Immunoglobulin G Molecules Research Articles

Silicon surface modification with a mixed silanes layer to immobilize proteins for biosensor with imaging ellipsometry

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (MTES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.

Macroscopic-scale surface properties of streptavidin and their influence on aspecific interactions between streptavidin and dissolved biopolymers

To avoid aspecific attractions between carrier surfaces for either ligand or receptor molecules in, e.g., immunoassays, or kinetic rate constant measurements, it has long been established that a background consisting of an aspecific, very hydrophilic carrier surface is generally quite effective. However, it is not often realized that one achieves such a non-reactive background, even with electrostatically neutral materials, at the price of creating a strong (polar) hydrophilic repulsion between dissolved biopolymer (e.g., protein) molecules and the non-adsorbing carrier surface. To investigate the quantitative effects of this type of repulsion in systems involving streptavidin, the surface properties of a streptavidin-coated glass plate were determined by contact angle measurements, from which the aspecific, macroscopic-scale free energies of repulsion between a streptavidin-coated surface and dissolved proteins such as immunoglobulin-G (IgG), and human serum albumin (HSA), could be derived. Streptavidin, even at neutral pH (at which it has virtually no electric surface charge as determined by electrophoresis) is very hydrophilic and strongly repels both IgG and HSA molecules. At neutral pH, molecules such as IgG and HSA, in aqueous solution, cannot approach a streptavidin layer more closely than to approximately 3.0 nm, which suffices to prevent IgG or HSA from any aspecific adherence to the streptavidin layer (as determined by extended DLVO analysis). This aspecific repulsion however also has the (usually unsuspected) effect of causing a decreased specific attachment between ligand and receptor molecules. In addition, it decreases the measured kinetic on-rate constants, often by about two decimal orders of magnitude. However, once the surface-thermodynamic properties of all the aspecific (macroscopic-scale) and specific (microscopic-scale) entities, as well as the specific equilibrium binding constant are known, the real kinetic on-rate constant between just the ligand and the receptor determinants can be determined, yielding the value it would have if the measurement of that constant were unhindered by the repulsive interactions exerted by the background of hydrophilic carrier molecules.

Purification of immunoglobulin G from human plasma by metal‐chelate affinity chromatography

AbstractA new group of specific‐affinity beads with metal chelates as ligands and synthetic polymer beads as support matrices was prepared and tested. This study focused on developing metal‐chelated poly(hydroxyethyl methacrylate) (PHEMA) gel beads in a spherical form (100–150 μm in diameter) for the separation of immunoglobulin G (IgG) from human plasma. Crosslinked PHEMA gel beads were prepared by suspension polymerization. The metal‐complexing ligand L‐histidine was then immobilized by covalent binding onto these gel beads. Different transition‐metal ions, including Zn(II), Ni(II), Co(II), and Cu(II), were chelated on these gel beads. An elemental analysis of immobilized L‐histidine for nitrogen was estimated to be 401.9 μmol of L‐histidine/g of PHEMA. The nonspecific IgG adsorption onto plain PHEMA gel beads was negligible (ca. 0.17 mg/g). Higher adsorption values (up to 3.5 mg/g) were obtained in which L‐histidine‐immobilized PHEMA gel beads were used from aqueous solutions. A remarkable increase in the IgG adsorption capacities were achieved from human plasma with L‐histidine‐immobilized gel beads (up to 44.8 mg/g). Further increases in the IgG adsorption capacities of the metal‐chelated gel beads were observed when human plasma was used (up to 79.6 mg/g). The metal‐chelate affinity gel beads allowed the one‐step separation of IgG from human plasma. The recognition range of metal ions for surface histidines from human plasma followed this order: Cu(II) > Ni(II) > Zn(II) > Co(II). IgG molecules could repeatedly be adsorbed and desorbed with these metal‐chelated PHEMA gel beads without a noticeable loss in their IgG adsorption capacity. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1567–1572, 2003

Cleavage of antigen-bound immunoglobulin G by SpeB contributes to streptococcal persistence in opsonizing blood.

Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenic speB mutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenic speB derivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding.

Multi-analyte analysis system using an antibody-based biochip.

A multi-analyte detection system using a unique antibody (Ab) biochip is described. The Ab-based biochip, also referred to as the protein biochip, uses a sensor array based on a complementary metal oxide silicon (CMOS) integrated circuit. The Ab-biochip has a sampling platform of four-by-four microarrays of antibodies deposited onto a Nylon membrane substrate. The micro-arrayed antibodies can be interrogated simultaneously or sequentially using the biochip sensing array detector with the use of a diffractive optical element illuminating each antibody spot individually. The usefulness of the Ab biochip is illustrated by the measurements of immunoglobulin G (IgG) used as the model analyte system. The detection limit for Cy5-labeled IgG molecules was 13 pg.

Binding of Rheumatoid and Lupus Synovial Fluids and Sera-Derived Human IgG Rheumatoid Factor to Degalactosylated IgG

Background Altered glycosylation of immunoglobulin G (IgG) has been demonstrated in patients with rheumatoid arthritis (RA). Specificity of IgG rheumatoid factor (RF) in recognizing degalactosylated IgG (Fab) 2 and Fc was analyzed in the present study. Methods (Fab) 2 and Fc fragments were prepared from IgG of normal healthy subjects. Enzymes, including peptide-N-glycosidase (PNGase), neuraminidase, and β-1,4-galactosidase, were used to digest (Fab) 2 and Fc fragments. Binding capacity of IgG RF from patients with RA, systemic lupus erythematosus (SLE), and from healthy subjects to (Fab) 2 and Fc treated with glycosidase, was measured with immunoassay. Results Treating (Fab) 2 and Fc with PNGase significantly diminished their binding capacity to IgG RFs. Degalactosylated (Fab) 2 revealed higher affinity to IgG RF in all tested groups. Fc lacking terminal galactose on oligosaccharide chains showed elevated binding with synovial IgG RF of RA patients. However, lesser binding was observed in sera of patients with RA and SLE. Conclusions Our data suggest that IgG molecules containing less terminal galactose on their oligosaccharide moieties are preferentially recognized by IgG RF. Furthermore, IgG RF display alternative binding specificity to degalactosylated (Fab) 2 and Fc in synovial fluids and sera of RA patients.

Isolation of peroxisomal subpopulations from mouse liver by immune free-flow electrophoresis

Peroxisomes (PO) are a heterogeneous population of cell organelles which in mammals are most abundant in liver and kidney. Commonly, differential and density gradient centrifugation are used for their isolation which, however, give only rise to the so-called "heavy" PO with a buoyant density of 1.22-1.24 g/cm(3). Subpopulations other than the heavy PO which are also present in both of these tissues have escaped adequate purification because of their sedimentation characteristics which are close to those of other major organelles, in particular microsomes. Since the purification of these subpopulations has become an essential task in view of the putative importance of peroxisomal subpopulations in the biogenesis of this organelle, alternatives to density gradient centrifugation are required. Recently, we have introduced such a novel approach, named immune free flow electrophoresis (IFFE). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of immunoglobulin G (IgG) molecules. pH-values other than 8.0 proved to be less efficient, probably because IgG molecules only focus at pH 8.0 but are scattered at any other. Applying IFFE to heavy and light mitochondrial as well as microsomal fractions of rat liver not only regular PO (rho = 1.22-1.24 g/cm(3)) but also other subpopulations could be isolated. To substantiate the validity of this approach, we now have subfractionated mouse liver homogenates accordingly. Of the PO subpopulations collected, mainly that obtained from the heavy mitochondrial fraction differed in its composition of matrix and membrane proteins as revealed by immunoblotting. This is in line with the data reported on rat liver thus confirming the potential of IFFE in the isolation of distinct subpopulations of hepatic PO.

Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors.

Recombinant adeno-associated virus type 2 (rAAV2) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for some applications, because it reduces the specificity of the gene transfer. To overcome this limitation, we sought to create a versatile rAAV vector targeting system which would allow us to redirect rAAV binding to specific cell surface receptors by simple coupling of different ligands to its capsid. For this purpose, an immunoglobulin G (IgG) binding domain of protein A, Z34C, was inserted into the AAV2 capsid at amino acid position 587. The resulting AAV2-Z34C mutants could be packaged and purified to high titers and bound to IgG molecules. rAAV2-Z34C vectors coupled to antibodies against CD29 (beta(1)-integrin), CD117 (c-kit receptor), and CXCR4 specifically transduced distinct human hematopoietic cell lines. In marked contrast, no transduction was seen in the absence of antibodies or in the presence of specific blocking reagents. These results demonstrate for the first time that an immunoglobulin binding domain can be inserted into the AAV2 capsid and coupled to various antibodies, which mediate the retargeting of rAAV vectors to specific cell surface receptors.

Adsorption of Native and Hydrophobically Modified Human Immunoglobulin G on Polyethylene Solid Films: Specific Recognition of Adsorbed Layers

The in situ adsorption/desorption studies of 14C-labeled human immunoglobulin G (IgG) were performed at the polyethylene (PE)/aqueous solution interface. The results reveal that the adsorbed protein molecules were predominantly packed in the end-on orientation. Desorption experiments showed that upon rinsing with buffer only minute fractions of adsorbed IgG could be removed from the PE surface. Both the competitive and the sequential adsorption measurements from the binary mixtures of hydrophobic IgG modified by attachment of 19 caprylic chains (19C8-IgG) and [14C]IgG confirm that native IgG strongly adhered to the PE films. The specific recognition ability of adsorbed human IgG layers was studied by using specific (goat anti-human) IgG and nonspecific (goat anti-rabbit) IgG. It was shown that the prevailing effect of the interaction of [14C]IgG adsorbed onto PE with the specific antibody was the increase in the surface radioactivity, which was attributed to the formation of a more densely packed layer of IgG molecules at the PE surface. It was also demonstrated that, whereas the specific antibody preadsorbed onto PE films retained the recognition ability relative to human IgG, its hydrophobic modification resulted in a substantial decrease in this ability.

Native and Hydrophobically Modified Human Immunoglobulin G at the Air/Water Interface: Sequential and Competitive Adsorption

The adsorption of human immunoglobulin G (IgG) at the air/water interface was monitored both by the in situ radiotracer technique using [14C] labeled IgG and by surface tension measurements. The results reveal that adsorption of IgG from single protein systems displays bimodality due to molecular rearrangements at the interface. Above the threshold value of 1.5×10−2 mg/ml solution concentration, adsorbed IgG molecules reoriented from the side-on to the end-on configuration. The existence of a lag time which did not appear in Γ=f(t) curves, was observed in Π=f(t) relationships at low protein concentrations and was due to the limits of the surface pressure technique to detect protein adsorption. The adsorption of native IgG was also carried out in the presence of a hydrophobized IgG obtained by grafting capryloyl residues to its lysine groups by reaction with N-hydroxysuccinimide ester of caprylic acid, which yielded 19 covalently bound alkyl chains to the IgG molecule (19C8–IgG). This modified IgG exhibited enhanced adsorption at the air/water interface, as manifested by its increased adsorption efficiency relative to the native protein. Sequential and competitive adsorption experiments from binary mixtures of native IgG and 19C8–IgG clearly demonstrate that the displacement of the native protein from the air/water interface strongly depended on the manner of how 19C8–IgG and native IgG competed with each other. When the two proteins competed simultaneously, 19C8–IgG predominantly occupied the available area but when native IgG was adsorbed first, for 2 h, the sequentially adsorbed 19C8–IgG was incapable of substantially displacing it from the interface.

Structural basis of the interaction between immunoglobulins and Fc receptors provided by NMR spectroscopy

Fc gamma Receptors (Fc gamma R) are membrane glycoproteins that bind the Fc portion of immunoglobulin G (IgG). The cross linking of Fc gamma R-bound IgG by multivalent antigens allows clustering of the Fc gamma R and initiates a variety of effector mechanisms which play a key role in immune defenses against pathogens. The Fc region is composed of two identical polypeptide chains, which are related to each other by a two-fold axis. Recent elucidation of the crystal structure of human Fc gamma RII provided two distinct views of modes of IgG-Fc gamma R interactions, which is controversial against each other. Nuclear magnetic resonance (NMR) spectroscopy provides a unique and irreplaceable tool to solve these issues. We recently studied the interaction between the Fc fragment of mouse IgG2b and the extracellular domain of mouse Fc gamma RII by this method. We showed that Fc gamma RII binds to a negatively charged area of the CH2 domain, corresponding to the lower hinge region, and that the binding of Fc gamma RII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. This conformational change may account for the 1:1 stoichiometry that we and others observed between Fc gamma R and Fc. We therefore propose a model that explains why the interaction between IgG molecules and Fc gamma R does not trigger cellular responses in the absence of cross linking by multivalent antigens and does not lead to spontaneous inflammatory responses that would be deleterious for the organism.

Mediated efflux of IgG molecules from brain to blood across the blood–brain barrier

Dextrans and albumin exit brain from blood following intra-cerebral injection by a slow process of convection with a halftime of 10–12 h in the rat. The present studies show that immunoglobulin G (IgG) molecules rapidly efflux from brain to blood across the blood–brain barrier (BBB) following intracerebral injection. The IgG efflux is rapid with a halftime of 48 min in the rat. The efflux of [ 3H]mouse IgG 2a from brain to blood is competitively inhibited by intracerebral injection of unlabeled mouse IgG molecules, but is not inhibited by intracerebral injection of comparable doses of unlabeled rat albumin. The IgG efflux system has characteristics of an Fc receptor, as the efflux from brain is competitively inhibited by Fc fragments but is not blocked by F(ab′) 2 fragments. Precipitation of brain homogenate by trichloroacetic acid indicates there is no significant metabolism of the IgG molecules during the experimental time period. In conclusion, these studies provide evidence for a BBB Fc receptor that mediates the reverse transcytosis of IgG molecules in the direction of brain to blood.

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent.

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.

C-Terminal Labeling of Immunoglobulin G with a Cysteine Derivative by Carboxypeptidase Y Catalyzed Transpeptidation

We describe a new method for the site-specific incorporation of an extrinsic cysteine to the C-termini of immunoglobulin G (IgG) using carboxypeptidase Y (CPase Y) catalyzed transpeptidation. The transpeptidase activity of CPase Y was employed to attach cysteine esters to the C-termini of the IgG molecule (cysteinylation) at alkaline pH. No CPase Y catalyzed transpeptidation products were found when native IgG was used as the substrate or when cysteine was used as the nucleophile. However, C-terminal labeling occurred when cysteine ethyl ester (CysOEt) or cysteine isobutyl ester (CysOiBu) was used as the nucleophile and IgG methyl ester as the substrate. When CysOiBu was used as the nucleophile, the maximal labeling yield obtained with IgG methyl ester as substrate was 25%, assuming all four C-termini in the IgG molecule were labeled equally. The C-terminal labeling pattern of cysteinylated IgG was determined by autoradiography followed by the integration of radiodensity. It revealed that both the C-termini of the heavy and light chains of IgG methyl ester were labeled with CysOiBu.

Orientations of Liquid Crystals on Mechanically Rubbed Films of Bovine Serum Albumin: A Possible Substrate for Biomolecular Assays Based on Liquid Crystals

We report the uniform planar anchoring of thermotropic liquid crystals on films of bovine serum albumin (BSA) covalently immobilized on the surface of glass microscope slides and mechanically rubbed using a cloth. The azimuthal orientations of the liquid crystals were measured to be parallel to the direction of rubbing. Following immersion and removal of these rubbed films of BSA from aqueous solutions containing either BSA, fibrinogen, lysozyme, anti-FITC immunoglobulin G (IgG), or antistreptavidin IgG, we measured liquid crystals placed onto these surfaces to largely retain their uniform alignment. In contrast, following immersion of a rubbed film of BSA into an aqueous solution of anti-BSA IgG, we observed liquid crystals on these surfaces to assume nonuniform orientations. We conclude that specific binding of anti-BSA IgG to the film of rubbed BSA erased anisotropy induced within the film of BSA by rubbing. This result suggests that the spatial scale of anisotropy within the rubbed film of BSA is comparable to or smaller than the size of the IgG molecule. Because the anisotropy within a rubbed film of a protein can be erased by specific binding of a second protein, we believe these types of substrates (rubbed films of proteins) have the potential to be useful in a variety of label-free biomolecular assays where specific binding of a target species to its ligand can be imaged through observation of the optical appearance of liquid crystal placed onto the surface.

Influence of matrix activation and polymer coating on the purification of human IgG with protein A affinity membranes

Nylon microfiltration membranes were coated with dextrans ( M r≈6000 and 40,000) and poly(vinylalcohol) (PVA) ( M r≈72,000) to produce affinity matrices with adequate chemical functionality and low non-specific protein binding characteristics. In all cases, a considerable reduction of non-specific adsorption was attained, with PVA being most effective. Also, the permeability was reduced, indicating the presence of coating polymers in the flow-through pores. The extent of permeability reduction was related both to differences in the molar mass of dextran, as well as to polymer characteristics. The highest reduction was observed with the coil-forming high-molecular weight dextran. Protein A was immobilized to the different membranes for the adsorption of human immunoglobulin G (IgG). The highest apparent association constants were obtained with PVA-coated membranes, followed by membranes which were coated with low-molecular weight dextran, and then by membranes, which were coated twice with high-molecular weight dextran. It is concluded that multiple covalent binding of protein A occurs in the open network of dextran coils, restricting the accessibility of the various binding sites of this ligand for the large IgG molecule, and therefore, weakening the ligand–ligate association. With the film-forming PVA, a better accessibility was obtained with comparable ligand density to commercial protein A chromatography supports.

Urinary loss of immunoglobulin G anti-F(ab′) 2 and anti-DNA antibody in systemic lupus erythematosus nephritis

The objective of this study was to determine whether the low levels of serum immunoglobulin G (IgG) anti-F(ab′) 2 seen in some patients with active systemic lupus erythematosus (SLE) were directly related to the deposition of antibody with this specificity in the kidney or alternatively to the urinary loss of IgG anti-F(ab′) 2. Serum Levels of IgG anti-F(ab′) 2, anti-tetanus toxoid, and anti-ds DNA antibody were measured in parallel with urinary excretion of these same 3 antibodies in 28 patients with SLE nephritis and in 28 control patients with other forms of chronic kidney disease. Low levels of both serum IgG anti-F(ab′) 2 or anti-tetanus antibody appeared to correlate with increased levels of urinary loss of these same antibodies in some patients with SLE and in control subjects with kidney disease. However, urinary loss could not account for low serum levels of either IgG antibody in many subjects. Quantitative 24-hour urinary losses of IgG anti-F(ab′) 2 and anti-DNA were much higher in patients with SLE than in control subjects with kidney disease ( P < .05), whereas amounts of IgG urinary loss of anti-tetanus were similar in patients with SLE and in control subjects. In nearly 1 third of SLE nephritis patients, 13% to 53% of total excreted urinary IgG showed anti-DNA enzyme-linked-immunosorbent assay reactivity. Urinary IgG in many patients with SLE showed both anti-DNA and anti-F(ab′) 2 reactivity, but dual anti-DNA/F(ab′) 2 specificity was more pronounced in affinity-isolated serum IgG anti-DNA or anti-F(ab′) 2 than in excreted urinary IgG molecules. The affinity of urinary IgG for either DNA or F(ab′) 2 was much lower than the same antibody activities measured either in serum or in kidney biopsy eluates. When the relative affinity of anti-DNA antibody in serum, urine, and kidney biopsy eluate was measured in parallel, the highest affinity antibody was found in kidney biopsy eluates, followed by serum antibody with urine antibody affinity showing the lowest values. These findings suggest a relative concentration of the highest affinity, doubly reactive IgG anti-DNA/F(ab′) 2 in SLE kidney tissues during SLE nephritis and implicate this process as an important factor in ongoing tissue damage.

Covalent polymyxin B conjugate with human immunoglobulin G as an antiendotoxin reagent.

Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 microg of bound functional PMB per 100 microg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 microg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.

Protein-A-mediated targeting of bacteriochlorophyll-IgG to Staphylococcus aureus: a model for enhanced site-specific photocytotoxicity.

A model for studying the efficiency of photodynamic action with a photosensitizer placed exclusively on the bacterial cell wall has been used. Bacteriochlorophyllide molecules, conjugated to rabbit immunoglobulin G (IgG), were synthesized. The conjugated pigment bacteriochlorophyll (Bchl)-IgG bound with high specificity to protein-A residues naturally exposed on the cell wall of the bacterium Staphylococcus aureus Cowan I. In bacterial suspensions the phototoxicity of the targeted conjugates (0.5-2.5 pigment per IgG molecule) was dose dependent (LD50 = 1.7 microM) in the presence of light (lambda > 550 nm) and inhibited by native IgG but not by ovalbumin, suggesting selective interaction with protein-A on the bacterial cell wall. No dark toxicity was noticed even with the highest conjugate concentration tested. In contrast, the photocytotoxicity of bacteriochlorophyll-serine (Bchl-Ser, LD50 = 0.07 microM) used as a nontargeted control was not inhibited by IgG. In spite of its lower apparent potency, Bchl-IgG was found to be 30 times more efficacious than Bchl-Ser: At LD50, only 66,000 Bchl-IgG molecules were bound per bacterium compared to 1,900,000 molecules of Bchl-Ser. The higher efficacy of Bchl-IgG is explained by its exclusive position on the bacterial cell wall. Consequently, photogeneration of oxidative species is confined to the cell wall and its vicinity, a seemingly highly susceptible domain for photodynamic action. In considering the design of cell-specific sensitizers for bacterial and cancer therapies, it would be beneficial to identify the more discretely sensitive subcellular domains as targets.

Favourable interaction between heavy and light chains arrests the undesirable oligomerization of heavy chains in the refolding of denatured and reduced immunoglobulin G.

Recently we developed a slow dialysis method that effectively refolds denatured and reduced immunoglobulin G (IgG) [Maeda, Ueda and Imoto (1996) Prot. Engng 9: 95-100]. This method allows both individual and simultaneous refolding of denatured and reduced H and L chains. Analysis by SDS-polyacrylamide gel electrophoresis revealed that some oligomers were formed through disulfide bonds when H chains were refolded individually. It was also shown that the extent of IgG obtained by rejoining the mixture of refolded H and L chains which had been refolded individually was similar to that obtained by refolding denatured and reduced whole IgG. The results indicated that a favourable interaction between H and L chains prevented formation of H-chain oligomers to yield intact IgG. The present results suggest a mechanism whereby individually folded chains might associate to form IgG molecules in vivo.