An Enhanced Direct Competitive Immunoassay for the Detection of Kanamycin and Tobramycin in Milk Using Multienzyme-Particle Amplification
- 171 Downloads
Kanamycin (Kan) and tobramycin (Tob) are widely found in many foods of animal origin, including milk. More rapid, simple, and sensitive methods are urgently needed to monitor antibiotic residues in milk. An enhanced direct competitive enzyme-linked immunosorbent assay (dcELISA) based on gold nanoparticles (AuNPs)/horse radish peroxidase-Kan (HRP-Kan) was developed. A monoclonal antibody (Mab) against Kan was developed by classic hybridoma technology. The Mab had higher cross-reactivity with Tob (99.07%) and no cross-reactivity with other related antibiotics (< 0.5%). A novel multienzyme probe was synthesized based on AuNPs modified using HRP-Kan. The Mab against Kan, fixed by a goat anti-mouse antibody, was competitively bound by AuNPs/HRP-Kan and Kan in samples. After optimization, the limit of detection of the enhanced dcELISA was 0.022 ng/mL, representing a fivefold improvement when compared to that of conventional dcELISA (0.13 ng/mL). The recoveries of Kan and Tob in milk samples varied from 81.0 to 121.0% and 86.4 to 123.9%, respectively. Kan or Tob was found to be present at concentrations of 0.352–0.548 ng/mL in five milk samples from local markets. The results by the enhanced ELISA and UPLC-MS/MS had good correlation. It was suggested that the enhanced dcELISA, based on AuNPs/HRP-Kan, has higher sensitivity and reliable reproducibility, and thus, this could be used to detect trace contaminants.
KeywordsKanamycin Tobramycin Monoclonal antibody Multienzyme-particle amplification
The authors would especially like to thank Dr. Fei Xu (Feed Research Institute, Chinese Academy of Agricultural Sciences) for her help in UPLC-MS/MS.
This work was supported financially by the National Natural Science Foundation of China (31502118 and 31570414), the China Postdoctoral Science Foundation (2013M541606), the Natural Science Fund project of Jiangsu Province (BK20130507), the Scientific Research Funds in Jiangsu University (13JDG016), and the Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment.
Compliance with Ethical Standards
Conflict of Interest
Ling Jiang declares that she has no conflict of interest. Dali Wei declares that he has no conflict of interest. Kun Zeng declares that he has no conflict of interest. Jie Shao declares that he has no conflict of interest. Fang Zhu declares that he has no conflict of interest. Daolin Du declares that he has no conflict of interest.
This article does not contain any studies with human participants performed by any of the authors. All mice used for experiment were obtained from the Comparative Medicine Center, Yangzhou University, China (Certificate No. SCXK (SU) 2007-0007). All of the experimental procedures were conducted in accordance with the principle of laboratory animal care and approved by the Jiangsu University Committee on Animal Care and Use (Certificate No. SYXK (SU) 2013-0035).
- European Commission (2010) Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off J Eur Union, L: Legislation (English Edition). 15:1–72, http://ec.europa.eu/health/files/eudralex/vol-5/reg_2010_37/reg_2010_37_en.pdf
- Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature 241:20–22Google Scholar
- Kaufmann A, Maden K (2005) Determination of 11 aminoglycosides in meat and liver by liquid chromatography with tandem mass spectrometry. J AOAC Int 88:1118–1125Google Scholar
- Li D, Cui Y, Morisseau C, Gee SJ, Bever CS, Liu X, Wu J, Hammock BD, Ying Y (2017) Nanobody based immunoassay for human soluble epoxide hydrolase detection using polymeric horseradish peroxidase (polyHRP) for signal enhancement: the rediscovery of polyHRP? Anal Chem 89:6248–6256CrossRefGoogle Scholar
- Liu L, Chen Y, Song S, Zheng Q, Wu X, Kuang H (2017) Development of an antibody-based colloidal gold immunochromatographic lateral flow strip test for natamycin in milk and yoghurt samples. Food Agric Immunol 28:1283–1292Google Scholar
- Nouws JF, Verdijk AT (1991) How can the incidence of antibiotic residues in meat animals be minimized? Tijdschr Diergeneeskd 116:833–840Google Scholar
- Nouws JFM, Loeffen G, Schouten J, Van Egmond H, Keukens H (1995) Improvement of the tube diffusion test with respect to detection of antibiotic residues and sulphonamides in raw milk. Arch Fuer Lebensmittelhygiene 46:140–141Google Scholar
- Su S, Zuo X, Pan D, Pei H, Wang L, Fan C, Huang W (2013) Design and applications of gold nanoparticle conjugates by exploiting biomolecule-gold nanoparticle interactions. Nano 5:2589–2599Google Scholar
- Xu F (2014) Study on screening and validation methods for determination of aminoglycoside drugs residue. Doctor thesis: China Agriculture UniversityGoogle Scholar