Development of a sensitive monoclonal antibody-based immunochromatographic strip for neomycin detection in milk

ABSTRACT

To improve the detection sensitivity of neomycin (NEO) in milk, we produced a sensitive monoclonal antibody (mAb) against NEO and developed a lateral flow immunoassay based on amorphous carbon nanoparticles (ACNPs-LFA). First, we conjugated NEO to carrier protein to prepare mAbs. We obtained six mAbs: mAb 1C6, mAb 1D3, mAb 2D3, mAb 4D5, mAb 5D1, and mAb 5H1. We characterised the mAbs by indirect competitive enzyme-linked immunosorbent assay and selected the most sensitive mAb based on the half-maximal inhibitory concentration (IC₅₀). We selected mAb 4D5 (IC₅₀ = 0.15 ng/mL) for the development of LFA. MAb 4D5 was labelled with ACNPs (ACNPs-mAb 4D5) by electrostatic absorption. Under optimised conditions, 5.4 μg mAb 4D5 coupling with 1 mL ACNPs, NEO-OVA at concentration of 80 μg/mL, 3 μL ACNPs-mAb 4D5 were used to develop LFA. The cut-off value was 8 ng/mL. Therefore, our developed ACNPs-LFA is suitable for on-site detection of NEO residues.

KEYWORDS:

• Lateral flow immunoassays
• amorphous carbon nanoparticles
• monoclonal antibody
• neomycin
• milk
• detection

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

BALB/c mice were purchased from the Experimental Animal Centre of Zhengzhou University and raised in the Animal Experimental Centre of the University of Traditional Chinese Medicine. Animal experiments were approved by the animal ethics committee and carried out in accordance with the guidelines of the animal experiment centre.

Additional information

Funding

This study was financially supported by the Key Research and Development Programme of Henan Province (No. 212102310390), Natural Science Foundation of China (No. 32172298 and No. 31802249), and Young Talents Foundation of Henan Agricultural University (No. 30500645 and No. 30500673).