The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Complementation of Der P 2-induced histamine release from human basophils sensitized with monoclonal IgE: not only by IgE, but also by IgG antibodies directed to a nonoverlapping epitope of Der p 2.

The interaction of free allergen with two (or more) IgE molecules bound to the high-affinity receptor for IgE (FcepsilonRI) on mast cells and basophilic granulocytes results in the release of inflammatory mediators. The role of allergen-specific IgG antibodies in the allergic reaction in human beings is less clear. We produced two chimeric IgE antibodies, hIgE-Dp2A and hIgE-Dp2B, directed to two nonoverlapping epitopes (A and B) of the house dust mite allergen Der p 2. Chimeric IgG1 and IgG4 variants of these antibodies were produced also. Basophil activation by the house dust mite allergen Der p 2 was induced after sensitization of basophils with a mixture of chimeric hIgE-Dp2A and hIgE-Dp2B antibodies but not after sensitization by the individual IgE antibodies alone. Basophil activation was also shown after sensitization with hIgE-Dp2A and stimulation with Der p 2 incubated with hIgG1-Dp2B or hIgG4-Dp2B antibodies. Both IgE and IgG antibodies directed to the other nonoverlapping epitope complemented the sensitization by the hIgE-Dp2A antibody. Nonsensitized basophils were not activated by the Der p 2/hIgG-Dp2 mixtures. These results indicate that allergen-specific IgG can complement an IgE-dependent reaction and therefore under certain conditions can act as an anaphylactic antibody.

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.

A chimeric human IgE monoclonal antibody was developed against the house dust mite allergen Der p 2. This chimeric antibody (hIgE-Dp2A) was composed of the heavy-chain variable domains and light chains of the original murine monoclonal antibody retaining its binding characteristics, whereas the heavy-chain constant domains were exchanged with the human IgE heavy chain. The chimeric IgE expression level was IgE 600 IU/ml (1 IU = 2.4 ng/ml). The binding of the chimeric hIgE-Dp2A to mite extract was indistinguishable from that of the original mouse monoclonal antibody. Parallel dose-response curves were found when the binding of hIgE-Dp2A to mite extract and anti-IgE coupled to sepharose were compared. Binding levels were not identical; however, hIgE-Dp2A bound significantly better to the mite-extract sepharose. This result indicates that the commonly used anti-IgE on solid phase calibration systems may lead to an overestimation of the amount of allergen-specific IgE present in the serum sample. The less efficient binding of the detector anti-IgE in case of the anti-IgE sepharose is likely to be because of the occupation of epitopes of the IgE by the sepharose-bound anti-IgE. Dose-response curves of serial dilutions of patient samples were parallel with the hIgE-Dp2A dose-response curve, which indicates that hIgE-Dp2A behaves like natural IgE antibodies in binding to allergen coupled to solid phase. This antibody is well suited for use as a reference reagent in the RAST and enables the expression of the amount of allergen-specific IgE present in a patient sample in absolute amounts.

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

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.

The relationship between RAST and skin test results in patients with asthma or rhinitis: a quantitative study with purified major allergens.

BACKGROUND: Study of the relationship between skin test results and IgE antibody levels is seriously hampered by the use of conventional allergen extracts because the precise amount of relevant allergen for each patient is unknown. OBJECTIVE: This study was designed to investigate skin reactivity with purified major allergens and to assess the relation with serum levels of IgE antibodies and to determine which additional factors contribute to the skin test result. METHODS: We used five purified major allergens (Der p 1, Der p 2, Fel d 1, Lol p 1, and Lol p 5) in skin tests, RASTs, and histamine release tests in 43 multisensitized patients with asthma or rhinitis. RESULTS: The differences in biologic activity of the five major allergens at a given level of specific IgE are within one order of magnitude. A significant residual variation remains in the correlation between skin test results and levels of IgE antibodies, which cannot be explained by imprecision of both tests (Pearson log skin test vs log specific IgE: r = 0.46-0.92). With similar levels of specific IgE, the amount of allergen that is required for a positive skin test result may differ by as much as a factor of 100 between patients. The amount of total IgE in serum contributes significantly to the skin test result. High values of total IgE are accompanied by a lower skin reactivity for allergen. Within individuals, allergens that cause skin test results that deviate from the prediction based on IgE antibody level often show a similar deviation in the histamine release test. This indicates that the type of IgE response (i.e., affinity or epitope recognition pattern) contributes significantly to the skin test result. Skin reactivity for histamine does not significantly influence the skin reactions expressed as allergen threshold. However, increased skin reactions with higher allergen dosages depend on histamine reactivity. CONCLUSION: The major allergens tested show similar biologic activities. In addition to IgE antibody level, total serum IgE and type of IgE antibody response contribute significantly to the skin test threshold for allergens. Even in a system with purified allergens, IgE antibody levels and skin test results are not interchangeable as an indicator of the degree of allergic sensitization.

Mouse/human chimeric IgG1 and IgG4 antibodies directed to the house dust mite allergen Der p 2: use in quantification of allergen specific IgG.

BACKGROUND AND OBJECTIVE: Chimeric mouse/human monoclonal IgG1 and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains of the original murine monoclonal antibody 2B12, whereas the heavy chain constant domains have been replaced by the human IgG1 or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 microg/mL, respectively. METHODS AND RESULTS: Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgG1 and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgG1 and IgG4 RAST and with reversed IgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. CONCLUSION: With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.