Immunoglobulin G Binding Research Articles

The fate of IgE bound to rat basophilic leukemia cells. IV. Functional association between the receptors for IgE.

Rat basophilic leukemia cells (RBL-2H3) have receptors for immunoglobulin E (IgE) and immunoglobulin G (IgG). These receptors for IgE mediate the endocytosis of chemically or immunochemically cross-linked IgE but not monomeric IgE. However, unoccupied receptors were endocytosed with cross-linked IgE. To further assess the degree and specificity of the observed coendocytosis, we exposed cells carrying monomeric rat IgE and monomeric mouse IgE anti-DNP to a DNP-protein conjugate. We found that up to 30% of the surface-bound monomeric rat IgE redistributed at 0 to 4 degrees C and was then internalized at 37 degrees C with the immunochemically cross-linked mouse IgE. To assess the specificity of the coendocytosis, we exposed cells carrying monomeric rat IgE to immunochemically cross-linked mouse IgG. We found that the binding, patching, and endocytosis of cross-linked mouse IgG had no effect on the monomerically bound rat IgE. The rate of coendocytosis was the same as the rate of endocytosis (t 1/2 3 to 5 min). The extent of coendocytosis depended on the extent of endocytosis but was relatively insensitive to changes in the ratio between mouse and rat IgE over a broad range. These results indicate that some of the receptors for IgE are associated in a specific fashion.

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).

Kinetics of intravenous saline infusion and selective IgG removal in rabbits.

Selective removal of approximately 60% of the plasma immunoglobulin G (IgG) mass in conscious rabbits was studied and compared with similar procedures combined with intravenous saline infusions equal to 7.5% body weight. Plasma concentrations of 125I-IgG and endogenous IgG were employed in model analysis to examine if saline infusions could be used to shift IgG from extra- to intravascular pool, thereby making more protein available for removal by extracorporeal plasma treatment. After IgG removal, the fractional metabolic clearance and the extra- to intravascular transfer coefficient were 40-50% lower than before IgG removal, and model simulations indicated that this may be caused by IgG binding. Saline infusion resulted in 40% more IgG mass in plasma 24 h after treatment compared with procedures without saline. Model analysis indicated that the increased IgG mass in plasma after saline procedures could be explained by a three- to fourfold increase in lymphatic clearance. Crystalloid infusions may be a method to increase the efficacy of repeated plasma exchange treatment.

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent.

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.

Properties of immunoglobulin G-Fc receptors from neonatal rat intestinal brush borders.

Newborn rats acquire immunity passively by receptor-mediated uptake of maternal immunoglobulin G (IgG) from the first milk. Specific IgG binding to brush borders and IgG transport across the gut increase concomitantly for 10-12 days after birth and then fall until closure at about 21 days. Cortisol acetate administration accelerates this decline. Two classes of binding site are resolved by their affinities (KA1 = 1.3 X 10(8) M; KA2 = 5.15 X 10(6) M). Persistence of the low-affinity site after closure precludes a transport role (see Rodewald et al, this volume). Target size analysis gives a preliminary Mr for the high-affinity site of 90 000-100 000. IgG recognition involves a small number of positively charged residues in the Fc region. An Fc binding activity is solubilized from intestinal brush borders by lithium 3,5-diiodosalicylate.

Immunoglobulin G receptors of intestinal brush borders from neonatal rats.

Intestinal absorptive cells of the neonatal rat display on their brush border membranes receptors for immunoglobulin G (IgG) which function in selective transfer of maternal IgG. Our Scatchard analysis of [125I]IgG binding to isolated brush borders has corroborated the presence of two classes of specific binding sites (KA1 = 2.4 X 10(7) M-1 and KA2 = 3.7 X 10(5) M-1) and the increase in overall binding with decreased buffer concentration, as shown by Wallace & Rees 1980. However, our Scatchard analysis of binding at different buffer concentrations indicates that the only significant effect of lowered buffer concentration is to increase the number of low-affinity sites. Neither the number nor the affinity of the high-affinity sites is affected. Furthermore, brush borders from rats at 21 days have only the low-affinity sites and at this age the selective transfer of IgG has ceased. Morphological experiments with tracers for both light and electron microscopy suggest that the high-affinity sites correspond to the specific IgG receptors on the apical membrane. The majority of the low-affinity sites are found within the terminal web and are likely not to be involved in selective transport of IgG.

Specificity of autoimmune monoclonal Fab fragments binding to single-stranded deoxyribonucleic acid.

Fab fragments from hybridoma HEd 10 [Lee, J. S., Lewis, J.R., Morgan, A.R., Mosmann, T.R., & Singh, B. (1981) Nucleic Acids Res. 9, 1707-1721] were prepared in large amounts by papain digestion of the immunoglobulin G (IgG) fraction from ascites fluid. Binding data were generated by a fluorescence quenching technique, and binding constants [K(0)] were estimated from Scatchard plots. The Fab fragments bound tightly to poly(dT) [K(0) = 12.7 X 10(6) M-1], and analysis of binding constants for the series p(dT)2 to p(dT)17 showed that the recognition sequence consisted of four consecutive residues. The effect of ionic strength on the interaction suggested that only two phosphates were involved. Binding constants for poly(dU), poly[d(brU)], poly[d(brC)], and poly(rU) were 1.0 X 10(6) M-1, 18.8 X 10(6) M-1, 0.5 X 10(6) M-1, and less than 0.5 X 10(6) M-1, respectively, implicating the involvement of the 3, 4, and 5 positions of the pyrimidine ring as well as the deoxyribose sugar in the recognition process. At high ionic strength (0.5 M) K(0) for whole IgG binding to poly(dT) was 75 X 10(6) M-1 compared to a value of 1.1 X 10(6) M-1 for the Fab fragment. These results may have implications for the tissue damage caused by DNA-containing immune complexes in systemic lupus erythematosus.

Effect of neuraminidase treatment of IgG and alpha 1-acid glycoprotein on their intestinal transport in suckling rats in vivo.

Binding and transport of rat immunoglobulin G (IgG), human alpha 1-acid glycoprotein, and their neuraminidase-treated derivatives were studied in vivo in the intestine of suckling rats. IgG and asialo-IgG binding was saturable, preceding active transport; the amounts of gut-associated alpha 1-acid glycoprotein and asialo-alpha 1-acid glycoprotein ("binding") were not saturable; and transport was passive. Binding of asialo-IgG to the intestine was significantly higher than that of native IgG, whereas transport rates were similar. No difference was found for both binding and transport of native and asialo-alpha 1-acid glycoprotein. These results suggest an interaction between the heteroglycan moiety of IgG and its intestinal receptor and exclude a role of sialic acid in the intestinal transport of a heterologous, passively absorbed glycoprotein.

Binding of human immunoglobulin G to protein A in encapsulated Staphylococcus aureus.

Recent studies of the mechanism of resistance to phagocytosis in encapsulated Staphylococcus aureus have suggested that the capsule is readily penetrated by high-molecular-weight proteins such as antibodies and complement components. S. aureus strains contain a cell wall protein, protein A, that reacts with the Fc portion of immunoglobulins. The binding of immunoglobulin G (IgG) to encapsulated and unencapsulated S. aureus strains has been studied to assess the penetrability of the S. aureus capsule by IgG. Encapsulated S. aureus strains M and Smith diffuse bound large amounts of human IgG which were comparable to amounts bound by the unencapsulated strains Cowan I, M variant, and Smith compact. Trypsin treatment of bacteria reduced their ability to bind IgG. Bound IgG was not removed by extensive washing of bacteria with buffer. A non-protein A-containing, coagulase-negative, encapsulated staphylococcal strain did not bind IgG. These observations suggest that IgG is binding to cell wall protein A in encapsulated S. aureus. No differences in the rates of IgG binding by encapsulated and unencapsulated S. aureus strains were observed. It is concluded that the S. aureus capsule is freely permeable to IgG. This is of importance in considerations of the mechanisms of resistance to phagocytosis and antigen masking in encapsulated microorganisms.

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.

Binding of immunoglobulin G to phospholipid vesicles by sonication.

Purified goat immunoglobulin G (IgG) does not bind to sonicated phospholipid vesicles. However, when IgG is sonicated together with phospholipids, 4 to 40% of the IgG can be bound to the vesicles, depending on the experimental conditions. The extent of binding depends on the period and power of sonication, the IgG to lipid ratio, and the lipid composition. Anionic phospholipids such as phosphatidylglycerol and phosphatidylserine, but not cholesterol, enhance binding about 50% over that obtained with the neutral phosphatidylcholine. Binding of IgG causes extensive aggregation of vesicles, as shown by electron microscopy, and aggregates can be separated from unbound IgG by molecular-sieve chromatography on Sepharose 4B or by sucrose density gradient centrifugation. The IgG-vesicle aggregates remain stable in either phosphate-buffered saline or 50% fetal calf serum up to 20 h at 37/sup 0/C, although substantial lipid degradation in 50% fetal calf serum was observed. The use of goat IgG containing antibody to a purified protein antigen allowed quantitation of antibody activity of these preparations. Immune IgG sonicated alone shows 100% of the original antigen binding capacity while vesicle-bound IgG retains 30 to 50%. Antigen binding capacity of bound IgG is not increased when vesicles are lysed by 1.5% NP-40, suggestingmore » all of the bound IgG is exposed on the outer surfaces of the vesicles. IgG's of human, mouse, and rabbit, as well as the purified goat F(ab')/sub 2/ fragments, also bind with vesicles by cosonication.« less

Liability to hydrophobic and charge interaction of smooth Salmonella typhimurium 395 MS sensitized with anti-MS immunoglobulin G and complement

Sensitization of smooth Salmonella typhimurium 395 MS bacteria with hyper-immune anti-MS immunoglobulin G (IgG) antibodies increased the liability to hydrophobic interaction as assessed by the affinity for a column of Octyl-Sepharose. After sensitization, the material originally eluted with 1 M (NH4)SO4 in a 0.01 M phosphate buffer (pH 6.8) was not desorbed until the ionic strength was reduced to nil, and 0.1% (vol/vol) Triton X-100 in the 0.01 M phosphate buffer was used as eluant. Furthermore, by including positively charged bis-trimethylamino-polyethylene glycol (PEG) or negatively charged bis-sulfoamino-PEG in an aqueous two-phase system of dextran T500 and PEG 6000, the partition of the IgG-sensitized bacteria was affected by either of the polymers, whereas that of the parent bacteria was not. The hydrophobic effect of IgG binding was enhanced by complement. With heat-inactivated complement, the effect of IgG was diminished. The F(ab')2 fragment showed a much lower capacity to promote a hydrophobic interaction than the complete IgG molecule.

Classical and alternative complement pathway activation by pneumococci.

Sixty-two strains of Streptococcus pneumoniae were studied for their abilities to consume selected components of classical and alternative complement pathways in human sera. The classical pathway was blocked by chelating calcium with ethyleneglycol-bios (beta-aminoethyl ether)-N,N-tetraacetic acid and by removing C4. The alternative pathway was blocked by removing factor B. Each strain's activation of the two pathways was compared with its nonimmune reactivity with the Fc region of immunoglobulin G (IgG). Activation of the classical complement pathway appeared to be independent of such Fc reactivity. Highly Fc-reactive strains, however, were shown to activate the alternative pathway more effectively than did less Fc-reactive strains. Since pneumococcal activation of the alternative pathway requires non-immunospecific IgG, these findings suggest that nonimmune binding of IgG on the pneumococcal surface endows it with complement-activating properties.

Cytomegalovirus Specific IgM and IgG Response in Humans Studied by Radioimmunoassay

An indirect solid phase micro-radioimmunoassay (RIA) was adapted for the measurement of anti-cytomegalovirus class-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). Cytomegalovirus (CMV) antigen (Ag) was added to the wells of microtiter plates and desiccated onto the bottom surface of the wells. Serial dilution of human CMV antisera were added and allowed to react with the Ag. The amount of viral antibody (Ab) was determined by measuring the specific binding of 125I-labeled goat anti-human IgG (125I-AHIgG) and/or IgM (125I-AHIgM). The multiple factors which influence the test were determined. Serial samples of sera from CMV-positive patients showed progressive increases in Ab titers on the basis of specific binding of 125I-AHIgG. The titers of IgM class anti-CMV Ab were also determined with the same sera, and enhancement of the titers was demonstrated when the incubation periods of the first Ag-Ab reaction were extended from 1 to 3 hr. The competition between the different Ig classes of Ab for CMV Ag and the involvement of rheumatoid factor (RF) were investigated. The anti-CMV IgG that may compete with the IgM determination was removed by adsorption of sera with Staphylococcus aureus (Cowan 1). The RF that would cause false anti-CMV IgM results was adsorbed with glutaraldehyde-insolubilized IgG. Our results indicate that this RIA is a practical, specific, and reproducible technique for detection of specific IgG and IgM Ab to CMV.

The Development by Cytomegalovirus-Infected Cells of Binding Affinity for Normal Human Immunoglobulin

After infection with human cytomegalovirus (CMV), cells develop an affinity for normal human immunoglobulin G (IgG). This was demonstrated using 125iodine-labeled purified IgG. It was further demonstrated that the immunoglobulin molecule binds to CMV-infected cells via its Fc portion, and competition for binding to infected cells occurred between purified preparations of human IgG and the Fc fragment of human IgG. Whole sera from individuals with or without a high titer of anti-CMV antibody were labeled with 125iodine and it was demonstrated that serum from individuals with no anti-CMV antibody had an affinity for CMV-infected cells which probably reflected binding of IgG via its Fc fragment. The possible significance of these results in immunologic studies of human CMV is considered.