title: Hapten synthesis and antibody production for the development of a melamine immunoassay.
abstract: The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC(50) of 70.6 ng mL(-1), a LOD of 2.6 ng mL(-1) and a LOQ of 7.6 ng mL(-1). Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.
authors.affiliation: The Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong, PR China.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Lei
authors.forename: Yudong
authors.initials: Y
authors.lastname: Shen
authors.forename: Lijun
authors.initials: L
authors.lastname: Song
authors.forename: Jinyi
authors.initials: J
authors.lastname: Yang
authors.forename: Olivier P
authors.initials: OP
authors.lastname: Chevallier
authors.forename: Simon A
authors.initials: SA
authors.lastname: Haughey
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Yuanming
authors.initials: Y
authors.lastname: Sun
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Elliott
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology; D000917:Antibody Formation; D002417:Cattle; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006241:Haptens / Q000737:chemistry / Q000276:immunology*; D008968:Molecular Conformation; D010047:Ovalbumin / Q000737:chemistry; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014227:Triazines / Q000032:analysis / Q000737:chemistry / Q000276:immunology*
chemical_list: D000906:Antibodies; D006241:Haptens; D014227:Triazines; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; C011907:melamine