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Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific.

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Presentation on theme: "Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific."— Presentation transcript:

1 Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE  Janine Schuurman, PhDa, Gerrard J. Perdok, MSca, Tom E. Lourens, MSca, Paul W.H.I. Parren, PhDb, Martin D. Chapman, PhDc, Rob C. Aalberse, PhDa  Journal of Allergy and Clinical Immunology  Volume 99, Issue 4, Pages (April 1997) DOI: /S (97) Copyright © 1997 Mosby, Inc. Terms and Conditions

2 Figure 1 Heavy-chain expression plasmid was prepared in vector pSV-SPORT and contained mouse IgH enhancer, IgH promoter, IgH leader sequence, VH 2B12 domain, intron fragment, and human genomic Cϵ gene.4,5,11 AMP, Ampicillin resistance gene. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

3 Figure 2 Comparison of binding of hIgE-Dp2A (open circles) and original mouse monoclonal antibody 2B12, mIgG2b-Dp2A (solid circles) to mite extract coupled to sepharose. Serial dilutions of antibodies were incubated with mite-sepharose. Detection of antibodies bound was done with 125I-labeled anti–light chain antibodies. Light chain of both antibodies is identical. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

4 Figure 3 Binding of hIgE-Dp2A (solid circles) and three patient samples (open circles, squares, triangles) to mite extract coupled to CNBr-activated sepharose. Sample dilutions (1 = undiluted) were incubated with sepharose and binding was detected with 125I–anti-IgE antibodies. IgE concentration in culture supernatant was 600 IU/ml (1 IU = 2.4 ng/ml). Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

5 Figure 4 Adsorbent in excess conditions. Several hIgE-Dp2A concentrations (squares 1.2 IU/ml, circles 3.7 IU/ml, inverted triangles 11 IU/ml, triangles 33 IU/ml, squares 100 IU/ml, circles 300 IU/ml) were incubated with anti-IgE (open symbols) or mite extract (solid symbols) coupled to sepharose. Binding was detected with use of 125I-labeled anti-IgE antibodies. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

6 Figure 5 Binding of chimeric hIgE-Dp2A to mite and anti-IgE coupled to sepharose. Serial diluted hIgE-Dp2A supernatant was incubated with mite extract (solid circles) or anti-IgE (open circles) coupled to sepharose. Bound IgE was detected with 125I-labeled sheep–anti-IgE antibodies. Results shown are representative for seven independent experiments. Journal of Allergy and Clinical Immunology  , DOI: ( /S (97) ) Copyright © 1997 Mosby, Inc. Terms and Conditions

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