Preparation of monoclonal mouse antibodies against two specific eu-melanin related compounds

Two eu-melanin precursors, 6-hydroxy-5-methoxyindole-2-carboxylic acid (HMI2C) and 5,6-dihydroxyindole-2-carboxylic acid (DHI2C) were synthesized and coupled to bovine serum albumin, hemocyanin and polylysine by the combined action of carbodiimide and succinimide. These indole-carrier conjugates served as antigens for the production of specific antibodies against DHI2C and HMI2C in BALB/c mice. The specificity of these antibodies was tested using a combination of affinity chromatography and ELISA procedures. Polyclonal mouse antibodies reacted with the indole-carrier conjugates, but not with the unbound indole compounds. Monoclonal antibodies from two hybridoma cell lines were obtained from a HMI2C-immunized mouse after a fusion with four subclonings. They reacted with free HMI2C and to a lesser extent with unbound DHI2C. One monoclonal showed 50% inhibition in the ELISA test at concentrations of 0.6 mumol.l-1 and 5 mumol.l-1 for HMI2C and DHI2C, respectively. These antibodies did not show any cross-reactivity with nine structurally related compounds and should be valuable reagents for the detection and quantification of HMI2C and other eu-melanin related compounds.