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.
Kammeyer
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Department of Dermatology and Clinical Chemistry, University of Amsterdam, Netherlands.
Oomen
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Pavel
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007211:Indoles / Q000032:analysis* / Q000276:immunology; D008297:Male; D008543:Melanins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D007211:Indoles; D008543:Melanins; C017408:6-hydroxy-5-methoxy-2-indolylcarboxylic acid; C030692:5,6-dihydroxy-2-indolylcarboxylic acid