Isolation and serological analyses of fungal melanins

Melanins are notoriously difficult to work with because of their unique phy sical and chemical properties. The study of melanins is hampered by the sca rcity of melanin-specific reagents and serological techniques. In this stud y we describe modifications to the standard method for the isolation of mel anins from in vitro-melanized fungal cells and detail the optimization of s erological techniques for the study of melanin compounds. The isolation pro cedure involves the digestion of melanized cells with a combination of prot eolytic and glycolytic enzymes, denaturant, organic extractions, and boilin g in 6.0 M HCl. Elemental quantitative analyses suggest that this procedure does not significantly affect the relative elemental composition of melani ns. For the serological assays, our goal was to achieve a homogenous distri bution of melanin particles on a solid support to maximize their recognitio n by melanin-binding antibodies. The results from enzyme-linked immunosorbe nt assays (ELISAs) demonstrate that melanins, in general, disperse more eff iciently on, and adhere better to, medium-binding polystyrene surfaces, esp ecially in the presence of trace amounts of salt. Blocking the melanin-coat ed ELISA plates with the commercially available SuperBlock Blocking Buffer for 4 h was more efficient at reducing non-specific binding of a negative c ontrol monoclonal antibody (mAb) compared to blocking with 2% bovine serum albumin (BSA) and 5% milk. Increasing the ionic strength of the antibody so lutions reduced binding to the melanins, indicating that binding is in part mediated by electrostatic interactions. These conditions were also applied to immunofluorescence (IF) analyses of melanins, and the results were cons istent with those obtained by ELISA. (C) 2000 Elsevier Science B.V. All rig hts reserved.