>b<p>ND not detectable.</p><p>Determination of milk samples collected from retail outlets in Beijing by the DLTRRC-CIA and traditional ELISA kit.</p

Kinetic measurement of chemiluminescence (CL) output intensity (RLU) for Super Signal and Visiglo Plus substrates catalyzed by HRP and ALP in the developed DLTRRC-CIA.

<p>Kinetic measurement of chemiluminescence (CL) output intensity (RLU) for Super Signal and Visiglo Plus substrates catalyzed by HRP and ALP in the developed DLTRRC-CIA.</p

A comparative analysis of the developed DLTRRC-CIA with various immunoassay formats and commercial kits for CAP and CLE detection in standard solution.

<p>A comparative analysis of the developed DLTRRC-CIA with various immunoassay formats and commercial kits for CAP and CLE detection in standard solution.</p

Normalized standard curve by developed DLTRRC-CIA for CAP (a) and CLE (b) under optimized conditions compared to the standard curve obtained by reverse competitive CL-ELISA and traditional ELISA method.

<p>Normalized standard curve by developed DLTRRC-CIA for CAP (a) and CLE (b) under optimized conditions compared to the standard curve obtained by reverse competitive CL-ELISA and traditional ELISA method.</p