Affinity Immunoglobulin Research Articles

Protein L: An Immunoglobulin Light Chain-binding Bacterial Protein: Characterization of binding and physicochemical properties

Protein L, a cell wall molecule of the bacterial species Peptostreptococcus magnus with affinity for immunoglobulin (Ig) light chains, was isolated after solubilization of the bacterial cell walls with mutanolysin or from the culture medium by a single affinity chromatography step on human IgG-Sepharose. A major protein band with an apparent molecular weight of 95,000 was obtained from both sources. The protein from the growth medium was size heterogeneous. From 1 ml of packed bacteria was prepared 0.92 mg of the mutanolysin-solubilized protein L (73% yield), whereas 4.1 mg of spontaneously released protein L (49% yield) was purified from the corresponding culture medium. The Mr of protein L was estimated to 76,000 by gel chromatography in 6 M guanidine HCl. Using this Mr value, the Stokes radius and the frictional ratio of protein L were determined to 4.74 nm and 1.70, respectively, suggesting an elongated fibrous molecule. No disulfide bond or subunit structure could be shown. The amino-terminal amino acid sequences of the whole protein and two internal non-IgG-binding tryptic fragments were determined and found to be unique. One of the tryptic fragments showed homology (40% identical residues) to a sequence within the cell wall-binding region of protein G, the Fc-binding protein of group C and G streptococci. The binding specificity of protein L was directed to the light chains of immunoglobulins; the affinity constant for polyacrylamide-coupled kappa-chains was 1.5 x 10(9) M-1 and for IgG, IgA, and IgM around 1 x 10(10) M-1. Maximal binding was achieved between pH 7 and 10. The binding to lambda-chains was too weak for determination of the affinity constant. 125I-Protein L was also shown to bind to mouse immunoglobulins. It could be used for detection of antigen-bound polyclonal and monoclonal antibodies in Western blots. This shows that the protein L/light chain reaction was not obstructed by occupation of the antigen-binding site. Finally, protein L and kappa-chains of human Ig formed precipitates upon double immunodiffusion analysis, an indication of at least two binding sites on protein L.

Expression of High-affinity Binding of Human Immunoglobulin E by Transfected Cells

The receptor with high affinity for immunoglobulin E (IgE) on mast cells and basophils is critical in initiating allergic reactions. It is composed of an IgE-binding alpha subunit, a beta subunit, and two gamma subunits. The human alpha subunit was expressed on transfected cells in the presence of rat beta and gamma subunits or in the presence of the gamma subunit alone. The IgE binding properties of the expressed human alpha were characteristic of receptors on normal human cells. These results now permit a systematic analysis of human IgE binding and a search for therapeutically useful inhibitors of that binding.

A cDNA presumptively coding for the .alpha. subunit of the receptor with high affinity for immunoglobulin E [Erratum to document cited in CA107(7):53218m

A cDNA presumptively coding for the .alpha. subunit of the receptor with high affinity for immunoglobulin E [Erratum to document cited in CA107(7):53218m

Isolation and characterization of cDNAs coding for the beta subunit of the high-affinity receptor for immunoglobulin E.

Among receptors that bind the Fc region of immunoglobulins ("Fc receptors"), only the one with high affinity for immunoglobulin E (IgE) is known to consist of more than a single polypeptide. In addition to the IgE-binding alpha chain, the receptor contains a single beta chain and two, disulfide-linked, gamma chains. From a cDNA library of a rat mucosal mast cell tumor, from which we recently cloned cDNAs coding for the alpha chain, we have now isolated cDNAs coding for the beta subunit. In vitro transcription-translation of the cDNA directed the synthesis of a polypeptide reactive with two distinctive anti-beta monoclonal antibodies and whose molecular weight was identical to that of authentic beta chains. Polyclonal antibodies to beta peptides expressed in Escherichia coli reacted with intact receptors and isolated beta chains. The gene encodes a protein of 243 residues with no leader sequence. A hydropathicity plot suggests that the polypeptide crosses the plasma membrane four times. The epitope recognized by one of the monoclonal antibodies was localized to the NH2 terminus; that by the other was localized to the COOH terminus. Since those antibodies react with membranes and not with intact cells, we suggest that both ends of the beta subunit are cytoplasmic. RNA transfer blots at high stringency failed to reveal mRNA for beta chains in a variety of cells (in particular, monocytes) that do not contain the high-affinity receptor for IgE.

Binding affinity of serum immunoglobulin G to cardiolipin and other phospholipids in patients with systemic lupus erythematosus and syphilis.

Qualitative and quantitative assays for human antibodies to cardiolipin and other phospholipids were used in tests for these reactions in sera from patients with systemic lupus erythematosus (SLE) and syphilis. Of 22 SLE serum samples tested by the qualitative assay, 8 showed positive staining to cardiolipin, phosphatidic acid, and/or phosphatidylserine. All 47 syphilitic sera reacted with these three phospholipids. The apparent affinity of anticardiolipin binding was estimated by normalizing absolute binding levels as a function of serum concentration to the maximum percent bound. It was evident that antibody affinity was four- to fivefold lower in the SLE sera than in the syphilitic sera. Twelve serum samples from patients with one or more features of the anti-cardiolipin syndrome demonstrated mean binding values which were not distinguishable from binding in other SLE sera. In sera from patients with active SLE, binding affinity for cardiolipin was somewhat greater than that in samples from patients with inactive disease, but the differences were not statistically significant. The low anticardiolipin binding affinity which was observed in patients with SLE compared with that in patients with syphilis casts doubt on a pathogenic role for these reactions.

Studies with a monoclonal antibody to the β subunit of the receptor with high affinity for immunoglobulin E

The receptor with high affinity for IgE consists of a tetrameric complex of polypeptides, one of which ( α), contains the binding site for IgE. The function of the other chains—a single and two disulfide-linked γ chains—is unknown. We report the cloning of a murine hybridoma that secretes an IgG1 antibody which specifically reacts with the subunit. Studies with this monoclonal antibody show that the subunit stoichiometry of the receptor is unaffected by the presence or absence of bound IgE. We also found that under certain conditions where the αβγ 2 complex dissociates, remains attached to the dimer of γ chains, indicating that these chains contact each other in the native receptor. In rat basophilic leukemia cells—a neoplastic line of mucosal-type mast cells—all of the subunits expressed by the cells appeared to be associated with the high affinity receptor. However, in at least one cell line which has no high affinity receptors—a putative rat lymphoma line— β or β-like polypeptides were also expressed.

How antibodies work: focus on Fc receptors.

It is increasingly appreciated that the part of an antibody not involved in the binding of antigen--the Fc region--plays an important biological role. It activates a variety of receptors not only on so-called effector cells such as macrophages and granulocytes, but also on lymphocytes, and it can thereby modulate the immune response itself. Over the past 2 years much new information has been gained about the structure of such receptors, in large part through molecular genetics. In this review we describe the structure and some aspects of the function of the most complicated of the cellular Fc receptors so far identified: the receptor with high affinity for immunoglobulin E (IgE) on mast cells. The structure of its IgE-binding chain is strikingly similar to the corresponding polypeptide of an immunoglobulin G receptor. Like the latter and like a receptor that binds polymeric immunoglobulin, segments of the protein resemble immunoglobulin sequences. It is surprising that other IgE-binding proteins that putatively serve related functions have completely different structures.

Calcium-independent phosphoinositide breakdown in rat basophilic leukemia cells. Evidence for an early rise in inositol 1,4,5-trisphosphate which precedes the rise in other inositol phosphates and in cytoplasmic calcium.

Aggregation of the receptor with high affinity for immunoglobulin E (IgE) in rat basophilic leukemia cells leads to a calcium-dependent and a calcium-independent hydrolysis of phosphoinositides. The increase in the levels of inositol phosphates induced in the absence of calcium is only 25% of that observed with 1 mM Ca2+. The inositol phosphates reach a new steady state level 2 min after stimulation in EGTA, whereas with calcium they continue to increase up to 15 min. A similar response is observed when the receptors are aggregated due to the interaction of bound IgE with antigen or with anit-IgE, or by the binding of IgE cross-linked chemically. The antigen-mediated response is inhibited by hapten and disruption of such antigen-antibody aggregates late after stimulation leads to a rapid decline in the levels of the inositol phosphates to basal values. Separation of the inositol phosphates by Dowex columns shows that there is a fast rise in inositol trisphosphate which peaks at 15 s and slowly declines to a lower plateau within 2 min. Analysis by high pressure liquid chromatography reveals a 5-fold increase in the levels of inositol 1,4,5-trisphosphate in less than 10 s after stimulation, which precedes any major change in the other inositol phosphates. Aggregation of the receptor in the absence of external calcium induces a transient increase in cytoplasmic calcium which reaches a maximum of approximately 25 nM over basal levels after activation. The onset of the rise in Ca2+ lags after the initial rise in the inositol 1,4,5-trisphosphate.

A cDNA presumptively coding for the alpha subunit of the receptor with high affinity for immunoglobulin E.

Rat mast cells and a neoplastic analogue such as rat basophilic leukemia (RBL) cells have receptors that have exceptionally high affinity for immunoglobulin E (IgE). When aggregated, these receptors induce cellular degranulation. The alpha chain of the receptor contains the binding site for IgE; the function(s) of the noncovalently associated beta and gamma chains is (are) still undefined. Using a cDNA library constructed from the mRNA of RBL cells, we have isolated a cDNA clone whose sequence predicts a putative 23-residue signal peptide, followed by a sequence that accurately predicts the amino acid composition, the peptide molecular weight, and six peptide sequences (encompassing 59 residues or 26% of the total number) determined for the alpha chain by direct analysis. These findings provide strong evidence that the cDNA codes for the alpha chain, even though expression has not been unambiguously achieved. The sequence suggests that the alpha chain contains a 180-residue extracellular portion with two homologous domains of approximately 35 residues, a 20-residue transmembrane segment containing an aspartic acid, and a 27-residue cytoplasmic portion containing 9 basic amino acids. The sequence shows no homology with the low-affinity receptor for IgE from lymphocytes but over 30% homology with an Fc gamma receptor.

Further characterization of the subunits of the receptor with high affinity for immunoglobulin E.

The alpha, beta, and gamma subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the alpha chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for alpha and beta. However, these putative homologies were not apparent when tryptic maps of the biosynthetically ([3H]leucine) labeled subunits were analyzed.

The receptor with high affinity for immunoglobulin E.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read More

The role of soluble protein a in chicken spleen cell activation

Soluble protein A (SpA) caused chicken spleen cell proliferation despite considerable evidence that SpA has no binding affinity for avian immunoglobulin (Ig). This biological activity was examined by several approaches. SpA-stimulated spleen cell cultures demonstrated no difference in proliferative responses regardless of the addition of human gamma globulin (HGG) or keyhole limpet hemocyanin. Adsorbents composed of HGG or hemoglobin were equally ineffective in abrogating the ability of SpA to induce spleen cell proliferation. In addition, ion exchange purified SpA was observed to induce comparable levels of spleen cell proliferation as ion exchange-affinity purified preparations. The lymphocyte subpopulation responsible for the observed proliferative response to SpA resides in the nylon wool adherent population. Nylon wool non-adherent lymphocytes failed to proliferate to SpA, but did proliferate in response to phytohemagglutinin as did nylon wool adherent cells. These data indicate that SpA is not responsible for the observed biological activity and suggest that components from Staphylococcus aureus other than SpA copurify during the preparation of SpA and are responsible for the activation of spleen cells.

The Receptor with High Affinity for Immunoglobulin E

The Receptor with High Affinity for Immunoglobulin E

Thiophilic adsorption - a new method for protein fractionation

Divinylsulphone-activated agarose to which mercaptoethanol is coupled showed very selective group adsorption of human serum proteins, in particular the immunoglobulins. The adsorption increases markedly in the presence of high concentrations of neutral water-structure forming salts and is distinct from adsorptions based on hydrophobic interaction. A characteristic feature of this new type of adsorbent is the structure of the groups attached to the polymer, XXX, i.e., R-S-CH 2-CH 2-SO 2-CH 2-CH 2-O-XXX, where R is a small aliphatic residue. Our results indicate that the thioether sulphur and the adjacent sulphone group act cooperatively and are apparently necessary to maintain the distinct behaviour of such absorbents. Divinylsulfone Immunoglobulin Affinity chromatography Hydrophobic interaction (salting-out) Serum fractionation

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.

Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies.

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q--immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15--0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9--12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338--344].

Affinity maturation of NZB and BALB/cV mice anti-fluorescyl response

Immunoglobulin affinity maturation of the anti-fluorescyl response was assessed with time and compared in groups (12 weeks old) of New Zealand Black (NZB) and BALB/cV mice, following intraperitoneal immunization with 100 μg of F(I) 220 KLH per 15g of body weight. Affinity measurements were based on the quantitation of fluorescence quenching of fluorescein when specifically bound by immunoglobulin, and were expressed as relative association constants ( Krs). Although Kr varied significantly within each group of mice, quantitative and qualitative differences in the pattern of affinity maturation between the two strains of mice were evident. NZB mice demonstrated a relatively more rapid increase in Kr and attained a higher average Kr for the primary response than BALB/cV mice. Thus, the NZB primary response resembled the BALB/cV secondary response kinetically and in the extent of affinity maturation. Secondary immunization of NZB mice did not result in an increase in affinity relative to the primary response. In contrast, reimmunization of BALB/cV mice produced a secondary anti-fluorescyl response of significantly higher affinity than the primary one. Comparison of affinity maturation patterns between the two mouse strains indicated a significant difference in responding lymphocyte populations. Since T s activity in young NZB mice is known to be defective, a possible role for T s function in the process of affinity maturation is discussed.

Immunoglobulin characterization by bacterial absorption of antibrain antibodies in multiple sclerosis.

Complement-fixing (CF) antibrain antibodies are frequently found in serum and CSF in multiple sclerosis (MS). They represent several specificities and appear to be synthesized on both sides of the blood-brain barrier. Five sera and six CSF samples from 11 MS patients, representing 6 different specificities of antibodies, were absorbed with a series of bacterial strains with affinity for various immunoglobulins. Reactivity with brain preparations was eliminated by absorption with the following IgG absorbents: Staphylococcus aureus strain Cowan I, group A streptococcus strain A R1, and group G streptococcus strain G 148, but not by absorption with strains with low or no affinity for IgG. The results indicate in all tested samples the IgG1 and/or IgG2 nature of the antibrain antibodies.

Antigen-Binding Lymphocytes in Guinea Pigs

Abstract The monofunctional antigen L-tyrosine-p-azophenyltrimethylammonium chloride, tyr(TMA), and the polyfunctional antigen, TMA-human γ-globulin (TMA-HGG), were used to investigate the antigen structural requirements necessary for clonal proliferation of B cells. This clonal expansion was characterized with respect to receptor immunoglobulin class and affinity maturation. Antigen-binding analysis revealed that inoculation of tyr(TMA), although only of m.w. 344, triggers clonal expansion of B lymphocytes 9-fold in the absence of any apparent antibody production. There does not appear to be any maturation with respect to antibody class since τ;90% of the tyr(TMA)-specific B cells bear the μ receptor in the nonimmune and immune state. However, the average avidity of the B cells for this antigen increases with time after immunization. In contrast, immunization with TMA-HGG results in an 18-fold increase in B lymphocytes with significant amounts of anti-TMA antibody production. With time after immunization, both maturation of average avidity and class of Ig receptor (μ → γ shift) occur. These findings indicate that the functionally T cell-specific antigen tyr(TMA) can trigger clonal B cell expansion and affinity maturation at the receptor level in the absence of detectable antibody production.

A study on the affinity of immunoglobulins from LDH anomaly for five human LDH isoehzymes

A study on the affinity of immunoglobulins from LDH anomaly for five human LDH isoehzymes