Article Figures & Data
Figures
- FIG. 1.
Immunohistochemical analyses of MAb EA6 in formalin-fixed paraffin tissue sections. (A) Immunohistochemical staining of lung tissue from a patient with invasive aspergillosis. Magnification, ×200. (B) Immunohistochemical staining of kidney tissue from the experimental rabbit model of IA. Magnification, ×400. No staining occurred with the control MAb.
- FIG. 2.
Indirect immunofluorescent analyses of MAb MA6 binding to the hyphae and the conidia of A. fumigatus. Confocal microscopy immunofluorescent images (A and C) and contrast bright images (B and D) demonstrated the binding of the MAb to the inner cell wall and intracellular membranes of A. fumigatus hyphae (A) and conidia (C). Similar labeling was seen with other MAbs, whereas no binding occurred with the control MAbs.
- FIG. 3.
Western blot analyses of the binding of MAbs to ConA-purified mannoprotein derived from A. fumigatus. Lanes: 1, Con1; 2, Con2; 3, MA7; 4, EA4; 5, EA5; 6, EA8; 7, MA4; 8, EA6; 9, EA7; 10, MA2; 11, MA3; 12, MA5; 13, MA6; 14, control MAb; 15, EB-A2; 16, EA1; 17, EA2; 18, EA3; 19, MA1; M, markers. The Western blot demonstrated the binding of MAbs EA4, EA5, EA8, MA4, EA6, EA7, MA2, and MA6 to proteins at 50 and 75 kDa. MAbs MA2 and MA6 also bound to several bands between 25 and 75 kDa, and the pattern of reactivity was similar to that seen in the rat anti-GM MAb EB-A2 supplied with the Platelia Aspergillus assay kit.
- FIG. 4.
Evaluation of the sensitivity and the specificity of the Aspergillus antigen-capture ELISA for the detection of culture filtrates of fungal antigens. Culture filtrates from five Aspergillus species gave a very strong positive signal following the dilution (approximately 1:10,000) of cell cultures containing 2 × 106 cells/ml. The culture filtrate from Penicillium marneffei was not detectable beyond a dilution of 1:2.
- FIG. 5.
Detection of antigenemia in the experimental rabbit model of IA by a two-step Aspergillus antigen-capture ELISA. Blood samples were drawn prior to the induction of infection (day 0) and daily thereafter until the death of each rabbit. The panels display the appearances of the infections caused by A. fumigatus (A), A. flavus (B), A. niger (C), A. nidulans (D), and A. terreus (E).
- FIG. 6.
Detection of antigenemia in the experimental rabbit model of IA by a one-step commercial GM detection ELISA (the Platelia Aspergillus assay). Blood samples were drawn prior to the induced infection (day 0) and daily thereafter until the death of each rabbit. The times of appearance of the infection in rabbits infected with A. fumigatus (A) and A. flavus (B) are shown.
Tables
- TABLE 1.
Reactivities of MAbs against Aspergillus by IFA, ELISA, and IHC
MAb Isotype Reactivity by IFAa A. fumigatus ELISA result (OD450)a Reactivity by IHCa,b A. fumigatus A. flavus A. niger A. terreus A. nidulans A B C Conidia Hyphae Conidia Hyphae Conidia Hyphae Conidia Hyphae Conidia Hyphae EAs MAs MA7 IgG3 ++++ ++++ +++ +++ − +++ − − +++ +++ 2.038 2.304 +++ ++ +++ EA4 IgM ++ +++ + ++++ + ++ − − + ++ 1.576 2.058 ++ +++ ++ EA5 IgM ++ +++ + ++++ + + − − ++ + 1.777 2.284 ++ ++ +++ EA8 IgM ++++ ++++ ++++ ++++ +++ ++ − − ++ + 1.945 2.333 ++ + +++ MA4 IgM ++++ ++++ ++++ ++++ ++ + − − ++ ++ 2.233 2.55 ++ ++ +++ EA6 IgM ++++ ++++ ++ ++++ ++ ++ − − +++ + 1.938 2.460 ++ ++ ++ EA2 IgG1 − ++ − − − − − + − − 2.261 2.861 +++ ++ + EA3 IgG1 − ++ − + − − − + − − 2.350 2.950 + + + MA1 IgG1 − ++ − − − − − − − + 1.620 2.358 + − + EA1 IgG1 + ++ − − − + − − − − 1.538 2.407 + +++ + Con1 IgM ++ ++ ++ +++ + + ++ +++ +++ +++ 1.731 2.548 + − − Con2 IgG3 +++ ++ ++ ++ ++ + +++ ++ ++ ++ 1.676 2.558 + − ++ EA7 IgM +++ +++ ++ ++++ ++ ++ ++ ++++ − +++ 2.343 2.302 + + − MA2 IgG1 ++ ++++ ++ +++ ++ ++++ +++ ++++ + ++ 2.432 1.957 ++ ++ − MA3 IgG1 + +++ + +++ ++ +++ +++ +++ − − 1.893 2.668 ++ +++ + MA5 IgG1 − ++ − + − + − + − − 1.841 2.513 + + + MA6 IgG1 ++ ++++ ++ ++ + +++ +++ ++++ + ++++ 2.344 2.344 ++ + − ↵ a The reactivity of each MAb was determined by IFA to the mycelia and conidia of Aspergillus species, by indirect ELISA to the MAs and EAs of A. fumigatus, and by immunohistochemical assay (IHC) to formalin-fixed paraffin section of tissues. −, no reactivity; +, weak reactivity; ++, intermediate reactivity; +++, strong reactivity; ++++, very strong reactivity.
↵ b A, kidney tissue from an experimental rabbit model of IA; B, lung tissue from a patient with IA; C, lung tissue from a patient with aspergilloma.
- TABLE 2.
Analysis of distinct epitope binding of MAbs by competition ELISA
MAb Epitope groupa % Inhibition of the following HRP-labeled MAbb: MA7 EA4 EA5 EA8 MA4 EA6 EA2 EA3 MA1 EA1 Con1 Con2 EA7 MA2 MA3 MA5 MA6 EB-A2c MA7 I 86 81 75 81 45 1 23 0 0 0 30 8 0 0 0 0 0 0 EA4 I 82 90 78 85 57 0 13 4 1 3 67 2 50 0 1 0 2 0 EA5 I 83 85 82 77 45 0 5 4 0 0 65 4 27 0 0 0 7 0 EA8 I 76 84 68 78 38 0 7 11 0 0 54 0 21 0 0 0 0 0 MA4 I 78 91 69 88 70 0 9 1 0 0 28 3 18 0 0 0 2 0 EA6 I 84 92 85 87 50 0 6 2 0 0 60 0 28 0 0 0 0 0 EA2 II 0 0 0 0 0 0 96 75 88 88 0 0 0 0 0 12 0 0 EA3 II 0 0 0 0 0 0 95 75 77 82 17 1 0 0 0 13 0 0 MA1 II 0 0 0 0 0 0 67 0 8 1 0 0 0 0 0 9 0 0 EA1 II 0 0 0 0 0 0 88 10 75 36 0 0 0 0 0 9 0 0 Con1 III 65 12 0 0 4 0 2 0 0 0 93 47 15 0 0 0 0 0 Con2 III 2 0 0 0 0 0 0 0 0 0 93 18 16 0 0 0 0 0 EA7 IV 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 MA2 V 0 0 7 0 1 0 0 0 0 0 0 0 29 0 2 0 0 0 MA3 VI 0 0 0 0 0 0 57 0 0 0 0 0 0 0 0 1 0 0 MA5 VII 0 0 17 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 MA6 VIII 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ↵ a The competition ELISA was performed to analyze the binding epitopes of the different MAbs. By use of the criteria described in Materials and Methods, 17 MAbs were divided into eight groups, with each group reacting with the same epitope or sterically overlapping epitopes on Aspergillus antigens. The experiment was repeated with similar results.
↵ b Competitive inhibition values ≥75% are in boldface.
↵ c EB-A2, a rat anti-GM MAb supplied with the commercial Platelia Aspergillus kit.
