Production of monoclonal antibodies for detection of a secreted aspartyl proteinase from Candida spp. in biologic specimens.
Secreted acid proteinases (SAP) constitute an important group of virulence factors in Candida albicans. In the present work, an acid proteinase from C. albicans was sequentially purified from the supernatant of a yeast culture by precipitation with ammonium sulfate, ion exchange chromatography, and molecular exclusion chromatography, yielding a specific enzymatic activity of 204.1 IU/mg on bovine serum albumin (BSA). The molecular mass of the purified proteinase was estimated at 43 kd after exclusion chromatography and at 41 kd by nondenaturating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteinase was able to degrade BSA at pH 2.5, but was not active on collagen, and it was significantly inhibited by pepstatin A. The immunization of BALB/c mice with the purified proteinase and later fusion of their spleen cells with myeloma cells resulted in 19 monoclonal antibody secreting hybridomas (MAbs) capable of detecting SAP in enzyme-linked immunosorbent assay (ELISA) assays. All MAbs obtained are isotype IgG1 kappa (kappa) immunoglobulins and develop a 41 kd protein band by Western blot (WB) in samples of SAP obtained from C. albicans (12-A) and C. dubliniensis (strain 778) crude extracts. The anti-SAP MAbs were used in capture ELISA and two combinations of these antibodies proved suitable for SAP detection, that is, MAP1 (1B1B3) or MAP2 (2D2C10) as coat antibodies, and biotinylated MAP3 (2A6E8) as detect antibody. Capture ELISA using these sets of MAbs detected over 32 ng/mL protein in purified SAP samples as well as in crude C. albicans and C. dubliniensis extracts. The results herein obtained allow for the prediction of how this set of antibodies can be useful for SAP detection in biologic specimens.
Rodrigues
Janaina Aparecida De Oliveira
JA
Area de Microbiologia e Imunologia, Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Unicamp, SP, Brazil.
Höfling
José Francisco
JF
Azevedo
Ricardo Antunes
RA
Gabriel
Dirce Lima
DL
Tamashiro
Wirla Maria Da Silva Cunha
WM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002175:Candida / Q000201:enzymology*; D002176:Candida albicans / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000378:metabolism*; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D016282:Aspartic Acid Endopeptidases