Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages.
A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.
Romero-Martínez
Rafael
R
Departamento de Microbiología-Parasitología, Facultad de Medicina, Universidad Nacional Autónoma México (UNAM), Mexico.
Curiel-Quesada
Everardo
E
Becerril-Luján
Baltazar
B
Flores-Carreón
Arturo
A
Pérez-Torres
Armando
A
Taylor
Maria Lucia
ML
D017104:Bacteriophage M13 / Q000235:genetics* / Q000276:immunology; D006658:Histoplasma / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library
D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv