Studies on Complement-Fixation Reaction in Equine Infectious Anemia

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

Studies on complement-fixation reaction in equine infectious anemia. II. Identification of complement-fixing inhibitors.

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors

Polysulfone membrane is used for the first time for the preparation of electrochemical immunosensors. A disposable immunosensor based on a porous conductor polymer graphite-polysulfone-electrode has been developed using a phase inversion technique for the determination of anti-rabbit IgG (anti-RIgG) as a model analyte. To construct the sensor, a conductor membrane was deposited on the surface of working graphite-epoxy composite (GEC) electrode. The membrane was characterized by SEM. This sensor was based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). Incubation parameters were optimized in this work. The immunological reaction was detected using an enzymatic-labeling procedure (HRP enzyme) combined with the amperometric detection using H(2)O(2) as substrate and hydroquinone as mediator. This sensor shows stability during a week and a good reproducibility. The current was monitored amperometrically at -0.1 V versus SCE and this method showed a linear range of the anti-RIgG from 1 to 6 microg/ml. The detection limit was determined to be 0.77 microg/ml.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

鼻咽癌患者血清中抗Epstein-Barr病毒早期和晚期膜抗原抗体的检测

对Epstein-Barr(EB)病毒抗原的研究,发现有淋巴细胞确定的膜抗原(Lydma)、早期抗原(EA)、壳抗原(VCA)、核抗原(EBNA)、早期膜抗原(EMA)和晚期膜抗原(LMA)。除了Lydma抗原外,鼻咽癌患者对上述抗原均产生相应的IgG和IgA抗体。因而研究这些抗体,对阐明EB病毒与鼻咽癌的关系及鼻咽癌的早期诊断都十分有价值。     

Rates and impact of human antibody glycation in vivo

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.     

The antibody response to the T1699 murine adenocarcinoma: antibody class and subclass heterogeneity detected in serum and in situ.

We have used indirect immunofluorescence to study antibody responses directed against membrane antigens expressed on in vitro and in vivo T1699 mammary adenocarcinoma cells. IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM antibodies were present in the serum of DBA/2 mice bearing T1699 tumors; IgG2a and IgG2b antibodies were readily detected on the cells in situ. Lesser amounts of the other classes and subclasses could be detected by indirect immunofluorescence measurements on in vivo tumor cells and with low pH eluates of in vivo cells tested on the in vitro line of T1699. The antigenic determinants on in situ tumor cells are not saturated with antibody as these cells demonstrated enhanced fluorescence of all immunoglobulin classes and subclasses when treated with autologous serum. Experiments with thymus-depleted mice indicated that immunoglobulin production was strongly dependent on thymus-derived cells for all immunoglobulin classes and subclasses except IgG2b. Our studies suggest that IgG2a may be active in the macrophage-mediated cytotoxic reaction and IgG2b in the immediate hypersensitivity reaction to T1699 cells. These results provide further evidence for an active role of tumor-specific antibody in the host defense to the T1699 adenocarcinoma in situ.     

Immunoassay Using Microfluid Filters Constructed by Deep X-Ray Lithography

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (φ40 μm) in 200 μm-thickness polymethylmethacrylate (PMMA) sheets (φ3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunogloblin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0–100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.     

Immunoassay using microfluid filters constructed by deep X-ray lithography

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.     

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG₁ (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS or Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m² histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 1 of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

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.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Clinical effect and some pathogenic mechanisms of high dose monomeric IgG therapy in childhood chronic idiopathic thrombocytopenic purpura

8 children with chronic idiopathic thrombocytopenic purpura received a high dose therapy with monomeric IgG. Before and after the treatment immunological investigations were carried out. All the children showed a positive clinical response, in 5 children there was an increase of thrombocytes to 111-340 X 10(9)/l. A clinico-haematological effect could be shown in those children with an increased percentage of marrow megakaryocytes with IgG fixed on the membrane before treatment. There was no haematological effect neither in cases with fixed IgG and IgA nor IgM and IgA combined, as well as in the case when there was no fixed IgG on the membrane. A steady clinical effect was provided if the number of bone marrow megakaryocytes with fixed IgG reached the norm. The suppression of the synthesis of antithrombocytic antibodies of the same immunoglobulin class can be regarded as a specific mechanism of high IgG doses.     

Protein A immunoaffinity hollow fiber membranes for immunoglobulin G purification: experimental characterization

Immunoaffinity adsorption is increasingly used for protein purification and medical applications. Synthetic membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. The goal of this study was to explore in detail the performance of microporous hollow fibers composed of modified polysulfone to which protein A was immobilized for adsorption of human IgG. The internal matrix was characterized by scanning electron microscopy. The binding equilibrium constant was measured using both static and dynamic methods. Break-through curves up to ligand saturation were measured and used to study the effects of IgG concentration, presence of contaminant albumin, flow direction, flow mode, and especially filtrate flow rate and maximum IgG binding capacity. The highest binding capacities studied were comparable with that attainable with bead matrices. All of the breakthrough curves could be represented on a single figure when plotted versus the dimensionless relative throughput (the mass of IgG loaded on the membrane divided by the mass that would be bound when the entire fiber is in equilibrium with the feed concentration), and the effect of operating variables on the position and shape of the individual breakthrough curves could be understood in terms of a dimensional performance parameter (the product of membrane volume and maximum binding capacity divided by the filtrate flow rate). The best breakthrough curves were obtained with the highest values of the performance parameter. Based on the results, membranes as solid supports for immunoadsorption can be a useful alternative to the use of traditional columns for protein separations. (c) 1995 John Wiley & Sons, Inc.     

Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification

Microporous membranes are an attractive alternative to circumvent the typical drawbacks associated to bead-based chromatography. In particular, the present work intends to evaluate different affinity membranes for antibody capture, to be used as an alternative to Protein A resins. To this aim, two Mimetic Ligands? A2P and B14, were coupled onto different epoxide and azide group activated membrane supports using different spacer arms and immobilization chemistries. The spacer chemistries investigated were 1,2-diaminoethane (2LP), 3,6-dioxa-1,8-octanedithiol (DES) and [1,2,3] triazole (TRZ). These new mimetic membrane materials were investigated by static and by dynamic binding capacity studies, using pure polyclonal human immunoglobulin G (IgG) solutions as well as a real cell culture supernatant containing monoclonal IgG(1). The best results were obtained by combining the new B14 ligand with a TRZ-spacer and an improved Epoxy 2 membrane support material. The new B14-TRZ-Epoxy 2 membrane adsorbent provided binding capacities of approximately 3.1mg/mL, besides (i) a good selectivity towards IgG, (ii) high IgG recoveries of above 90%, (iii) a high Pluronic-F68 tolerance and (iv) no B14-ligand leakage under harsh cleaning-in-place conditions (0.6M sodium hydroxide). Furthermore, foreseeable improvements in binding capacity will promote the implementation of membrane adsorbers in antibody manufacturing.     

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH‐20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm‐zona binding in other species. Anti‐macaque PH‐20 IgG, anti‐pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD‐46, which is also located on the inner acrosomal membrane, but has no known function in sperm‐zona pellucida interaction. After labeling with anti‐acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH‐20 and CD‐46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti‐acrosin IgG nor anti‐CD‐46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti‐PH‐20 IgG (100 μg/ml) was present during sperm‐oocyte interaction. Ultrastructural observations of oocytes incubated with anti‐PH‐20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti‐PH‐20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm‐zona binding, rather than primary sperm‐zona binding or the zona‐induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 Wiley‐Liss, Inc.     

Rabbit peroxidase-antiperoxidase complex (PAP) as a model for the uptake of immunoglobulin G by the human placenta

Summary Rabbit peroxidase-antiperoxidase complex (PAP) has been shown to bind to IgG receptors on the human placental syncytiotrophoblast microvillar membrane. Its binding characteristics suggest that it is suitable as a probe for studies on the uptake of IgG by the human placenta.A novel assay system was developed to measure the dissociation constants (K _d) of the binding of PAP and of unlabelled human IgG to purified placental microvillar membranes. TheK _d for PAP was found to be 54 nM, while that for unlabelled IgG was found to be 17.5 nM.The uptake of PAP by placental tissue slices was observed using peroxidase histochemistry and electron microscopy. In initial experiments, reaction product was confined to the peripheral regions of the syncytiotrophoblast. Assaying a placental homogenate for catalase activity showed that it contained 250 units of activity per g wet weight of tissue (compared with 680 units/g for rat liver). Treatment of fixed tissue with the catalase inhibitor 3-amino-1, 2, 4-triazole allowed the localization of peroxidase reaction product in deeper regions of the syncytiotrophoblast. Based on observations of the localization of reaction product, we propose that PAP is taken up in coated pits, transferred into large apical multivesicular bodies, segregated into small vesicles which then transport it to the Golgi. From here the PAP is directed to the basal membrane by a mechanism as yet unknown.     

Fate of plasma membrane during endocytosis. I. Uptake and processing of anti-plasma membrane and control immunoglobulins by cultured fibroblasts

The uptake and processing by cultured rat embryo fibroblasts of control rabbit immunoglobulins (C IgG) or IgG directed against plasma membrane constituents (anti-PM IgG), and labeled with fluorescein (F) or with radioactive acetate (A), have been investigated by cell fractionation and immunological techniques. Both F and A anti-PM IgGs become bound to the cell surface, by a process that is slow, but largely temperature-independent. In the presence of an excess of high-affinity antibodies, binding reaches an absolute limit which corresponds to extensive coating of the plasma membrane. The anti-PM IgGs remain attached to the membrane for at least several days, even at 37 degrees C, with no significant transfer to lysosomes or degradation. In contrast, C IgGs are handled very differently by the fibroblasts, and their fate is strikingly affected by the type of labeling used. AC IgG is taken up slowly, at a rate proportional to its concentration, and is subsequently broken down in what appears to be lysosomes. Part of the AC IgG also binds to the plasma membrane. FC IgG is taken up many times faster than AC IgG, though with the same strict linearity as a function of concentration. Most of the FC IgG taken up is stored in cytoplasmic granules which behave like lysosomes. For reasons that are not understood, only about half of the stored FC IgG can be broken down. Cells exposed simulatnaously to AC IgG and FC IgG, or to A anti-PM IgG and FC IgG, handle each type of IgG in its characteristic fashion. Kinetic analysis of these results indicates that Ac IgG could be taken up by fluid endocytosis, but that FC IgG must be interiorized by a selective mechanism, presumably adsorptive in nature. That anti-PM antibodies remain stably bound to the plasma membrane and do not interfere with the uptake of FC IgG is interpreted to indicate either that two distinct membrane domains are involved in the two phenomena, or that membrane patches coated with anti-PM IgG participate in endocytosis, and are recycled back to the cell surface after delivering their contents intracellularly.