Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis

Abstract

IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen‐binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self‐antigens, enhanced binding to non‐self‐antigens (bacterial), an increased ability to engage in F(ab′)2/F(ab′)2 (idiotype/anti‐idiotype) interactions and an increased functional antigen‐binding affinity are reported here. The newly acquired ‘induced polyreactivity’ of low‐pH buffer‐exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low‐pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen‐binding behavior and biological activity. Low‐pH buffer‐treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer‐exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide‐induced septic shock.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

“…This scenario may not be the case, since the licensed therapeutic immunoglobulins, produced using a fractionation step at pH 4.0, have been shown to possess an increased potential to bind to self-antigens (12). Importantly, this increased reactivity to antigens correlates with different functional acti vity of immu noglobulin preparation (13,14). Thus, the administration of the acid pH-treated IVIg, but not of the same unmodified preparation, significantly decreased mortality in animals with endotoxemia (12,13).…”

“…Importantly, this increased reactivity to antigens correlates with different functional acti vity of immu noglobulin preparation (13,14). Thus, the administration of the acid pH-treated IVIg, but not of the same unmodified preparation, significantly decreased mortality in animals with endotoxemia (12,13). Previous studies by our group and others have proven that, in addition to low pH buffers, the exposure to a number of other substances (for example, ferrous ions, heme, reactive oxygen species, and so on) also increases the antigen-binding polyspecificity of some IgG molecules (5,(15)(16)(17).…”

Antibody Polyreactivity in Health and Disease: Statu Variabilis

“…In addition to naturally polyreactive Abs, all healthy individuals contain a fraction of circulating Igs that in their native state express low Ag-binding activity but acquire polyreactive characteristics after exposure to certain protein-destabilizing agents (40)(41)(42)(43). Thus, the transient contact of polyclonal or monoclonal Igs with chaotropic agents, low pH (i.e., ,4), or high salt concentrations results in molecular modifications of the Ig and leads to transition toward a multispecific Ag-binding state.…”

“…Importantly, in many studies of polyreactive Abs, the Igs have been subjected to acidic pH during their purification prior to analysis of their Ag binding. Because this exposure is a strong inducer of polyreactivity in the case of sensitive IgG and IgM Abs (41)(42)(43), it may turn out that many Abs considered as polyreactive would not express multispecific Ag binding when purified under different conditions.…”

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo

“…However, it has been reported that exposure of low -affinity polyclonal IgG to low pH may result in structural changes or even partial denaturation of antibodies which may generate artefactual increases in apparent activity against endogenous antigens [15, 16]. In vitro assays such as ELISAs employing IgG isolated from human plasma using methods that require a treatment at low pH may therefore artefactually under- or over- estimate IVIG activity against endogenous antigens [17–19]. …”