Jinting Liu

Preparation of Monoclonal Antibody for Melamine and Development of an Indirect Competitive ELISA for Melamine Detection in Raw Milk, Milk Powder, and Animal Feeds

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

One-Pot Green Synthesis and Bioapplication ofl-Arginine-Capped Superparamagnetic Fe3O4 Nanoparticles

Water-solublel-arginine-capped Fe3O4 nanoparticles were synthesized using a one-pot and green method. Nontoxic, renewable and inexpensive reagents including FeCl3,l-arginine, glycerol and water were chosen as raw materials. Fe3O4 nanoparticles show different dispersive states in acidic and alkaline solutions for the two distinct forms of surface bindingl-arginine. Powder X-ray diffraction and X-ray photoelectron spectroscopy were used to identify the structure of Fe3O4 nanocrystals. The products behave like superparamagnetism at room temperature with saturation magnetization of 49.9 emu g−1 and negligible remanence or coercivity. In the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, the anti-chloramphenicol monoclonal antibodies were connected to thel-arginine-capped magnetite nanoparticles. The as-prepared conjugates could be used in immunomagnetic assay.(See supplementary material 1)

Preparation of anti-melamine antibody and development of an indirect chemiluminescent competitive ELISA for melamine detection in milk

An indirect chemiluminescent competitive ELISA (CL-ELISA) method was developed to detect residues of melamine in milk. A high-quality polyclonal antibody towards melamine cyanurate (MC) was prepared based on synthesis of a novel immunogen. The method is applicable over the range of 0.5–7.0 µg.mL−1 of MC, with an IC50 value of 1.7 µg.mL−1. The developed method was used in the detection of melamine residue in milk with the detection limit of 1 ng.mL−1. There was no cross-reactivity with commonly used veterinary drugs. The CL-ELISA method developed provides an alternative to chromatography spectrometry for regulatory analysis of melamine in milk and could be promisingly used to improve the sensitivity of the available ELISA test kits.

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC50 of 0.4 ng mL−1 and a detection limit of 0.001 ng mL−1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.

Preparation of Anti-Lomefloxacin Antibody and Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Lomefloxacin Residue in Milk

Lomefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a complicated instrumental method to detect lomefloxacin residue in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen of bovine serum albumin-lomefloxacin showed high sensitivity toward lomefloxacin with an IC50 value of 0.35 ppb in PBS buffer and was suitable to be used as a screening assay to detect lomefloxacin residue in food products. The ELISA (Enzyme-Linked ImmunoSorbant Assay) developed in this study was compared with a commercial ELISA kit and significant improvement was achieved in terms of sensitivity and specificity. The antibody prepared showed excellent specificity with certain cross-reactivity with only two drugs including norfloxacin (17.5%) and fleroxacin (8.8%) among commonly used (fluoro)quinolones. The assay measured drug residue in defatted milk spiked with lomefloxacin with an inter-assay coefficient of variation <22.2% and an intra-assay coefficient of variation <18.2%. The average recovery rates at 0.1, 0.3, 0.6, 1.0, and 2.0 ppb were in the range of 120–85% for inter-assay and in the range of 113–90% for intra-assay.

Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine

To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC50) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products.