Ulla-Britt Hansson

Comparison of Surface Properties of Human IgA, IgE, IgG and IgM Antibodies with Identical and Different Specificities

In this paper, the authors report the use of liquid‐liquid partition chromatography (LLPC) in an aqueous polyethylene glycol (PEG)/dextran two‐phase system to compare the surface properties (partition properties) of human antibodies and fragments thereof. The surface properties of all the monoclonal antibodies of different classes and subclasses investigated were within the same broad range as that observed for the polyclonal antibodies and no relationship was found between the exposed surfaces of the immunoglobulins (Ig) and their heavy chain isotype. Moreover, Fc fragments from various IgG1, 2 and 4 myeloma proteins were found to exhibit similar surface properties. Employing chimeric antibodies with identical variable regions the authors found that intact IgG1, 2 and 4 displayed identical surface properties, while the corresponding IgA1, IgA2, IgG3, IgE and IgM antibodies differed both from each other and from the IgGs. The surface properties of chimeric IgG3 could be made similar to those of the IgG1, 2 and 4 chimers by partially reducing the length of the hinge section, but new differences in surface properties appeared when their hinges were of similar length. Thus, LLPC can be used to detect differences or similarities in the surface properties of the antigen‐binding regions as well as the Fc part in the various isotypes. This can shed light on biological activities such as antigen binding and effector function.

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pHs where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.

Antigen-binding sites dominate the surface properties of IgG antibodies

A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen-binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties.

A simple routine method for detecting hidden rheumatoid factors

A new and simple routine method is described for detecting hidden rheumatoid factors in human serum. EDTA glycine and NaCl were used to liberate hidden rheumatoid factors and to inactivate complement before rheumatoid-factor activity was determined in a glycine--NaCl solution. Forty-nine out of 97 sera from individuals with seronegative rheumatoid arthritis gave positive reactions by this method. Rheumatoid sera with low titres by standard tests gave higher titres with the new method. The new method detects both IgM and IgG rheumatoid factors and is simple and suitable for use in routine medical laboratories. Used in parallel with the classical tests, it facilitates detection of hidden rheumatoid factors.

Liquid‐Liquid Partition Chromatography as a Method to Examine Surface Properties of Antibodies and Antigen‐Antibody Complexes

We demonstrate liquid‐liquid partition chromatography in aqueous two‐phase systems (LLPC) as a simple method for examining the surface properties of immunoglobulins and antigen antibody complexes in solution. LLPC separates molecules with respect to the properties of the exposed surfaces. As an example, the method may be used to detect changes in the conformation of IgG following chemical modification like acylation or iodination. We have studied the partitioning of antibodies and antigen antibody complexes, modelled by rabbit antibodies against three human serum proteins, in aqueous polyethylene glycol/dextran two‐phase systems at pH 7. Analysis of both polyclonal and monoclonal antibodies against various antigens suggested that the partition properties of immunoglobulins are related mainly to their antigen specificity and not to subclass‐specific structures. Furthermore, experiments indicated that changes in the surface properties of antigen and/or antibody following complexation may be detected. Thus, LLPC may prove to be a new way of studying the relation between antibody structure and function in solution.

Detection of Monoclonal IgM Subunits in Macroglobulinemia Waldenström

A serum containing an M-component of IgM type gave on immunoelectrophoresis a biarcuate precipitation line. The anodal arch was produced by subunits of IgM, the other, by polymeric (17 S) IgM. The subunits combined in other optimal proportions with anti IgM than polymeric IgM as shown by e.g. immunoelectrophoresis and antigen-antibody crossed electrophoresis. Changes in immunoprecipitation patterns after reduction with mercaptoethanol of the cathodal arch to a mobility more equal to the anodal arch is one way to trace IgM subunits in plasma.

Electrophoretic and immunologic differences between monoclonal IgM molecules and their subunits in native plasma.

Abstract Naturally occurring IgM subunits had a higher negative charge than those obtained after mercaptoethanol reduction of 19 S IgM and higher polymers. Both types of subunits regularly had a stronger negative charge than the pentamers. Extension of the immunoelectrophoretic IgM precipitation line on the anodal side of the 19 S M-component component indicated occurrence of subunits if trailing could be excluded. The precipitation patterns were easier to interpret when electroendoosmotic inert support media were used for electrophoresis. In most cases antigen-antibody crossed electrophoresis allowed semiquantitative estimation of proportions between mono- and pentamers. Of 32 sera with increased monoclonal IgM, 25 were found to contain measurable amounts of 7 S IgM. Quantitative precipitin and gel diffusion studies showed that maximal amounts of precipitates with anti-IgM were obtained in antigen excess for 19 S IgM, but an equivalence for 7 S IgM.

Supports for liquid-liquid partition chromatography in aqueous two-phase systems: A comparison of LiChrospher and LiParGel

Abstract The ability of Superdex 200, a gel filtration matrix consisting of dextran-grafted beads, to act as a support for liquid-liquid partition chromatography (LLPC) in aqueous polyethylene glycol (PEG)-dextran two-phase systems was examined. The gel adsorbed the dextran-rich bottom phase readily and retained it during elution with the PEG-rich top phase. In contrast to LiParGel 650, a matrix designed for LLPC, the entire Superdex matrix seemed to form an immobilized stationary phase. Ideal partitioning of proteins was observed only for molecules partitioning towards the stationary phase on Superdex and for those favouring the mobile phase on LiParGel. Hence, the choice of matrix depends on the separation problem at hand.

Surface Properties of Antigen–Antibody Complexes

In this paper, the authors show that liquid–liquid partition chromatography in an aqueous two‐phase system offers unique possibilities of comparing the overall surface properties of intact antibodies in solution before and after binding of antigen. The authors demonstrate that the surface properties of antigen–antibody complexes are dependent on the variable regions of the antibodies, the nature of the antigen and/or possible conformational changes induced by antigen binding. Thus, each antigen–IgG antibody pair formed one type of complex with respect to the exposed dominant surface. The antigen‐binding sites of IgG antibodies were exposed and dominant even after binding of hapten or hapten‐carrier. In contrast, the antibody‐combining sites were concealed upon protein binding and the exposed surfaces of the protein–antibody complexes were related mainly to those of the antigen. IgA1, IgA2, IgE and IgM formed, in comparison to the IgG, hapten–antibody complexes which exhibited surface properties that could be related to both the antigen‐binding sites and Fc parts of the antibodies. Moreover, the results indicated that antigen‐induced conformational changes occurred in either IgA1, IgA2, IgE, or IgM, but not in IgG1, ‐2, ‐3 and ‐4, making the surfaces of their heavy chain constant regions more similar.

Conformational isomerism of IgG antibodies

The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid-liquid partition chromatography (LLPC) in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen-antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed.