Preparation of Monoclonal Antibody for Melamine and Development of an Indirect Competitive ELISA for Melamine Detection in Raw Milk, Milk Powder, and Animal Feeds

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 mu g L-1 MEL in buffer solution, with an IC50 value of 22.6 +/- 1.9 mu g L-1. The MAbs showed high specificity with low cross-reactivity 1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L-1 for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L-1 for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.