Human monoclonal or polyclonal antibodies recognize predominantly discontinuous epitopes on bee venom phospholipase A2 Theres Schneidera, Alois B. Lang,

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Presentation transcript:

Human monoclonal or polyclonal antibodies recognize predominantly discontinuous epitopes on bee venom phospholipase A2 Theres Schneidera, Alois B. Lang, PhDb, José M. Carballido, PhDa, Luis F. Santamaria Babia, Thomas Dudlera, Martin K. Kägi, MDc, Kurt Blaser, PhDa, Mark Suter, DVMa Journal of Allergy and Clinical Immunology Volume 94, Issue 1, Pages 61-70 (July 1994) DOI: 10.1016/0091-6749(94)90072-8 Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 1 Analysis of the antigen specificity of BVA2. A, Antigen-specific ELISA with native (squares) or denatured PLA (triangles) coated to the solid phase. Serial twofold dilutions of BVA2 are plotted against the resulting OD. B, Inhibition ELISA with native (squares) or denatured PLA (triangles) as inhibitor for solid-phase–bound native PLA. Denatured PLA was produced as described by Dudler et al.24 Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 2 A, Western blot analysis of native PLA (lanes 1 and 2) or deglycosylated PLA (lanes 3 and 4) probed with BVA1. Protein was separated by SDS-PAGE under reducing (lanes 1 and 3) or nonreducing conditions (lanes 2 and 4). B, SDS-PAGE of native PLA (lane 1) and deglycosylated PLA (lane 2) separated under nonreducing conditions. Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 3 Binding inhibition of biotinylated BVA2 to solid-phase–bound PLA by hpAbs from a typical beekeeper. With an antigen-specific ELISA, serum of a beekeeper was added to the antigen coat in serial twofold dilutions from 1:20 to 1:640 as shown on the x axis. Subsequently, constant amounts of biotinylated BVA2 were added. The binding of BVA2 to solid-phase–coupled PLA was detected with alkaline phosphatase–coupled streptavidin. BVA2 binding with (squares) or without (triangles) previous incubation of hpAbs was compared. Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 4 A, Cross reaction of BVA1 with BVA2. With an antigen-specific ELISA, BVA1 (squares) or BVA2 (triangles) each titrated separately and BVA1 cotitrated with BVA2 (circles) were added to the PLA-coated plates in serial twofold dilutions. The antibody dilution was plotted against the resulting OD. B, Cross reaction of BVA2 with a representative murine mAb 3F3. The antigen-specific ELISA was as described in A. 3F3 (triangles) and BVA2 (squares) each titrated separately; BVA2 cotitrated with 3F3 (circles). Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 5 Enzymatic activity of native and deglycosylated PLA. The activity of native PLA was considered to be 100 units. The control sample contained denatured PLA.24 Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions

FIG. 6 Comparison of antibody binding to native or deglycosylated PLA assayed in an antigen-specific ELISA. Human mAbs or hpAbs were added to solid-phase–bound native PLA (filled symbols) or deglycosylated PLA (open symbols) in serial twofold dilutions. A, The binding of BVA1 to the two PLA preparations. B, The binding of IgE (squares), IgG1 (triangles), and IgG4 serum antibodies (circles) of a representative beekeeper to native and deglycosylated PLA. The serum was diluted 1:10 to 1:1280 for the detection of PLA-specific IgE or 1:80 to 1:10240 for the detection of PLA-specific IgG1 and IgG4, respectively. C, The PLA-specific binding of hpAbs of a representative allergic individual. Symbols are as described in B. The serum was diluted 1:5 to 1:160 or 1:20 to 1:640 for the detection of PLA-specific IgE or IgG1 and IgG4, respectively. D, PLA binding of hpAbs of allergic patient 2786 with preferential binding to the sugar residue on the antigen. Symbols and serum dilutions are as described in C. Journal of Allergy and Clinical Immunology 1994 94, 61-70DOI: (10.1016/0091-6749(94)90072-8) Copyright © 1994 Mosby, Inc. Terms and Conditions