Preparation and Characterization of Monoclonal Antibody Against Melatonin

Abstract

Abstract

Anti-melatonin monoclonal antibodies (MAb) were prepared following coupling melatonin to bovine serum albumin (BSA) by Mannich reaction. Balb/c mice were immunized via injection of the melatonin-BSA intraperitonally. The spleen cells producing high titer of antibody were fused with myeloma cells of SP2/0 origin. After two limiting dilutions, two stable clones (AS-H10 and AS-D26) exhibiting best properties were selected for further studies. The class and subclass of two MAbs were found to be IgG₁ and IgG_2a with λ and κ light chains, respectively. Antibodies secreted by these two clones showed high affinity of about 10⁹M⁻¹. Study of the specificity criteria showed that these clones had no cross reactivity with indolic, aromatic, and imidazole ring-containing compounds, and had high specificity towards melatonin. The calibration curve was constructed with a sensitivity range of 10 ng/mL to 10 μg/mL. In conclusion, these MAbs may be useful for immunoassay of melatonin.