A new approach to the depletion of albumin and immunoglobulin G from human serum

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients’ blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HSA and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography was obtained. The developed sorbents were used to prepare serum for proteomic analysis.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

Concerning the axial rotational flexibility of the Fab regions of immunoglobulin G

By electron microscopy, we have observed immunocomplexes with both negative stain and in amorphous ice using monoclonal antibodies directed against one of the 24 subunits of scorpion haemocyanin. A copy of this subunit occurs at each of the corners of the square-shaped haemocyanin molecule. Three distinct orientations of adjacent haemocyanin molecules may be observed in immunocomplex pairs or chains using both the above-mentioned methods. These observations, coupled with low-resolution computer simulations of immunocomplex formation, argue strongly in favour of the existence of a considerable degree of rotational flexibility within the IgG molecule and around the long axis of the Fab arms, as was suggested by previous observations with negative stain. We find that the arms can rotate by up to 180 degrees with respect to the Fc region.     

Ultrastructural localization of gamma-chain and immunoglobulin G in human lymphocytes using enzyme-labeled Fab fragment.

By the use of rabbit antibodies against the heavy chain of human immunoglobulin G (IgG), the gamma-chain and IgG molecules were successfully localized at the ultrastructural level in human peripheral lymphocytes. The rabbit Fab fragment was coupled to horseradish peroxidase by means of glutaraldehyde and the resulting conjugate could penetrate the intact plasma membrane. Discernible reaction product was observed in cisternae of the nuclear envelope, rough endoplasmic reticulum and Golgi apparatus as well as on the surface of the lymphocytes. In normal human individuals under no specific antigenic stimulation, only a few peripheral lymphocytes showed a rare positive intractoplasmic reaction. Reaction product may represent either the whole IgG molecule, the half molecule consisting of one heavy and one light chain or nascent gamma-chain.     

Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum

All four subclasses of human serum IgG contain a single N-glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core-fucosylated and partially truncated oligosaccharides, that may carry a bisecting N-acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N-glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N-glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N-glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass-specific analysis of IgG glycosylation from clinical samples.     

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.     

A micropreparation of mitochondria from cells using magnetic beads with immunoaffinity

Mitochondrial preparation is a key technique in the study of mitochondria. Growing evidence has demonstrated that mitochondrial proteins are tissue or cell type dependent. Locating the proteins in the global presence of mitochondrial membranes is a primary consideration in adopting antibodies for affinity enrichment of mitochondria on a micro scale. Two proteins located on the outer membrane of mitochondria, cytochrome b5 type B (CYB5B) and synaptojanin-2-binding protein (SYNJ2BP), were selected as candidates based on a survey of databases and the literature. The polyclonal antibodies against the truncated CYB5B and SYNJ2BP exhibited specific recognition to mitochondria and wider sensitivity to several tested mouse tissues and cell lines, whereas the antibody 22-kDa translocase of the outer mitochondrial membrane (TOM22) nearly missed detection of mitochondria in the liver and responded minimally to mitochondria from H9C2 and L-02 cells. Through the affinity enrichment for cellular mitochondria using magnetic beads coated with anti-CYB5B or anti-SYNJ2BP, we found that the anti-CYB5B beads could enrich mitochondria more efficiently even on a scale of 10,000 cultured cells. For the integrity and protein components, the enriched mitochondria on anti-CYB5B were carefully examined and were accepted in further functional study. We propose that an anti-CYB5B immunomagnetic approach is feasible in the micropreparation of mitochondria from cultured cells.     

Electron microscopic evidence for the axial rotation and inter-domain flexibility of the Fab regions of immunoglobulin G

The Fab arms of immunoglobulin G (IgG) have long been known to hinge about their joint with the Fc subunit. Using monoclonal antibodies bound to influenza haemagglutinin (HA) as position markers, we now show that these arms can also rotate about their long axis with respect to Fc. We also show that when two IgGs are bound cyclically with two HA molecules, the arms can bend between the variable and constant domains to accommodate bond angle constraint.     

Hemagglutinin 1-specific immunoglobulin G and Fab molecules mediate postattachment neutralization of influenza A virus by inhibition of an early fusion event

In standard neutralization (STAN), virus and antibody are reacted together before inoculation of target cells, and inhibition of almost any of the processes concerned in the early interaction of virus and cell, including inhibition of virus attachment to cell receptors, can be the cause of neutralization by a particular monoclonal antibody (MAb). To simplify the interpretation of antibody action, we carried out a study of postattachment neutralization (PAN), where virus is allowed to attach to target cells before neutralizing antibody is introduced. We used influenza virus A/PR/8/34 (H1N1) and monoclonal immunoglobulin G (IgG) molecules and their Fabs specific to antigenic sites Sb (tip), Ca2 (loop), and Cb (hinge) of the hemagglutinin 1 (HA1) protein. All IgGs and Fabs gave PAN, although with reduced efficiency compared with STAN. Thus, bivalent binding of antibody was not essential for PAN. By definition, none of these MAbs gave PAN by inhibiting virus attachment, and they did not elute attached virus from the target cell or inhibit endocytosis of virus. However, virus-cell fusion, as demonstrated by R18 fluorescence dequenching or hemolysis of red blood cells, was inhibited in direct proportion to neutralization and in a dose-dependent manner and was thus likely to be responsible for the observed neutralization. However, to get PAN, it was necessary to inhibit the activation of the prefusion intermediate, the earliest known form on the fusion pathway that is created when virus is incubated at pH 5 and 4 degrees C. PAN antibodies may act by binding HA trimers in contact with the cell and/or trimers in the immediate vicinity of the virus-cell contact point and so inhibit the recruitment of additional receptor-HA complexes.     

Synthesis of immunoglobulin conjugates with dehydrogenases

Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.     

Binding and activation of plasminogen on immobilized immunoglobulin G. Identification of the plasmin-derived Fab as the plasminogen-binding fragment

We have found that tissue plasminogen activator catalyzes the binding of plasminogen (Pg) to immunoglobulin G (IgG) immobilized on a surface. This enhancement is due to the formation of plasmin, since plasmin treatment of immobilized IgG produced a 20-fold increase in Pg binding. Pg binding is lysine site dependent and reversible. The augmentation of Pg binding by plasmin is specific as other proteases produced significantly less or no effect. Immobilized plasmin-treated IgG also specifically binds Pg in plasma. IgG-immobilized Pg is activated by tissue plasminogen activator, and a significant portion of the plasmin formed remains bound to the IgG. The Pg reactive species in a plasmin-treated IgG digest was identified as the Fab fragment by chromatography utilizing the immobilized high affinity lysine-binding site of plasminogen. Specificity of the interaction was further demonstrated by immunoblot-ligand analysis which demonstrated that the plasmin-derived Fab fragment bound Pg whereas papain-derived Fab or plasmin-derived Fc fragments did not. These data suggest that Pg binds to the new COOH-terminal lysine residue of the plasmin-derived Fab. Pg also binds to an immobilized immune complex following plasmin treatment. These findings indicate that surface-bound IgG localizes plasminogen thus extending the spectrum of activity of the plasmin system to immunologic reactions.     

Cleavage of human serum immunoglobulin G by an immobilized pepsin preparation

In order to obtain an efficacious and safe immunoglobulin G (IgG) preparation for intravenous use, the digestion of IgG with an immobilized pepsin (EC 3.4.23.1) preparation was studied. Thus, pepsin was immobilized onto glutaraldehyde-activated AH-Sepharose 4B under acidic conditions. THe enzymatic properties, such as proteolytic activity, pH-activity profile and heat stability, of the immobilized pepsin preparation were examined. The immobilized pepsin retained more than 40% of its proteolytic activity toward N-acetyl-L-phenylalanyl-L-3,5-diiodo-tyrosine and more than 30% toward IgG, and also remarkable stability as compared with free pepsin. The immobilized pepsin thus prepared was efficiently used for the limited cleavage of IgG and the gel-filtration effect of the column made it easily possible to yield the F(ab')2-rich fraction for intravenous use.     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Immunodepletion of albumin and immunoglobulin G from bovine plasma

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.     

Common peptides from the N-terminal half of heavy chain of immunoglobulin G from normal rabbit serum and a specific antibody

Fragment C-1, the N-terminal half of the heavy chain of rabbit immunoglobulin G, was prepared by cyanogen bromide cleavage from the heavy chain of immunoglobulin G obtained both from the pooled serum of normal rabbits and from specific anti-dinitrophenyl antibody. Tryptic digestion of fragment C-1 after the lysine residues had been allowed to react with S-ethyl trifluorothioacetate led to the isolation of six peptides from inert immunoglobulin G and specific antibody that appear to account for most of this section of the heavy chain. This approach should make possible comparative sequence studies of the Fd section of the heavy chain from different allotypes and from specific antibodies.