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 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

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

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

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

CR of CAP and CLE in DLTRRC-CIA with some structurally related and unrelated compounds.

<p>CR of CAP and CLE in DLTRRC-CIA with some structurally related and unrelated compounds.</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

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