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MICROBIAL IMMUNOLOGY

Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity

Wei Hao, Yu-Xian Pan, Yan-Qing Ding, Sha Xiao, Kai Yin, Ya-Di Wang, Li-Wen Qiu, Qing-Lin Zhang, Patrick C. Y. Woo, Susanna K. P. Lau, Kwok-Yung Yuen, Xiao-Yan Che
Wei Hao
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Yu-Xian Pan
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Yan-Qing Ding
2Department of Pathology, School of Basic Medical Science, Southern Medical University Guangzhou, China
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Sha Xiao
3Department of Pathology, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Kai Yin
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Ya-Di Wang
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Li-Wen Qiu
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Qing-Lin Zhang
2Department of Pathology, School of Basic Medical Science, Southern Medical University Guangzhou, China
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Patrick C. Y. Woo
4Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
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Susanna K. P. Lau
4Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
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Kwok-Yung Yuen
4Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
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Xiao-Yan Che
1Center of Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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  • For correspondence: linche@pub.guangzhou.gd.cn
DOI: 10.1128/CVI.00362-07
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  • FIG. 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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

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  • TABLE 1.

    Reactivities of MAbs against Aspergillus by IFA, ELISA, and IHC

    MAbIsotypeReactivity by IFAaA. fumigatus ELISA result (OD450)aReactivity by IHCa,b
    A. fumigatusA. flavusA. nigerA. terreusA. nidulansABC
    ConidiaHyphaeConidiaHyphaeConidiaHyphaeConidiaHyphaeConidiaHyphaeEAsMAs
    MA7IgG3++++++++++++++−+++−−++++++2.0382.304++++++++
    EA4IgM+++++++++++++−−+++1.5762.058+++++++
    EA5IgM++++++++++++−−+++1.7772.284+++++++
    EA8IgM+++++++++++++++++++++−−+++1.9452.333++++++
    MA4IgM+++++++++++++++++++−−++++2.2332.55+++++++
    EA6IgM++++++++++++++++++−−++++1.9382.460++++++
    EA2IgG1−++−−−−−+−−2.2612.861++++++
    EA3IgG1−++−+−−−+−−2.3502.950+++
    MA1IgG1−++−−−−−−−+1.6202.358+−+
    EA1IgG1+++−−−+−−−−1.5382.407+++++
    Con1IgM++++++++++++++++++++++1.7312.548+−−
    Con2IgG3+++++++++++++++++++++1.6762.558+−++
    EA7IgM++++++++++++++++++++++−+++2.3432.302++−
    MA2IgG1+++++++++++++++++++++++++++2.4321.957++++−
    MA3IgG1+++++++++++++++++++−−1.8932.668++++++
    MA5IgG1−++−+−+−+−−1.8412.513+++
    MA6IgG1++++++++++++++++++++++++++2.3442.344+++−
    • ↵ 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

    MAbEpitope groupa% Inhibition of the following HRP-labeled MAbb:
    MA7EA4EA5EA8MA4EA6EA2EA3MA1EA1Con1Con2EA7MA2MA3MA5MA6EB-A2c
    MA7I 86 81 75 81 45123000308000000
    EA4I 82 90 78 85 570134136725001020
    EA5I 83 85 82 77 45054006542700070
    EA8I 76 84 68 78 380711005402100000
    MA4I 78 91 69 88 70091002831800020
    EA6I 84 92 85 87 50062006002800000
    EA2II000000 96 75 88 88 000001200
    EA3II000000 95 75 77 82 1710001300
    MA1II0000006708100000900
    EA1II000000 88 10 75 3600000900
    Con1III651200402000 93 471500000
    Con2III2000000000 93 181600000
    EA7IV000000020000000000
    MA2V0070100000002902000
    MA3VI0000005700000000100
    MA5VII0017040000000000000
    MA6VIII006000000000000000
    • ↵ 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.

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Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity
Wei Hao, Yu-Xian Pan, Yan-Qing Ding, Sha Xiao, Kai Yin, Ya-Di Wang, Li-Wen Qiu, Qing-Lin Zhang, Patrick C. Y. Woo, Susanna K. P. Lau, Kwok-Yung Yuen, Xiao-Yan Che
Clinical and Vaccine Immunology Feb 2008, 15 (2) 194-202; DOI: 10.1128/CVI.00362-07

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Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity
Wei Hao, Yu-Xian Pan, Yan-Qing Ding, Sha Xiao, Kai Yin, Ya-Di Wang, Li-Wen Qiu, Qing-Lin Zhang, Patrick C. Y. Woo, Susanna K. P. Lau, Kwok-Yung Yuen, Xiao-Yan Che
Clinical and Vaccine Immunology Feb 2008, 15 (2) 194-202; DOI: 10.1128/CVI.00362-07
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    • ABSTRACT
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KEYWORDS

Antibodies, Fungal
Antibodies, Monoclonal
Antigens, Fungal
Aspergillosis
Aspergillus
Cell Wall
Enzyme-Linked Immunosorbent Assay

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