Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose? solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20 mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1 ± 0.25 mg Fab/ml moist resin displaying an affinity constant K_d = (2.6 ± 0.3) × 10^?6 M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH₁-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.     

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.     

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

High affinity binding of immunoglobulin G fragments to human polymorphonuclear leukocytes

Human neutrophil elastase splits IgG into Fc, Fabc, and Fab fragments. The Fc and Fabc fragments bind with high affinity (KD 2.1 and 2.5 nM respectively) to a small number of binding sites (1175 and 1370 sites/cell respectively) on untreated human polymorphonuclear leukocytes. Molecular mass determination of the binding site by crosslinking of Fc fragments to the neutrophils followed by SDS electrophoresis yields one band corresponding to a molecular mass of 67 kDa for the binding site. Incubation of neutrophils with rIFN-gamma (50 ng/ml, 18 h, 37 degrees C) enhances the expression of binding sites by about 6 fold to about 14,500 sites/cell, while the binding affinity and the molecular mass of the ligand receptor complex remain constant. By comparison with known affinities of leukocyte Fc receptors it is concluded that IgG fragments bind to the high affinity FcRI receptor of human neutrophils.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.     

Specific recognition of a tetrahedral phosphonamidate transition state analogue group by a recombinant antibody Fab fragment

Summary In order to obtain antibodies able to catalyse a peptide synthesis, a naive combinatorial library of human Fab antibody fragments was screened with the phosphonamidate transition state analogue of the reaction. Several Fab fragments were able to bind the analogue. Competitive binding studies performed with molecules containing representative parts of the hapten showed that two Fabs were able to recognize specifically the tetrahedral phosphorus present in the hapten.     

Stability of DNA/anti-DNA complexes.

Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.     

人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面表达技术,获得人源和中性抗滩滩病毒汉滩型Ｇ１基因工程单克隆抗体Ｆａｂ段基因及其表达,并同时获得抗汉滩病毒核蛋白的Ｆａｂ抗体。从能综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中,提取了部细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法,用一组人ＩｇＧ Ｆａｂ基因特异性引物,从合成了ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因,将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３,ｄｎａｌｆ ｖｆ     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.     

Identification of a unique IgG Fc binding site in human intestinal epithelium

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine‐based affinity ligands

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine‐based ligands immobilized onto agarose‐derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2‐ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine‐based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.