Isolation of FAB and Fc fragments from murine immunoglobulin G1]

Two methods of isolating Fab- and Fc-fragments from mouse immunoglobulin G1 are presented. The first method involves fractionation of papain protein hydrolysate on a column with DEAE- (or DE-32)-cellulose adjusted with 0.005 M K-phosphate buffer, pH 8. The Fab-fragment was eluted from the column with the starting buffer. The Fc-fragment was eluted, with the buffer ionic strength being increased to 0.4 M. Another method involves protein fractionation on an ion exchanger adjusted with 0.004 M Tris-H3PO4 buffer, pH 8.5. All the protein was column bound. The Fab-fragment was eluted with 0.04 M Tris-buffer containing a 0.004 M mixture of K-phosphates, pH 8.6. The Fc-fragment was eluted, with ionic strength being raised to 0.4 M with phosphates. As none of the methods assures isolation of absolutely pure Fab- or Fc-fragments, it is requird that cross absorption of antisera with respective immunosorbents may be carried on in order to obtain monospecific antisera to these fragments.     

Chaperone Skp from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> exhibits immunoglobulin G binding ability

A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM^?1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.     

Effect of dehydration on interdomain interactions in immunoglobulin G and its fragments

Influence of urea on the structure of human IgG and isolated Fab and Fc-fragments was investigated by temperature-perturbation difference spectroscopy and circular dichroism. It was shown that 2M urea caused non-denaturational changes of IgG quaternary structure, localized in Fab-fragments. The same changes occur in solutions with ethylene glycol and glycerine as well. Apparently the main cause of these changes is dehydration. It is possible that the investigated effects model the conformational changes which appear in immunoglobulins after antigen binding.     

Effect of immunoglobulins and IgG-fragments on the human erythrocyte aggregation, studied by a rheoscope combined with image analyzer

Using a rheoscope, combined with a TV image analyzer and a computer, the effects of immunoglobulins and IgG-fragments on the process of human erythrocyte aggregation were determined. The immunoglobulins accelerated the aggregation; the effect increased with their molecular weight, i.e., IgG congruent to gamma-globulin less than IgA less than IgM. An empirical relationship, expressing the dependence on the immunoglobulin concentrations, was proposed. F(ab')2-fragment accelerated the aggregation, more effectively than IgG, while Fab- and Fc-fragments did not. Therefore, two Fab-portions are presumably needed to form the aggregates, and the flexibility between two Fab-portions may be important. A case of multiple myeloma showed an increased aggregation, due to the increased myeloma protein.     

Determination of the subclasses of monoclonal human immunoglobulins G and of the light chain type with the aid of specific antisera]

Sera of 86 patients suffering from G-myeloma were studied for the purpose of determination of subclasses of monoclone IgG. Investigations were carried out by means of antisera to subclasses IgG by the double diffusion method in gel after Ouchterlony. The following distribution of myeloma Ig was revealed: G1--70%, G2--17%, G3--11%,and G4--2%. In typing of the light igG chains by the method of immunoelectrophoresis, using antisera to the light chains of immunoglobulins of the chi and lambda type it was found that IgG1 chi was encountered more frequently than IgG1 lambda (3:1 ratio). The amount of the sera with the IgG2, IgG3, and IgG4 was insufficient for the reliable conclusion of their distribution by the type of light chains.     

New data on the biological action of normal immunoglobulins

Experiments on 2,520 CBA mice (CBA X X C57BL) F1 nice have shown that the injection of homologous serum immunoglobulins (obtained from intact and blood-stimulated animals), made 2 hours after gamma irradiation from a 60Co source, prevents the development of intestinal dysbacteriosis and endogenous infection. The injection of mouse and human immunoglobulins to nonirradiated mice improved their resistance to experimental infection with Escherichia coli live culture, increased the expression of receptors to the Fc-fragments of IgG in peritoneal macrophages and enhanced the physical working capacity of the animals. The preparations containing normal antitissular antibodies have proved to be particularly effective. In mice, rabbits and dogs the preparations under test have produced no changes in the general state of the animals, no local reactions and no disturbances in the cardiovascular activity.     

Immunoglobin-binding proteins in human placenta]

Phenomena of the binding of poor-soluble placenta proteins (PSPP) with pregnant women sera IgG as well as placenta blood IgG were studied. PSPP were extracted from the placenta tissue, washed out from soluble proteins, by the use of 3M KCl solution containing 0.005 M PMSF. PSPP were separated by the use of two-dimensional isoelectrofocusing and SDS-PAG electrophoresis and more than 30 different polypeptides were visualized. Having used various ELISA procedures with pregnant women sera IgG, placenta blood IgG as well as its Fab and Fc-fragments we have shown that both the receptor-type and the antigen-antibody-like interaction of PSPP took place. Both the polypeptide compositions and the isoelectrofocusing points ranges of the antigen-antibody-like interacting IgG-binding PSPP were determined by the use of the peroxidase conjugated Fab-fragments of the placenta blood IgG.     

Isolation and characterization of the immunoglobulin-binding protein from Yersinia pseudotuberculosis

A high molecular weight immunoglobulin-binding protein localized on the surface of bacterial cells has been isolated from the protein fraction of the outer membrane of Yersinia pseudotuberculosis, and its properties are described. The immunoglobulin-binding protein is a trypsin-resistant and temperature-sensitive -structured protein. As shown by MALDI-TOF mass spectrometry, after heating at 100°C the molecular weight of the protein constituted 37.5 kD. The native protein is capable of interacting with human and rabbit IgG but looses the ability to bind the immunoglobulins after the temperature denaturation. The immunoglobulin-binding protein binds to the Fc-fragments of the immunoglobulins and binding depends on the presence of calcium ions.     

The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate

Human IgG subclasses 1, 2, and 4, as well as proteins of the IgG3 subclass that are allotype G3m (s+t+), bind avidly to staphylococcal protein A by means of their Fc portion. Proteins of the IgG3 subclass that are allotype G3m (s-t-) do not bind. The importance of a histidine residue at position 435 has been implicated from comparison of amino acid sequences of immunoglobulins that bind with those that do not bind to staphylococcal protein A, as well as from crystallographic data. Modification of histidines at a low concentration of diethylpyrocarbonate successfully and reversibly alters the binding of immunoglobulins to staphylococcal protein A with only minimal change in the antigenic properties. This method provides strong evidence for the critical importance of histidine in the binding of immunoglobulins to staphylococcal protein A.     

Immunological and functional characteristics of the blast cells in acute monoblastic and acute myeloblastic leukemias

The increase in the expression of membrane receptors of monoblast cells to the Fc-fragment of immunoglobulins of classes IgG, IgA, IgM, to the C3-component of complement and FcH receptor in 8 patients with acute monoblastic leukemia and in 3 patients with acute myelomonoblastic leukemia, compared to results obtained for 11 patients with acute myeloblastic leukemia. Leukemic cells in the cases of acute myelomonoblastic and monoblastic leukemia maintained such properties of the phagocytosis (restoring Nitro-blue tetrazolium). Distinctions in degrees of expression of membrane receptors and functional activity of monoblastic and myeloblastic cells may be used as criteria of differential diagnosis of acute monoblastic and acute myeloblastic leukemia.     

Nephelometry determined serum immunoglobulin isotypes in healthy Thai children aged 2-15 years

Knowledge of what constitute normal serum immunoglobulin (Ig) concentrations are important for the diagnosis of immunologic disorders. Data on normal Ig evaluated by nephelometry are limited in healthy Asian children, none being available for Thai children. One hundred and forty-eight healthy Thai children aged 2-15 years were tested for serum immunoglobulins G, A, M, G1, G2, G3, and G4 (Ig G, A, M, G1, G2, G3, and G4) by nephelometry. Sixty-three percent were girls of median interquartile range age 6.9 (4.8-9.7) years. The geometric means for each Ig were summarized and categorized by age. Statistical analyses were used to compare Igs between sexes and age groups, and to compare IgG in this study with data from other published studies. The average ratios of IgG subclasses/IgG for Ig G1:2:3:4 were 66:22:5:7%. IgG, IgA, IgG2, and IgG3 concentrations showed a gradual increase with increasing age. There were no significant sex differences for any immunoglobulin isotype (P= 0.971). Our mean IgG concentration was lower than that measured by the radial diffusion method in healthy Thai children (P < 0.05). In all age groups, the mean IgG concentration in our study was significantly higher than that reported in Turkish and USA children, evaluated by the nephelometric and radial diffusion techniques, respectively (both P < 0.001). This study provides information about normal Ig concentrations measured by nephelometry in healthy Asian children and illustrates the importance of ascertaining normal Ig values for age- and ethnic-matched controls using the same assay to diagnose immunologic disorders correctly.     

Further characterization of thyrotropin-displacing-activity (TDA): evidence of restricted heterogeneity

For further characterization immunoglobulins G (IgG) of 14 patients with Graves' disease were fractionated on Protein A bound Sepharose. Sufficiently enriched IgG-subclasses were obtained despite incomplete separation. TSH displacing activity (TDA) was not homogeneously distributed in the subclasses, however IgG1 was most predominant in TDA followed by IgG3, IgG2 being essentially inactive. In two subjects TDA in whole IgG gave positive results in subclasses after fractionation, thus providing evidence, that it would be theoretically possible to move sensitivity of the method close to 100% after subfractionation. TDA was not enriched together with thyroid cell antibodies. We conclude from these results that TDA-IgG is characterized by restricted heterogeneity notwithstanding the incomplete separation of the IgG-subclasses.     

Structural heterogeneity of sugar chains in immunoglobulin G. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases

The heterogeneous asparagine-linked sugar chains of bovine and human immunoglobulins G were separated into 12 components by reversed-phase high performance liquid chromatography, and their structures were determined by 1H NMR spectroscopy. Both immunoglobulin (Ig) G sources contained eight non-bisected biantennary complexes and four bisected biantennary complexes. In the non-bisected sugar chains of bovine IgG, galactosylation of the Man alpha 1-3 branch predominated over that of the Man alpha 1-6, whereas in the bisected complexes galactosylation of the Man alpha 1-6 branch predominated. This difference can be explained by the substrate specificities of the galactosyl-transferases and of the N-acetylglucosaminyltransferase III involved in their synthesis. The sugar chains of human IgG1 differs in the distribution of its galactose residues from bovine IgG and human IgG2. The Man alpha 1-6 branch of all IgG1s was more highly galactosylated than the Man alpha 1-3 branch even in the non-bisected complexes. Such findings are in conflict with the substrate specificities of galactosyltransferases. Whereas these enzymes derivatized more of the Man alpha 1-6 branch of native human IgG1, in denatured protein more of the Man alpha 1-3 branch was galactosylated. Thus, protein conformation may influence the structure of its sugar chains.     

The interaction of protein A and Fc fragment of rabbit immunoglobulin G as probed by complement-fixation and nuclear-magnetic-resonance studies.

Protein-A-Fc-fragment complexes were observed in sedimentation-velocity experiments by ultracentrifugation. The interaction was studied by protein-fluorescence-quenching titrations of the Fc fragment with protein A, allowing the dissociation constant to be determined under a variety of conditions. The first component of the complement pathway, C1, is activated by complexes of protein A with rabbit IgG (immunoglobulin G), and the structural basis for this interaction was studied by using n.m.r. (nuclear magnetic resonance). The four Fc-fragment binding sites on protein A were shown to contain aromatic amino acids, and to be connected by mobile hydrophilic regions. Neither n.m.r. nor proton-relaxation-enhancement studies show evidence of a large conformational change of the Fc fragment on binding protein A, and this suggests that the cross-linking of the Fc fragments may be primarily responsible for the activation of component C1. This is supported by the inability of a univalent tryptic fragment of protein A to activate complement fixation by rabbit IgG.     

A multigene deletion in the human IGH constant region locus involves highly homologous hot spots of recombination

A simultaneous absence of the IgG1, IgG2, IgG4, and IgA1 immunoglobulins (Ig) was unambiguously demonstrated in six healthy individuals of two different families (family HASS and family TOU). These individuals were shown to be homozygous for a large deletion in the immunoglobulin heavy chain constant region locus. This deletion, which encompasses the G1-EP1-A1-GP-G2-G4 genes, allowed us to predict an order for the IgCH genes and to localize GP between A1 and G2. In this paper, we study the deletion-recombination point in the IGH locus of individual EZZ from the TOU family. We show that the distance between the G3 and the E genes on the EZZ recombinant chromosome is 24.7 kb and that the multigene deletion in the IgCH locus involves two highly homologous regions (hsg3 and hsg4) which are hot spots of recombination, outside of the switch sequences.     

Mass Spectrometry Detection of G3m and IGHG3 Alleles and Follow-Up of Differential Mother and Neonate IgG3

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.     

Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG

The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.     

Levels of immunoglobulins of isotype IgG and IgG2 in subjects lacking formation of IgG antibodies against lipopolysaccharide of Chlamydia

In some patients with antibodies against LPS antigen of Chlamydia, specific immunoglobulins G are not present. The findings of isolated anti-LPS IgA/IgM antibodies are to be considered as possibly non-specifically or "false" positive. The aim of this study was to investigate if there is any difference in the level of total immunoglobulins of isotypes IgG and IgG2 in probands with isolated positivity anti-LPS IgA (i.e. without simultaneous presence of specific IgG; n = 34) as compared with a control group of subjects presenting positive anti-LPS IgA and IgG antibodies (n = 44). Antibodies against LPS antigen of Chlamydia were determined by ELISA method ("Chlamydien--rELISA", medac, Germany). Total immunoglobulin levels were determined by nephelometry using the following kits: "Immunoglobulin G Reagent, ARRAY Systems", Beckman Coulter, USA and "Human IgG2 Subclass Beckman ARRAY Kits", The Binding Site, UK. The measured values were related to the age-dependent laboratory standard values and the differences between the tested groups were statistically evaluated (chi(2) test). Decreased total IgG have been demonstrated in 4 (11.8 %) probands and in 5 (11.4 %) subjects of the control group; increased total IgG were found in 2 (5.9 %) probands and in 1 (2.3 %) subject of the control group. Decreased levels of total IgG2 were not determined in any proband and were found in 1 (2.3 %) subject of the control group. Increased levels of IgG2 were registered in 12 (35.3 %) probands and in 15 (34.1 %) control subjects. No statistically significant differences were found between the two groups. It can be concluded that no relationship was proved between the levels of total IgG and IgG2 and the absence of formation of specific anti-Chlamydia-LPS IgG. Further research will be necessary for the elucidation of this phenomenon (e.g. the presence of specific anti-LPS IgG in immunocomplexes).     

Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5’- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.