Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody

Cox, Rebecca A.; Huppert, M.; Starr, Phoebe Betty; Britt, L A

doi:10.1128/iai.43.2.502-507.1984

Cited by 22 publications

(24 citation statements)

References 24 publications

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“…The latter possessed two antigenic species not present in the mycelial preparation (or present in concentrations too low to be detected by twodimensional immunoelectrophoresis). Cox et al (7) have recently added to the information indicating that antigen 2 has reactivity conforming to the ID-TP antigen, indicating, as did Collins et al (3), that this antigen is associated with the cell wall. Cole et al (4) demonstrated the presence of antigen 2 in the walls of arthroconidia and spherules.…”

Section: Discussionmentioning

confidence: 94%

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis

Zimmer¹,

Pappagianis

1986

Infect Immun

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The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28-and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band. * Corresponding author. t Present address: American Micro/Scan, West Sacramento, CA 95691.

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Section: Discussionmentioning

confidence: 94%

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis

Zimmer¹,

Pappagianis

1986

Infect Immun

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“…Solid-phase immunoadsorption was used to isolate the antibody fractions reactive with the purified 120-kDa TP-Ag from a pool of five ID-TP-positive patient serum samples used in the TP assay of this same antigen in an earlier report (24). Coupling of antigen to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) and the subsequent washing procedure were conducted as reported by Cox and coworkers (15). The gel was transferred to a column (0.8 by 15 cm) and equilibrated with wash buffer (phosphate-buffered saline [PBS; pH 7.4] containing 0.15 M NaCl and 0.005% [wt/vol] thimerosal).…”

Section: Methodsmentioning

confidence: 99%

“…We also show that reactivity of the antigen with serum samples from patients with coccidioidomycosis is retained after pronase digestion or heating (100°C, 5 min) but is lost after periodate oxidation. Examination of the carbohydrate composition of the TP antibody-reactive antigen (TP-Ag) by gas chromatography-mass spectroscopy revealed 3-0-methylmannose (3-0-MM), a sugar that was previously shown to be a component of immunoreactive fractions of C. immitis (3,15,17,28,(35)(36)(37) and possibly unique to this pathogen among the systemic fungal pathogens (3,37). In this report, we also further examine the serologic reactivity of the 120-kDa TP-Ag and, in particular, the significance of 3-0-MM in precipitin binding to this macromolecule.…”

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confidence: 99%

Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis

et al. 1990

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Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100°C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-0-methylmannose (3-0-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-0-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-0-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.

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“…44 Another protein, Antigen 2 (Ag2), was first identified in 1978 by two-dimensional immunoelectrophoresis (IEP) from the crude antigen preparations coccidioidin and spherulin, 45 and also found in the alkali-soluble, water-soluble mycelial and spherule extracts. 46 Ag 2 was not sequenced until 1996. 47 A parallel antigen discovery of the prolinerich antigen (PRA) was identified from a toluene spherule lysate.…”

Section: Application Of Proteomic Analysis To Fungal Systemsmentioning

confidence: 99%

The Application of Proteomic Techniques to Fungal Protein Identification and Quantification

Rohrbough

Galgiani

Wysocki

2007

Annals of the New York Academy of Sciences

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The number of sequenced genomes has increased rapidly in the last few years, supporting a revolution in bioinformatics that has been leveraged by scientists seeking to analyze the proteomes of numerous biological systems. The primary technique employed for the identification of peptides and proteins from biological sources is mass spectrometry (MS). This analytical process is usually in the form of whole-protein analysis (termed "top-down" proteomics) or analysis of enzymatically produced peptides (known as the "bottom-up" approach). This article will focus primarily on the more common bottom-up proteomics to include topics such as sample preparation, separation strategies, MS instrumentation, data analysis, and techniques for protein quantification. Strategies for preparation of samples for proteomic analysis, as well as tools for protein and peptide separation will be discussed. A general description of common MS instruments along with tandem mass spectrometry (MS/MS) will be given. Different methodologies of sample ionization including matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) will be discussed. Data analysis methods including database search algorithms and tools for protein sequence analysis will be introduced. We will also discuss experimental strategies for MS protein quantification using stable isotope labeling techniques and fluorescent labeling. We will introduce several fungal proteomic studies to illustrate the use of these methods. This article will allow investigators to gain a working knowledge of proteomics along with some strengths and weaknesses associated with the techniques presented.

show abstract

Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody

Cited by 22 publications

References 24 publications

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis

Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis

The Application of Proteomic Techniques to Fungal Protein Identification and Quantification

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