TY - JOUR
T1 - Isolation and serological analyses of fungal melanins
AU - Rosas, Ángel L.
AU - Nosanchuk, Joshua D.
AU - Gómez, Beatriz L.
AU - Edens, William A.
AU - Henson, Joan M.
AU - Casadevall, Arturo
N1 - Funding Information:
Arturo Casadevall is supported by NIH grants AI33774, AI13342, and HL59842, and is a recipient of a Burroughs Wellcome Fund Scholar Award in Experimental Therapeutics. Ángel L. Rosas is supported by NIH grant 5T32GM07491. Beatriz L. Gómez is supported by the UNESCO-ASM 1999 Travel Award. Joshua D. Nosanchuk is supported by NIH grant AI01489. William A. Edens and Joan M. Henson are supported by the U.S. Army Research Office grant DAAH04-96-1-0194 and the National Science Foundation grant MCB-9977922.
PY - 2000/10/20
Y1 - 2000/10/20
N2 - Melanins are notoriously difficult to work with because of their unique physical and chemical properties. The study of melanins is hampered by the scarcity of melanin-specific reagents and serological techniques. In this study we describe modifications to the standard method for the isolation of melanins from in vitro-melanized fungal cells and detail the optimization of serological techniques for the study of melanin compounds. The isolation procedure involves the digestion of melanized cells with a combination of proteolytic and glycolytic enzymes, denaturant, organic extractions, and boiling in 6.0 M HCl. Elemental quantitative analyses suggest that this procedure does not significantly affect the relative elemental composition of melanins. For the serological assays, our goal was to achieve a homogenous distribution of melanin particles on a solid support to maximize their recognition by melanin-binding antibodies. The results from enzyme-linked immunosorbent assays (ELISAs) demonstrate that melanins, in general, disperse more efficiently on, and adhere better to, medium-binding polystyrene surfaces, especially in the presence of trace amounts of salt. Blocking the melanin-coated ELISA plates with the commercially available SuperBlock(®) Blocking Buffer for 4 h was more efficient at reducing non-specific binding of a negative control monoclonal antibody (mAb) compared to blocking with 2% bovine serum albumin (BSA) and 5% milk. Increasing the ionic strength of the antibody solutions 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 consistent with those obtained by ELISA. Copyright (C) 2000 Elsevier Science B.V.
AB - Melanins are notoriously difficult to work with because of their unique physical and chemical properties. The study of melanins is hampered by the scarcity of melanin-specific reagents and serological techniques. In this study we describe modifications to the standard method for the isolation of melanins from in vitro-melanized fungal cells and detail the optimization of serological techniques for the study of melanin compounds. The isolation procedure involves the digestion of melanized cells with a combination of proteolytic and glycolytic enzymes, denaturant, organic extractions, and boiling in 6.0 M HCl. Elemental quantitative analyses suggest that this procedure does not significantly affect the relative elemental composition of melanins. For the serological assays, our goal was to achieve a homogenous distribution of melanin particles on a solid support to maximize their recognition by melanin-binding antibodies. The results from enzyme-linked immunosorbent assays (ELISAs) demonstrate that melanins, in general, disperse more efficiently on, and adhere better to, medium-binding polystyrene surfaces, especially in the presence of trace amounts of salt. Blocking the melanin-coated ELISA plates with the commercially available SuperBlock(®) Blocking Buffer for 4 h was more efficient at reducing non-specific binding of a negative control monoclonal antibody (mAb) compared to blocking with 2% bovine serum albumin (BSA) and 5% milk. Increasing the ionic strength of the antibody solutions 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 consistent with those obtained by ELISA. Copyright (C) 2000 Elsevier Science B.V.
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U2 - 10.1016/S0022-1759(00)00255-6
DO - 10.1016/S0022-1759(00)00255-6
M3 - Research Article
C2 - 11033020
AN - SCOPUS:0034693357
SN - 0022-1759
VL - 244
SP - 69
EP - 80
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -