Fractionation of &lt;i&gt;Aspergillus fumigatus&lt;/i&gt; Antigens by Hydrophobic Interaction Chromatography and Gel Filtration

Culture filtrate antigens of Aspergillus fumigatus were fractionated by isoelectric focusing using a pH gradient of 4–6.5. Three fractions, namely, 18, 19 and 20 were pooled and subjected to immunochemical analysis. It contained a concanavalin A binding glycoprotein. Antibody raised against this component was used to prepare an affinity column of IgG-Sepharose and was used to purify crude culture filtrate antigens. This component produced three precipitin arcs in crossed immunoelectrophoresis using anti-A. fumigatus rabbit serum. This fraction has an isoelectric point of 6.5 and showed three components in two-dimensional electrophoresis with approximate molecular weights of 20, 40 and 80 kilo daltons. This antigen reacted with patient sera in the biotinavidin linked immunosorbent assay and showed high levels of anti-A. fumigatus IgG and IgE antibodies in allergic bronchopulmonary aspergillosis and IgG antibodies in aspergilloma. Both controls and Aspergillus skin test positive asthmatics showed only low levels of specific antibodies. Because of the purity of this antigen, its potential use as a standardized antigen in the detection of antibody is discussed.

Section: Discussionmentioning

confidence: 99%

A Partially Purified Glycoprotein Antigen from Aspergillus fumigatus

Kurup¹,

John²,

Resnick³

et al. 1986

“…In a previous study, a 70,000-dalton A. fum igatus antigen fraction prepared by gel filtra tion revealed high ELISA antibody activity in pa tients with aspergillosis and only insignificant activity in patients with nonfungal lung disease and healthy controls [18]. A further purification o f A. fum igatus antigens have been obtained by combined hydrophobic interaction chrom atography and gel filtration [20]. With a small number o f sera several fractions seemed to detect antibodies of particular diagnostic significance.…”

Section: Introductionmentioning

confidence: 98%

IgG Antibodies to Purified Aspergillus fumigatus Antigens Determined by Enzyme-Linked Immunosorbent Assay

Schønheyder¹,

Andersen²

1984

Self Cite

IgG antibodies to three purified Aspergillus fumigatus antigens were determined by enzyme-linked immunosorbent assay (ELISA) in sera from 26 patients with definite pulmonary aspergillosis (group 1), 23 patients with suspected pulmonary aspergillosis (group 2), 8 patients with precipitating antibodies to A. fumigatus of undetermined clinical significance (group 3), and 113 healthy blood donors (group 4). With a 470,000-dalton antigen fraction ELISA values exceeded the upper range of group 4 (0.72) in 92, 91 and 13% of cases in groups 1, 2 and 3, respectively. With a 250,000-dalton antigen fraction the figures were 96, 78 and 13%, respectively (upper range of group 4 = 0.25). With an antigen fraction of 25,000–50,000 daltons the figures were 96, 65 and 13%, respectively (upper range of group 4 = 0.18). In 48 of 49 cases of definite or suspected aspergillosis (groups 1 + 2) ELISA values with at least one antigen exceeded the upper range of controls (group 4). It appears that antibody determination with these three antigen fractions may have a high diagnostic sensitivity.

“…Possibly, the IgG and IgE binding activity o f this fraction is represented by different proteins, because we could not find a major antigen at pi 6.5 in our extracts. Further investigation is needed to determine whether or not the antigens and allergens identified in this study correspond to these and other described antigens and allergens [9,[40][41][42][43][44][45][46], By the Western blot technique, IgG reactivity against proteins ofbetween 12 and 100 kD has been demonstrated in ABPA patients [12], Our results show that binding to pro teins with molecular weights above 45 kD is mainly caused by IgG2 and IgG3, while reactivity to lower molecular weight proteins is caused by IgGl and IgG4.…”

Section: %mentioning

confidence: 78%

Analysis of IgG Subclass and IgE Response in Allergic Disease Caused by Aspergillus fumigatus by Immunoblotting Techniques

Becker

Schlaak

1994

Analysis of sera from patients suffering from allergic asthma, extrinsic allergic alveolitis (EAA) due to Aspergillus fumigatus or allergic bronchopulmonary aspergillosis (ABPA) by means of IEF immunoprint and Western blot showed charcteristic IgG subclasses and IgE responses. Asthmatic patients revealed specific IgE antibodies. IgG subclass reactivities in asthmatics and healthy blood donors were weak and uncharacteristic. Major allergens recognized by asthmatic patients have isoelectric points (pI) of pI 4.4 and 4.8 and molecular weights of 20, 29, 34, 38 and 45 kD. Patients suffering from EAA typically demonstrated strong IgG subclass reactivities with an emphasis to IgG1 and IgG2, while no specific IgE was found. Various major antigens could be identified. Reaction to a pI 6.0 antigen seemed to be characteristic for EAA. ABPA patients showed strong reactivities of all IgG subclasses and of IgE. An intense IgG4 reaction appeared as a typical marker for ABPA. Several major antigens and allergens could be determined. Especially the IgG4 reactivities to proteins with pi 4.6, 5.0, 5.2, 5.4 and 6.5 and molecular weights of 14, 20, 29, 34, 38 and 45 kD seemed to be a characteristic pattern in ABPA. Furthermore, IgE antibodies to culture filtrate components of 34 and 38 kD appeared to be typical in ABPA.