Isolation and serological analyses of fungal melanins

Ángel L. Rosas, Joshua D. Nosanchuk, Beatriz L. Gómez, William A. Edens, Joan M. Henson, Arturo Casadevall

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)69-80
Number of pages12
JournalJournal of Immunological Methods
Volume244
Issue number1-2
DOIs
StatePublished - Oct 20 2000

Fingerprint

Melanins
Enzyme-Linked Immunosorbent Assay
Antibodies
Polystyrenes
Bovine Serum Albumin
Static Electricity
Osmolar Concentration
Fluorescent Antibody Technique
Digestion
Buffers
Milk
Peptide Hydrolases
Salts
Monoclonal Antibodies

Keywords

  • ELISA
  • Immunofluorescence
  • Isolation
  • Melanins
  • Monoclonal antibodies
  • Quantitative elemental analysis

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Rosas, Á. L., Nosanchuk, J. D., Gómez, B. L., Edens, W. A., Henson, J. M., & Casadevall, A. (2000). Isolation and serological analyses of fungal melanins. Journal of Immunological Methods, 244(1-2), 69-80. https://doi.org/10.1016/S0022-1759(00)00255-6

Isolation and serological analyses of fungal melanins. / Rosas, Ángel L.; Nosanchuk, Joshua D.; Gómez, Beatriz L.; Edens, William A.; Henson, Joan M.; Casadevall, Arturo.

In: Journal of Immunological Methods, Vol. 244, No. 1-2, 20.10.2000, p. 69-80.

Research output: Contribution to journalArticle

Rosas, ÁL, Nosanchuk, JD, Gómez, BL, Edens, WA, Henson, JM & Casadevall, A 2000, 'Isolation and serological analyses of fungal melanins', Journal of Immunological Methods, vol. 244, no. 1-2, pp. 69-80. https://doi.org/10.1016/S0022-1759(00)00255-6
Rosas, Ángel L. ; Nosanchuk, Joshua D. ; Gómez, Beatriz L. ; Edens, William A. ; Henson, Joan M. ; Casadevall, Arturo. / Isolation and serological analyses of fungal melanins. In: Journal of Immunological Methods. 2000 ; Vol. 244, No. 1-2. pp. 69-80.
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