AlphaLISA Detection Method for Quinolone Residues in Milk

This article aimed to establish an amplified luminescent proximity homogeneous assay linked immune sorbent assay (AlphaLISA) for detecting quinolones (QNS) residues in milk. The EDC/NHS active ester method was used to connect 6-aminocaproic acid as the spacer arm on the norfloxacin molecule to prepare biotin-modified norfloxacin hapten (Biotin-6AS-NOR), and QNS monoclonal antibody was coupled on the receptor microsphere. Biotin-6AS-NOR could compete with QNS in the sample to bind quinolone antibodies. A competitive indirect AlphaLISA for QNS residues in milk was established by optimizing the parameters including QNS antibody coupling amount on receptor microspheres, Biotin-6AS-NOR, the dosage of donor/receptor microspheres, and dilution schemes for milk samples. By using the 1:5 milk sample dilution scheme, the limit of detection and the limit of quantification of the established AlphaLISA for QNS in milk were 0.19 ng/mL and 0.46 ng/mL, respectively. The recovery values ranged from 85.97% and 110.11%, and the intraday and interday variation were both less than 10%. The cross-reactivity values for norfloxacin, enrofloxacin, ciprofloxacin, ofloxacin, pefloxacin, ofloxacin, and lomefloxacin were all higher than 70%. In conclusion, the established AlphaLISA showed high sensitivity, accuracy and good repeatability, without the need of tedious wash steps throughout the detection process; therefore, it is easy to achieve automated detection of chemical hazards residue in milk and has high application and promotion value.