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.

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.

Enzyme conformational alterations detected by partition column chromatography.

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.

Modulation of antigen-antibody complexations by immunoglobulins.

In this investigation, the modulating effects of non-immune human IgG and rheumatoid factors (RFs) on antigen-antibody complexations were studied. Non-immune human IgG, as well as RF, were found to inhibit the binding of antigen to specific antibodies of both human and rabbit origin. In addition, human immunoglobulins were also able to modify the composition of preformed antigen-antibody complexes. The effects were detected by immunological methods in two different antigen-antibody systems (human serum albumin-rabbit anti-HSA and tetanus toxoid-human anti-TT). Changes in biological activities could be followed by employing enzymes (glucose-6-phosphate dehydrogenase and human placental alkaline phosphatase) as antigens. The outcome of the effects was found to be dependent on the ratio of antigen to antibody, the antigen-binding properties of the antibody and its origin, and on the properties of the immunoglobulins added. The observed changes could not be explained only by the presence of specific antibodies in the immunoglobulin preparations. The ability of immunoglobulins to modulate antigen-antibody complexations may provide a rationale for the large amounts of non-specific immunoglobulins in the circulation by preventing premature precipitation and promoting the elimination of antigenic molecules.

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 new approach to examine conformational changes occurring upon binding of ligand by biomolecules.

Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques.

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.

The effect of non-immune IgG on antigen-antibody complexation.

Two preparations of human IgG, one acylated with beta-propiolactone (acylated IgG) and one treated at pH 4 with traces of pepsin (pH 4-IgG), were used to study the effect of non-immune IgG on antigen-antibody interactions in the antigen excess zone. Employing two immunological methods together with size-exclusion chromatography, we found that the formation of human albumin-rabbit anti-human albumin complexes was inhibited in the presence of human IgG. In addition, IgG seemed to promote the aggregation of already formed complexes. Thus, non-immune IgG may modulate immune complexation by direct molecular interactions. The effect was dependent on the size and composition of the immune complexes as well as on the conformation of the IgG molecules with respect to their shape, isotype, charge, and other surface properties. Some possible mechanisms for the reactions are discussed.

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 pH's 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.

Effects of pH on the stability of the indium-113m blood protein complex and the selective binding of indium-113m to transferrin.

Indium-113m (t1/2 = 100 min; gamma-emission of 393 keV) in trace amounts was injected i.v. in rats. Blood was collected by heart puncture 15 min after the injection, and blood plasma was separated by centrifugation. Gel filtration of plasma on Sephadex G-25M equilibrated with glycine/HCl (pH 2.2-3.6), NaHCO3/CO2 (pH 4.0-11.0) glycine/NaOH (pH 8.6-10.6) or sodium acetate/acetic acid (pH 3.0-5.0) was used to separate free indium from indium bound to macromolecular proteins. Determination of radioactivity in eluted fractions showed that more than 85% of the plasma indium was bound to macromolecules at pH values between 5.0 and pH 10.6. However, dissociation of the indium plasma protein complexes occurred at pH values below 5.5, and more than 90% of the indium radioactivity was found in the low molecular weight fraction at pH 2.2. Affinity chromatography using immobilized antibodies to rat transferrin was used to isolate transferrin at pH 7.4 and 5.5. Immunodiffusion and electrophoresis were used to identify the proteins in fractions obtained by affinity chromatography. It was found that the indium-113m activity was correlated with the content of transferrin and that 80%-90% of this activity was found in fractions that had affinity to antitransferrin. These fractions contained transferrin exclusively at pH 7.4, but additional protein fractions of albumin and alpha1-globulin mobility at pH 5.5. At pH 7.4 and 5.5, 10%-20% of the indium activity was detected in molecular fractions that had no affinity to antitransferrin. Immunologic analyses showed that these fractions contained transferrin.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG with a deviant conformation in serum and synovial fluid from rheumatoid arthritis patients.

Specific rabbit antisera were prepared against an IgG with a special conformation (IgG spec.) previously detected in some sera from patients with rheumatoid arthritis. The antibodies had no affinity to normal human IgG and were not anti-idiotypic to human rheumatoid factor. The affinity of IgG spec. to the antibodies could not be explained by an antiglobulin activity to rabbit IgG. The amount of protein with affinity to immobilized specific IgG F(ab')2 of the antibodies was determined in serum and synovial fluid from patients with various joint diseases. A relationship between the content of IgG spec. and the diagnosis of seropositive rheumatoid arthritis was found on analysis of serum samples. IgG spec. also occurred in synovial fluid from some individuals with seropositive rheumatoid arthritis. Differences in the serum content of IgG spec. could not be explained by differences in the normal IgG content. Circular dichroism analysis of isolated IgG spec. showed that in the region(s) close to tyrosine residue(s) this polyclonal protein had similarities to heat-aggregated IgG.

Factors affecting IgA related hyperviscosity.

Sera from 14 patients with an IgA M-component, six of whom had myelomatosis and eight with benign monoclonal gammopathy (BMG) were analysed. All six sera from patients with high IgA (greater than 40 g/l) and total protein (greater than 100 g/l) concentrations were hyperviscous (HV). Four of these six patients also had hyperviscosity syndrome (HVS). There was no correlation between the quantity of IgA dimers or polymers and the presentation of HV and HVS. The binding between IgA and albumin and alpha 1-anti-trypsin was not covalent. Differences in the microenvironment of S-S bonds or of aromatic amino acids between isolated monoclonal monomeric and dimeric IgA were demonstrated with circular dichroism. Besides that, differences in hydrophobicity (exposure of aromatic amino acids) between IgA from normal serum and monomeric and dimeric IgA from a myeloma serum were revealed using hydrophobic interaction chromatography. The significance of hydrophobic interactions involving IgA and the influence of such forces on the circulation of the molecules are discussed.

Circular dichroism of immune complexes, IgG and Fab gamma with unique antigenic determinants from rheumatoid serum.

IgG-IgG and IgG-IgM complexes were isolated from one rheumatoid arthritis (RA) serum by affinity chromatography to immobilized F(ag')2 gamma of specific antibodies against unique determinants in the complexes. Both IgG nd IgM, when isolated from these complexes, contained the unique determinants. The circular dichroism (CD) spectrum of IgG differed from that of normal IgG at neutral pH. At pH 3 both IgG and IgM displayed normal CD spectra, and only one third of the molecules now had affinity for the immobilized ligand. The molecules with affinity at pH 3 exhibited an abnormal CD spectrum at pH 3, and a normal CD spectrum was obtained only of those components that lacked affinity. One-third of the Fab gamma isolated from the IgG and IgG complexes with the unique determinants contained the unique determinants that were lacking in the rest of the same principal the Fc gamma fragments. The CD of the two Fab gamma preparations showed the same principal differences as the CD of the molecules with and without affinity to the ligand at acidic pH.

Circular dichroism of immune complexes with rheumatoid factor activity.

The circular dichroism (CD) of IgG complexes with RF activity and that of monomeric IgG without this activity, and isolated from the same individual, were compared. The IgG complexes had a significantly deviant CD spectrum in the near u.v. region, both before and after dissociation, whereas the monomeric IgG had a normal spectrum. Immunoglobulins were isolated from the same serum with the use of specific antiserum against unique determinants in some IgG complexes with RF activity. Both before and after dissociation the CD spectrum in the far u.v. region of these immunoglobulins differed significantly from that of the above-mentioned preparations. The results confirmed that the structure of IgG in the RF-active complexes differed from that of normal IgG. The immunoglobulins with the unique determinants had, in turn, a structure that was not found in the pool of the RF-active IgG molecules or in normal IgG.

Quantitative precipitin analysis of free and hidden RF IgM.

Quantitative precipitin analysis using rabbit IgG anti-human Fc mu was performed with 16 RF IgM, six macroglobulinaemic IgM and four normal IgM. Abnormal precipitin curves were obtained for all RF IgM, even when the latter were not readily demonstrated with conventional serological tests for RF, and for macroglobulinaemic IgM with sedimentation rates greater than 19S. These IgM formed significantly more precipitates with IgG anti-Fcmu in the antigen excess zone than did normal IgM, but the precipitin curves for the other zones were similar for all IgM. The underlying mechanisms of some of the reactions were studied and discussed. Because the divergence in the precipitin reaction for normal IgM and RF IgM was so pronounced, a model precipitin curve was constructed. This could be used to detect RF IgM, even when not readily demonstrable with conventional serological tests for RF, by direct analysis of serum. The results obtained for RF IgM suggested that the method might be applied to RF IgG and intermediate complexes comprised of IgG. The mechanisms demonstrated here might be used to develop immunological methods for routine use.

Isolation and characterization of intermediate complexes and other components with common antigenic determinants.

Fractions with affinity for heat-denatured human IgG (HDG) were isolated from four sera that contained intermediate complexes (IC). These IC fractions contained part of the 75 IgG, all IC, and part of the rapidly sedimenting complexes (RC) found in the sera. The IC consisted of IgG1 or IgG3 and the RC of IgG and IgM with kappa and lambda light chains. The IgG in the IC fractions contained an abnormally large amount of neuraminic acid. No correlation between IgG subclass or content of neuraminic aid and complex formation was found. There were indications that the formation of IC was not only the result of self-association of IgG molecules with anit-gamma-globulin activity. Specific rabbit antisera were prepared against two of the IC fractions. Affinity chromatography wtih immobilized IgG and F(ab')2gamma from these antisera confirmed the presence of common antigenic determinants in the sera. These determinants occurred mainly in 7S components in one individual, in IC in one and in RC in another. Only a minor part of the serum components with affinity for HDG contained the determinants. RF activity was found in the components that lacked and in those that contained the common antigenic determinants.

Effect of IgM fractions with or without rheumatoid factor activity on antigen-antibody reactions.

The effect of isolated IgM fractions, with or without rheumatoid factor (RF) activity, on reactions between human albumin and rabbit anti-human albumin, human IgG and rabbit anti-human IgG, and tetanus toxoid and human anti-tetanus toxoid was assessed in the antigen excess zone. RF-active fractions increased and RF-negative IgM fractions decreased the amount of free antigen. The immune complexes that were affected by RF IgM fractions differed in composition from those affected by normal IgM fractions.

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.