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Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, Guangdong Province, China
Institute of Agri-Food and Land Use, Queen’s University Belfast, Belfast BT9 5AG, Northern Ireland, UK
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, Guangdong Province, China
* Authors to whom correspondence should be addressed.
Received: 17 May 2011; in revised form: 13 June 2011 / Accepted: 15 June 2011 / Published: 30 June 2011
Abstract: An indirect competitive enzyme-linked immunosorbent assay (icELISA) with enhanced specificity for melamine in milk was developed. Three haptens of melamine with different spacer-arms were used to prepare different plate coating antigens. It was found that the icELISA show best sensitivity and specificity to melamine when using the coating antigen prepared by coupling 3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio)propanoic acid (Hapten C) with ovalbumin (OVA). The 50% inhibitory concentration (IC50) value was 35.4 ng·mL−1, the limit of detection (LOD) was 8.9 ng·mL−1 and the detectable working range (20–80% inhibitory concentration) was from 14.9 to 108.5 ng·mL−1, respectively. Compared to the ELISA results previously reported, the developed icELISA in the present study showed a much lower cross-reactivity to cyromazine, a fly-killing insecticide widely used in vegetables and stables. Recoveries obtained from milk samples in this study were in agreement with those obtained using the HPLC-MS method, indicating the detection performance of the icELISA could meet the requirement of the residue limit set by the Codex Alimentarius Commission. Therefore, the developed immunoassay can be applied for the analysis of melamine presented in milk.
Keywords: melamine; ELISA; specificity; milk
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Lei, H.; Su, R.; Haughey, S.A.; Wang, Q.; Xu, Z.; Yang, J.; Shen, Y.; Wang, H.; Jiang, Y.; Sun, Y. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk. Molecules 2011, 16, 5591-5603.
Lei H, Su R, Haughey SA, Wang Q, Xu Z, Yang J, Shen Y, Wang H, Jiang Y, Sun Y. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk. Molecules. 2011; 16(7):5591-5603.
Lei, Hongtao; Su, Rui; Haughey, Simon A.; Wang, Qiang; Xu, Zhenlin; Yang, Jinyi; Shen, Yudong; Wang, Hong; Jiang, Yueming; Sun, Yuanming. 2011. "Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk." Molecules 16, no. 7: 5591-5603.