Normal human immunoglobulin G4 is bispecific: it has two different antigen-combining sites

Abstract

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In this paper we show that the apparent monovalency of circulating IgG4 is caused by asymmetry of plasma IgG4. A large fraction of plasma IgG4 molecules have two different antigen-binding sites, resulting in bispecificity. Sera from patients with IgG4 antibodies to both house dust mite and grass pollen induced cross-linking of Sepharose-bound grass pollen antigen to radiolabelled house dust mite allergen Der p I. This bispecific binding activity was not observed in sera with IgG4 antibodies to either grass pollen or house dust mite exclusively. Depletion of IgG4 antibodies resulted in disappearance of the bispecific activity. By size exclusion chromatography we excluded the possibility that bispecific activity was caused by aggregation of IgG4 antibodies. These results indicate that circulating (polyclonal) IgG4 antibodies have two different antigen-binding sites and therefore are functionally monovalent antibodies.

Figure 2

The functional valency of chimeric IgG4, plasma #178 and affinity-purified IgG4 from plasma #178. (a) Binding of ¹²⁵I-labelled Der p II to serial dilutions of chimeric IgG4 (17 μg/ml, •), plasma #178 (▵) and affinity purified IgG4 from plasma #178 (○) to Sepharose-coupled Staphylococcal Protein A were tested and used to determine the amount of Der p II-specific IgG₁₂₄ in the plasma. (b) The cross-linking of Sepharose-coupled mite-extract to ¹²⁵I-labelled Der p II by chimeric IgG4 (•), plasma #178 (▵) or affinity-purified IgG4 from plasma #178 (○) was expressed as the amount of radioactivity bound relative to the amount of radioactivity added.

Figure 1

The relative contribution of IgG4 to the total IgG₁₂₄ antibody activity with respect to the binding of ¹²⁵I-labelled Der p of plasma #178 was deduced from the comparison between Sepharose-coupled anti-IgG4 (○) and Sepharose-coupled Staphylococcal Protein A (•); the results obtained with the chimeric IgG4 antibody, 17 μg/ml, are also shown (anti-IgG4, ▵, Protein A, ▴).

Figure 3

The bispecific binding is not caused by Der p I contamination of the grass pollen–Sepharose. Serial dilutions of rabbit polyclonal antiserum to Der p (○, □) and plasma #741B (•, ▪) were incubated with protein A–Sepharose (○, •) or with grass pollen–Sepharose (□, ▪). The binding was detected using radiolabelled Der p I. The results were expressed as the amount of radioactivity bound relative to the amount of radioactivity added.

Figure 4

Gel filtration of serum #741. Serum #741, 200 μl was fractionated on an ACA 34 column. The fractions were tested for total amounts of IgE (180 000 MW, broken line), Der p II-specific IgG (solid line) and for the binding in the heterologous binding assay (•). The void volume is indicated by ▾. The results are expressed as percentage of starting material.

Figure 5

Depletion of IgG4 antibodies from serum #741. Serum #741 was depleted for IgG4 (▴). As control, a sample was also depleted for IgE (□) and incubated with control Sepharose (○). The three samples were tested for the amount of Der p I-specific IgG (a) and for the binding activity in the heterologous binding assay (b).