An Enhanced Direct Competitive Immunoassay for the Detection of Kanamycin and Tobramycin in Milk Using Multienzyme-Particle Amplification



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.


Kanamycin 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.

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.

Ethical Approval

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).

Informed Consent

Not applicable.

Supplementary material

12161_2018_1185_MOESM1_ESM.doc (8.1 mb)
ESM 1 (DOC 8.13 mb)


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of the Environment and Safety EngineeringJiangsu UniversityZhenjiangChina

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