N-Glycanase treatment of F(ab′)2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity

Irina Mathov, Lilian Plotkin, Luis Squiquera, Carlos A. Fossati, Ricardo A. Margni, Juliana Leoni

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies ( 194 2, 194 5, 194 6 and 194 12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab′ glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab′ glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab′)2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab′)2 fragments.

Original languageEnglish (US)
Pages (from-to)1123-1130
Number of pages8
JournalMolecular Immunology
Volume32
Issue number14-15
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Immunoglobulin G
Carbohydrates
Glycosylation
Monoclonal Antibodies
Antibody Binding Sites
Antibodies
Lectins
Fluorescence
Binding Sites
Ligands

Keywords

  • asymmetric
  • glycosylation
  • IgG
  • mouse
  • N-glycanase

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

N-Glycanase treatment of F(ab′)2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity. / Mathov, Irina; Plotkin, Lilian; Squiquera, Luis; Fossati, Carlos A.; Margni, Ricardo A.; Leoni, Juliana.

In: Molecular Immunology, Vol. 32, No. 14-15, 1995, p. 1123-1130.

Research output: Contribution to journalArticle

Mathov, Irina ; Plotkin, Lilian ; Squiquera, Luis ; Fossati, Carlos A. ; Margni, Ricardo A. ; Leoni, Juliana. / N-Glycanase treatment of F(ab′)2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity. In: Molecular Immunology. 1995 ; Vol. 32, No. 14-15. pp. 1123-1130.
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AU - Margni, Ricardo A.

AU - Leoni, Juliana

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