Lysine Residues in Bee Venom Phospholipase A₂ Are Important for Binding to Human Monoclonal or Polyclonal Antibodies of the lgG4 Isotope

Abstract

Discontinuous antigenic sites on bee venom phospholipase A₂ (PLA) have been mapped using human monoclonal antibodies or human polyclonal serum antibodies (hpAbs) of the IgG4 isotype from beekeepers or of the IgE isotype from individuals allergic to PLA. Lysine residues of PLA have been specifically modified by acetylation or acylation by treatment with citraconic anhydride of their ε-amino groups to analyze their role in antigen-antibody binding. After the modifications, the binding of PLA to the human monoclonal antibodies is lost, whereas the binding to IgG4 hpAbs is significantly decreased. In contrast, the effect on the binding of PLA to IgE hpAbs appears to be more heterogeneous. The data indicate the importance of lysine residues as being part of B cell epitopes in PLA-specifíc antibodies of the IgG4 isotype, but less so for those of the IgE isotype.

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy

“…A human mAb against Api m 1 (BVA2) was used to characterize further the Api m [1/2] fusion protein. 16 Immunoblotting For Western blot analysis, equimolar amounts (1.5 mmol/L) of the 3 recombinant proteins, Api m 1, Api m 2, and Api m [1/2], were electrophoresed by 4% to 12% Bis-Tris polyacrylamide NuPAGE gels (Invitrogen, Basel, Switzerland) under denaturing, reducing conditions and transferred to nitrocellulose membrane (Amersham, Little Chalfont, United Kingdom). 17 The binding of IgE in pooled serum from 4 individuals allergic to BV was detected with rabbit antihuman IgE (Dako Inc, Burlingame, Calif) and developed by goat antirabbit biotin conjugated IgG antibody (Dako Inc) and streptavidin-horseradish peroxidase (Sigma Chemical Co, St Louis, Mo).…”

“…A human mAb against Api m 1 (BVA2) was used to characterize further the Api m [1/2] fusion protein in dot blot assays. 16 Peroxidase-conjugated rabbit antihuman IgG antibody (Dako Inc) was used as a second antibody.…”

Determinants and Mechanisms of Human Immune Responses to Bee Venom Phospholipase A₂

“…Two huMabs from the heteromyelomas BVA1 and BVA2 [37], and one from the EBV–B cell line BUB1 [21]showed identical epitope specificity involving Lys 25 of PLA. By using lysine–modified and point–mutated PLA it was demonstrated that this residue is an essential constituent of a major binding site for human anti–PLA antibodies [37, 38, 39]. In ELISA, binding of both BVA1 and BVA2 to PLA was inhibited by each other and about 90% of serum anti–PLA IgG4 antibody binding could be inhibited by the huMabs.…”

Comparison of the antibody response to bee venom phospholipase A2 induced by natural exposure in humans or by immunization in mice

“…To address these issues directly, we have compared the epitopes on phospholipase A 2 (PLA), the main allergen present in bee venom (Kuchler et al, 1989;Scott et al, 1990a), recognized by human monoclonal antibodies (hmAbs) (Schneider et al, 1994b) or murine monoclonal antibodies (mmAbs) (Müller et al, 1993). Two hmAbs of the IgG 4 isotype were made from lymphocytes of a nonallergic beekeeper naturally exposed to bee venom.…”

“…Two hmAbs of the IgG 4 isotype were made from lymphocytes of a nonallergic beekeeper naturally exposed to bee venom. These hmAbs were shown to be part of the polyclonal immune response of beekeepers who have been exposed for years to bee venom via bee stings (Dudler et al, 1994;Schneider et al, 1994aSchneider et al, , 1994b. ␥-Globulins from such individuals were previously shown to be protective when given to bee venom allergic individuals before exposing them to bee venom (Lessof et al, 1978;Müller et al, 1986;Bousquet et al, 1987).…”