A calcium-dependent cryoglobulin IgM kappa/polyclonal IgG.

The mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood. A mixed cryoglobulin IgM kappa/IgG (type II) of a patient with Sj?gren's syndrome was studied because of its unique properties. This cryoglobulin precipitated in serum but not in serum containing 10 mM EDTA. The cryoprecipitation was shown to require calcium (Ca) and was optimal at 1 mM of free Ca. Cryoprecipitation was also induced by Ba, Mn, and Sr, but not by Mg and Co. Purified IgM kappa/IgG complexes precipitated in the presence of Ca, but not IgM kappa alone. There was no significant binding of 45Ca to the purified IgM kappa, IgM kappa/IgG complexes formed with purified components, and the cryoprecipitate. The relative affinity of the radiolabeled [125I]IgM kappa for IgG was 3.6 x 10(3) liters/mol at 37 degrees C as assessed by sucrose density gradient ultracentrifugation, and increased to 1.7 x 10(4) liters/mol at 4 degrees C. The addition of Ca produced no change in the affinity at 37 degrees C and 4 degrees C. The absence of a direct effect of Ca on the Ag/antibody reaction was confirmed in experiments using polyethylene glycol as precipitating agent. In conclusion, two independent steps were responsible for the precipitation of this cryoglobulin. The first step was an efficient formation of soluble immune complexes as produced by a drop in temperature. The second step was caused by a change in the physicochemical conditions--the presence of Ca--which induced polymerization of the IgM kappa/IgG complexes.     

Nonimmunospecific protein-protein interactions of IgG: studies of the binding of IgG to IgG immunoadsorbents.

Nonimmunospecific interactions of IgG and IgG-agarose columns were systematically studied under varying conditions. Nonimmunospecific binding to the columns was primarily due to protein-protein interactions. These nonimmunospecific protein-protein interactions of IgG were enhanced with heat-induced or chemical aggregation of IgG, low pH, low ionic strength (at pH above 4), or low temperature. Conversely, this binding was decreased with proteolytic fragmentation of IgG, high ionic strength (at pH above 4), or temperatures above 4 degrees C. Chemical modification of IgG by acetylation, formalinization, carbamylation, or reaction with 1,2-cyclohexanedione significantly decreased these interactions. These observations suggest that above pH 4, ionic interactions caused the protein-protein binding. Below pH 4, hydrophobic interactions presumably play a major role. These results permit the development of rational methodology for avoiding nonimmunospecific protein-protein interactions in immunologic procedures for detection, isolation, or quantification of rheumatoid factors and other antibodies to IgG.     

Subclasses of human IgG. I. Production of antisera to IgG subclasses]

Guinea pigs were used for preparing antisera to human IgG subclasses for anti-IgG1, and rabbits--for anti-IgG2, anti-IgG3, and anti-IgG4. Schemes of laboratory animals immunization with myeloma paraproteins of four IgG subclasses were determined. Methods of antisera absorption for bringing them up to strict monospecificity were worked out. Antisera specificity were determined by the precipitation test after Ouchterlony with standard myeloma proteins in the concentration of 1 mg/ml, and in the passive hemagglutination test with erythrocytic antigenic diagnostic agents. Precipitating antisera to four human IgG subclasses were obtained.     

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG.

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.     

A genetically engineered human IgG mutant with enhanced cytolytic activity.

A mutant chimeric anti-5-dimethylaminonaphthalene-1-sulfonyl human Ig gamma that exhibited augmented effector function was constructed. Utilizing directed mutagenesis, a serine residue near the carboxyl terminus of the human IgG1 H chain (Ser444) was replaced by a cysteine. Novel intermolecular disulfide bonds between Cys444 residues linked pairs of Ig "tail-to-tail" to form covalent dimers ((H2L2)2). These dimers were 200-fold more efficient, compared with monomeric human IgG1, at antibody-dependent complement-mediated cytolysis of hapten-bearing erythrocytes. The ability to enhance the cytolytic activity of an mAb by genetic engineering may be of value in immunotherapy.     

A simplified process for large-scale isolation of IgG from goat serum.

Immunoglobin G (IgG) is routinely used in many immunoassay systems. Whilst various laboratory-scale procedures exist for the isolation of IgG from host serum few are appropriate for scale-up. We have previously reported the improvement of an existing process for isolation of IgG from goat serum at laboratory scale (Schwartz et al., 1986) and in this study we took those improvements to pilot-scale and significantly reduced the process time and associated costs. Media fouling was reduced and product yield enhanced by utilisation of a guard column of the appropriate geometry. Isolation of pure IgG by anion-exchange chromatography was improved by selection of a suitable mobile phase and column loading parameters. Using a 10 1 column of QA52, 36 g of immunoelectrophoretically pure IgG was isolated from 7.2 1 of goat serum at an overall yield of 58%. Product recovery and purity were reproducible at pilot-scale under these conditions. A procedure for media clean-in-place (CIP) is described which also effects sterilisation and depyrogenation of the column and media.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.     

Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4-restricted response

Labeled antigen-binding tests were used to determine quantitatively the contribution of IgG4 antibodies to the total IgG antibody response in humans. In agreement with literature, we found no IgG4-restricted antibody responses with tetanus toxoid or streptococcal carbohydrate. In the serum of individuals immunized for several years with phospholipase (PLA) from honey bee venom, grass pollen allergen, or house dust mite allergen, we often found that more than 50% of the total antigen-binding capacity was due to IgG4 antibodies. In the case of beekeepers, it could clearly be shown that during prolonged immunization a shift in the IgG4:IgG1 antibody ratio occurs that finally results in an IgG4-dominated antibody response. Evidence is provided that antigen-binding assays may even underestimate the contribution of IgG4 antibodies, because in contrast to IgG1 antibodies, IgG4 antibodies act as monovalent antibodies in being unable to cross-link immunosorbent-bound antigen and radiolabeled antigen.     

A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins.

Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.     

Modulation of the immune response by passive antibodies. III. Effects of IgG1 and IgG2 anti-carrier antibodies.

The effects of IgG₁ and IgG₂ anti-carrier antibodies were studied on cellular and humoral reactions induced by immunization with a hapten-carrier complex. IgG₁ was shown to depress both delayed hypersensitivity reactions (DHR) to the carrier and anaphylaxis to the hapten whereas IgG₂ had no activity. A mixture of IgG₁ and IgG₂ depressed only DHR to the carrier. The modulating effects of passive anti-carrier antibodies were shown to depend on their immunoglobulin class and the concentration used.     

Rabbits immunized with mixtures of staphylococcal protein A and autologous IgG produce anti-human IgG antibodies

The results of this study provide evidence that protein A may render IgG immunogenic in the autologous host. Antibodies to human but not rabbit IgG were detected in sera of rabbits immunized with a mixture of autologous serum and protein A. Anti-human IgG antibodies appeared within 2 weeks at which time the antibodies were of the IgM class. Upon further immunization, both IgM and IgG antibodies were produced with the IgG class predominating. The antibodies elicited by a mixture of protein A with autologous IgG resembled those which arise in response to autologous IgG that has been denatured by physicochemical means, in that they react mainly with foreign species IgG and weakly, if at all, with IgG of rabbit origin.     

A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Stimulation of eryptosis by anti-A IgG antibodies.

Anti-A IgG antibodies have previously been shown to stimulate Ca(2+) entry into red blood cells. Increased cytosolic free Ca(2+) concentration is known to trigger eryptosis, i.e. suicidal erythrocyte death, characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether anti-A IgGs trigger phosphatidylserine exposure of erythrocytes. Phosphatidylserine exposure was estimated from annexin-V binding as determined in FACS analysis. Exposure to anti-A IgGs (0.5 microg/ml) indeed significantly increased annexin-V binding in erythrocytes with blood group A, but not in erythrocytes with blood group 0. According to Fluo3 fluorescence, anti-A IgGs increased cytosolic Ca(2+) concentration. Whole cell patch clamp recordings revealed the activation of a Ca(2+)-permeable cation channel following treatment with anti-A-IgGs. Annexin-V binding following anti-A IgG exposure was blunted by Ca(2+) removal while anti-A IgG-stimulated cation channel activity was not dependent on extracellular Ca(2+). Osmotic shock (exposure of erythrocytes to 850 mOsm) increased annexin binding, an effect further enhanced by exposure to anti-A IgGs. In conclusion, anti-A IgGs activate erythrocyte cation channels leading to Ca(2+) entry and subsequent erythrocyte cell membrane scrambling. The effect most likely contributes to the elimination of erythrocytes following an immune reaction against the A antigen.     

Detection of pyrogens in intravenous IgG preparations.

Five different intravenous IgG (i.v. IgG) preparations were assessed for their capacity to modify the pyrogenic response to bacterial lipopolysaccharide (LPS) of rabbits under the conditions of a pharmacopoeal test. Four of the five preparations were found to mitigate the reaction rendering the result "non-pyrogenic" with an LPS dose proved pyrogenic when administered in saline or in albumin. Bacterial LPS was found readily detectable by a simple Limulus amoebocyte lysate (LAL) gelation test. Four of six brands of i.v. IgG were found reactive in the test under conditions adjusted to detect the FDA limit. The reaction obtained upon addition of standard LPS to the negative preparations supported the validity of the assay. The LAL reactivity of two of the reactive preparations was inhibited by laminarin, a compound known to inhibit Limulus lysate gelation by beta-D-glucan, but not by Polymyxin B. Specific detection of bacterial endotoxins in i.v. IgG solutions requires inhibition of the beta-D-glucan pathway of the Limulus lysate coagulation. Using an appropriate inhibitor, the LAL gelation test is suitable to detect a potential endotoxin contamination in i.v. IgG which might have not been unravelled by the in vivo test for pyrogens.     

IgG subclass alterations in adult asthma.

Immunoglobulin levels were measured in serum samples of 12 black adult non-smoking asthmatic patients, 11 females and 1 male, and compared with 15 age-, sex-matched normal controls. Their total IgG, IgA and IgM levels were within the normal range. However, on quantitation of subclasses, IgG1 levels were significantly above normal, while IgG2 and IgG3 levels were significantly lower than those of controls. No significant differences were found between the two groups when IgG4 levels were compared. These studies as well as those of others suggest that immunoglobulin administration, particularly of individual subclasses, might prove to be a beneficial addition in the management of this condition.     

Structure of human myeloma IgG3 Kuc.

An electron microscopy study of human myeloma IgG3 Kuc has shown that the hinge region in an intact molecule is in a compact state. The subunits are not fixed rigidly and are very mobile. These data are supported by results of ultracentrifugation and microcalorimetry. Non-extremal denaturating effects (pH 4.0, 20 degrees C or pH 7.8, 65 degrees C) lead to 'unfolding' of the hinge region which has a rod-like shape in electron micrographs.