Development of ELISA for melamine detection in milk powder

A sensitive ELISA method was developed based on a monoclonal antibody to detect melamine in milk powder. Two haptens were prepared using the melamine structural analogue, 2-chloro-4, 6-diamino-1, 3, 5-triazine. The obtained antibody belonged to the IgG1κ family and had high affinity for melamine (affinity constant was 3.09×10⁹). The antibody could also recognise cyromazine (cross-reactivity was 131%) and had low cross-reactivity toward the haptens. The effects of pH, ionic strength and organic solvents on ELISA performance were evaluated. The lowest IC₅₀ value (6.0±0.55 ng/mL) and the limit of detection in milk powder, 50 ng/g, were achieved under optimised conditions. Milk powder spiked at two levels of melamine (100 ng/g and 500 ng/g) was analysed with this ELISA method. Good recovery was obtained (76.2±7.3% at the 100 ng/g spiked level and 91.4±5.0% at 500 ng/g).