IgG Immunoreactivity Research Articles

Effect of Storage Time on the Immunoreactivity of IgG Physically Adsorbed or Chemically Bound to Latex Beads

The main purpose of the present work is to check whether the immunoreactivity of IgG molecules, chemically bound or adsorbed on latex, shows alteration after 6 months of storage. For this purpose, agglutination tests of sensitized carboxylated beads, measuring the immunoreactivity by turbidimetric monitoring after 5 min of immunoaggregation reaction with human C-reactive protein in a stopped-flow spectrophotometer, were repeated. The results show that while the covalently bound IgG maintains and in some cases improves on its previous immunological response, the passively adsorbed IgG shows loss of activity due to a decreased amount of adsorbed protein.

Clinical and immunohistochemical comparison of in vivo injected anti‐Hu and control IgG in the nervous system of the mouse

Anti‐Hu antibodies are usually present in patients with paraneoplastic encephalomyelitis/sensory neuropathy (PEM/SN), and anti‐Hu IgG is found in the nucleus of neurons in the autopsy of these patients. To investigate the clinical effect and distribution of anti‐Hu IgG in the nervous system in an experimental animal model, we injected intraperitoneally anti‐Hu IgG from five patients with PEM/SN to mice daily for 1 or 2 weeks. The IgG distribution in the nervous system was analyzed by an avidin‐biotin immunoperoxidase technique in animals sacrificed 24 h after the last injection. In one group of mice the nervous system was fixed by perfusion and in another (autopsy group) by immersion after keeping the dead animal 16 h at 4°C. None of the mice showed clinical or pathological abnormalities. IgG immunoreactivity was similar in the nervous system of mice injected with anti‐Hu or control IgG. In the perfusion‐fixed mice, IgG was present in leptomeninges, choroid plexus and extracellular space of Gasserian and dorsal root ganglia (DRG). In the autopsy group, there was IgG immunoreactivity in the cytoplasm of neurons of many areas of the brain and in more than 90% of neurons of DRG. Neuronal nuclear IgG deposits were only rarely observed. We conclude that anti‐Hu antibodies alone probably are not responsible for the PEM/SN syndrome. IgG diffusion into the cytoplasm of neurons is a post‐mortem artifact, but this model did not reproduce the predominant nuclear IgG staining observed in autopsies from PEM/SN patients.

Impact of the high tyrosine fraction in complementarity determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity

Although iodine-131 is the most widely used radionuclide for radioimmunotherapy, direct radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. We have studied the amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementarity determining regions (CDR), in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. In contrast, lysine residues that can be used for ligand attachment were evenly distributed throughout the IgG. HuM195 was first trace labeled with 111In and then labeled with stable 127I at various specific activities. The immunoreactivity of each product was determined using the 111In tracer. The immunoreactivity measured after varying levels of iodination fit a theoretical curve that was generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Similar theoretical curves for antibody fragments (F ab, F v) suggest an even faster decrease in immunoreactivity with increasing iodination. A review of the sequences of other therapeutic IgG shows that a similar overabundance of tyrosine residues is found in the CDRs. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The common abundance of tyrosine in the CDR of IgG and its correlation with loss of function have important implications for therapeutic use of high specific activity radioiodinated monoclonal antibodies or fragments.

The findings indicate that HSP70 expression is closely related to ischemic brain injury, and that HSP70 expression is not related to either breakdown of the blood-brain barrier or reactive axonal changes

The relationship between the 70-kilodalton hear shock protein (HSP70), morphological changes in neurons, protein extravasation, and reactive axonal changes was evaluated in rats with fluid percussion injuries to the right side of the brain. (4.8-5.6 atm, 20 ms). From 1 hour to 2 weeks after injury (or sham injury), serial sections of the brains were immunostained with antibodies to HSP70, rat IgG, and 68-kilodalton neurofilament. Ischemic changes in neurons in the injured cortex were noted in samples taken from 6 to 48 hours after injury, and macroscopic hemorrhages were noted in the corpus callosum and external capsule. Reactive axonal swelling was observed in the brain stem in samples taken from 6 to 48 hours after injury. Immunostaining of HSP70 was positive in the injured cortex from 6 hours to 1 week after injury. Maximal immunostaining was observed by 48 hours after injury in the injured cortical layers II and III. The region of HSP70 expression at 24 and 48 hours postinjury was the same as the region of ischemic cell changes. There were discrepancies between the region of HSP70 expression and the regions of IgG immunoreactivity and reactive axonal changes. These findings indicate that HSP70 expression is closely related to ischemic brain injury, and that HSP70 expression is not related to either breakdown of the blood-brain barrier or reactive axonal changes.

Experimental immune-mediated motor neuron diseases: Models for human ALS

Amyotrophic lateral sclerosis is an idiopathic, utlimately fatal disease, clinically manifest as progressive weakness and spasticity, associated with the loss of motoneurons. Circumstantial evidence supports a role for autoimmune processes in the progression of this human disorder. Two immune-mediated animal models have been developed in our laboratory for motor neuron loss. Experimental autoimmune motor neuron disease is a lower motor syndrome induced in guinea pigs by the repeated injection of a purified bovine spinal motor neuron antigen. Affected animals demonstrate extremity weakness, associated with electromyographic and morphologic evidence of denervation, a loss of spinal cord motor neurons, high antibody titers against motor neurons, and localization of IgG immunoreactivity to the neuromuscular junction and motor neuron cytoplasm. Experimental autoimmune grey matter disease is a more acute and severe disorder involving both upper and lower motor neurons, induced in guinea pigs by inoculation of a bovine ventral spinal cord homogenate, in which scattered foci of denervation are observed in the motor cortex and ventral spinal cord. Similarities between these diseases and human ALS are reviewed.

Cochlear IgG in the C3H/ lpr autoimmune strain mouse

The inner ear of the C3H/ lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6–10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/ lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/ lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.

Characterization of the Immune Barrier in Human Olfactory Mucosa

Immunologic defense factors in the human olfactory mucosa were localized immunohistochemically. Olfactory epithelium was identified with an antiserum to olfactory marker protein, specific for olfactory receptor neurons. Constituents of the secretory immune system, including IgA, IgM, secretory component, and J chain, were localized in the acinar and duct cells of Bowman's glands and in the mucociliary complex. In addition, B lymphocytes in the lamina propria near Bowman's glands displayed immunoreactivity for IgA, IgM, and J chain. Immunostaining also localized other humoral factors. Immunoreactivity for IgG was present throughout the stroma and in B lymphocytes in the lamina propria. Antibody to IgD stained numerous B lymphocytes clustered below the basement membrane. Antibody to IgE stained similarly distributed cells; toluidine blue staining demonstrated that many were mast cells. In addition, antibodies to IgD and IgE stained occasional intraepithelial B lymphocytes or mast cells. Two antimicrobial proteins, lactoferrin and lysozyme, were localized in Bowman's glands and the mucociliary complex. Thus, the human olfactory mucosa, which provides a direct neural route for pathogens to the brain, is a site for synthesis and secretion of immune and other defense factors.

Analytical control procedures of immunoreactivity for IgG and Fab fragments specific to haptens.

This study investigates immunoreactivity control procedures, i.e., specificity, affinity constant (Ka), and specific active binding sites (SABS), for polyclonal anticolchicine, monoclonal antidigitoxin IgG and Fab fragments, and antidigoxin Fab fragments (Digidot). Preliminary control procedures for IgG and Fab fragment purity indicated that all reagents were immunologically pure. All IgG and Fab fragments exhibited similar cross-reactivity and Ka. No decrease in percentage of Fab fragment SABS was observed after papain cleavage of anticolchicine and antidigitoxin IgG. Nevertheless, only 4.3 +/- 1.2% of nonimmunopurified anticolchicine polyclonal Fab fragments and 76.2 +/- 2.3 to 88.7 +/- 2.5% of different batches of immunopurified anti-digoxin Fab (Digidot) were active, the latter percentage being in the range of the 85% specified by the manufacturer. Only 58 +/- 3% of digitoxin-specific monoclonal IgG was active and 67 +/- 7% of its Fab fragments. Results show the importance of determining the ratio of SABS to presumed total specific binding sites for pharmaceutical monoclonal and polyclonal antibody preparations against haptens.

Fetal neocortical transplants in rats: blood-brain barrier development and partial permeability to IgG in long-surviving grafts

In previous work, allografts of fetal (E13-16) neocortical tissue transplanted into neocortical lesion cavities in newborn (PND 0-1) rats developed an impermeable blood-brain barrier (BBB) as shown by intravascular administration of horseradish peroxidase and by immunohistochemical staining for endogenous IgG. The present study examines the time course for the formation of the graft BBB by staining for endogenous IgG and also looks at transplants with extended survival times of 1-2 years. At two weeks post-transplantation, the grafts of all ten animals of this group showed evidence of IgG immunoreactivity within the graft parenchyma. This was greatest at the pial surface and adjacent to the ventricular surface of the transplant. By three weeks after transplantation, only four of nine grafts showed graft vessel permeability and this was confined to the area under the pia. At four weeks survival, one of nine grafts showed a small rostral patch of IgG reactivity, and a second animal showed very light, diffuse transplant labeling. The remaining seven animals were devoid of transplant IgG reactivity. At 11.5-28 months, three out of seven grafts had reaction product in the graft, indicating BBB permeability to IgG. In two of these older transplants, permeability was confined to the area around larger blood vessels, while one additional animal (28 months) showed dense labeling immediately below the pial surface. As in normal rats, host brain labeling was only found in circumventricular organs. These results show that circulating macromolecules are excluded from most CNS grafts within the first month of transplantation. This process progresses from the center of grafts and requires the longest time to complete in subpial regions possibly associated with healing of the pia. Some older grafts show leakage of protein which may relate to aging of the transplant or to a low-level graft rejection.

Effect of intraventricular injection of an anti-Purkinje cell antibody (anti-Yo) in a guinea pig model

Female guinea pigs had intraventricular injections of either IgG from a patient with parancoplastic cerebellar degeneration (PCD) and anti-Purkinje cell antibodies (anti-Yo IgG) or control IgG. In animals that received a single injection of control or anti-Yo IgG and were killed at different time intervals, IgG immunoreactivity was present in the cytoplasm of Purkinje cells at 2 h and persisted at 24 h. In guinea pigs injected for 15 days with control or anti-Yo IgG and sacrificed 24 h after the last injection, IgG was detected into the Purkinje cells in both groups, whereas animals killed 7 and 30 days after the last injection had no staining for IgG in the Purkinje cells. Clinical or pathologic evidence of cerebellar involvement was not seen in any of the animals. This study suggests the anti-Yo antibody alone may not be the cause of the Purkinje cell loss in PCD.

Detection of the anti‐Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy

We studied the nervous systems and tumors of five patients with anti-Hu-positive paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN) to determine if the autoantibody found in the serum and CSF was also present in those tissues. Immunohistochemical studies of the nervous system revealed the presence of IgG bound predominantly to the nuclei of most of the neurons and the cytoplasm of some glial cells. IgG was also present to a lesser degree in the neuropil. In brains of patients who died of cancer without the paraneoplastic syndrome, IgG was present in the immediate perivascular areas and to a very limited degree in the neuropil. There was no IgG in neurons, and in only some of the controls a few glial cells showed IgG immunoreactivity in the cytoplasm. The amount of anti-Hu IgG relative to total IgG in various brain regions and tumor was determined by quantitative Western blot analysis. The proportion of anti-Hu IgG was greater in some areas of the brain and tumor than in serum and CSF. Control brains did not contain anti-Hu IgG. There was a limited correlation among (1) the principal clinical symptoms, (2) regions of major tissue injury, and (3) the quantitative anti-Hu IgG distribution. We conclude that although the role of the antibody in the pathogenesis of the disease is still uncertain, its specific localization in the nervous system and tumor suggests an immunologic etiology of this paraneoplastic syndrome.

Use of Patients' Sera for Immunoperoxidase Demonstration of Infectious Agents in Paraffin Sections

Using patients' sera diluted from 1:10 to 1:1,000 as the primary antibodies in indirect immunoperoxidase staining, the authors visualized a variety of infectious agents in formalin-fixed, paraffin-embedded tissue sections. The target lesions included 1) pyoderma caused by Staphylococcus aureus, 2) cryptococcal infection, 3) dermal sporotrichosis, 4) colon ulcer caused by amebic dysentery, 5) cutaneous leishmaniasis, and 6) chronic liver abscess containing ova of Ascaris lumbricoides. The infectious agents were clearly identified in the respective lesions. Paraffin sections of other kinds of infectious lesions served as controls to clarify the specificity of the immunostaining. While the sera of patients with bacterial and fungal infection showed a wide range of cross-reactivity against bacteria and/or fungi, those with parasitic infection exhibited a relatively good specificity for the pathogen. Almost no immunoreactivity of endogenous human IgG in the paraffin sections was demonstrated under the conditions of this study. This approach can be used in diagnostic pathology, particularly when specific heteroantisera or monoclonal antibodies are unavailable.

Evidence of Serum-Protein Leakage Across the Blood-Brain Barrier in the Acquired Immunodeficiency Syndrome

Lesions of the central nervous system (CNS) in 23 patients with the acquired immunodeficiency syndrome (AIDS) were compared with control cases for the presence of serum protein by immunoperoxidase staining. A sensitive immunostaining kit in conjunction with rabbit antisera to human serum, albumin, immunoglobulins and complement component 3c was used to demonstrate specific immunoreactivity in paraffin-embedded sections. Most AIDS patients with CNS lesions had serum protein immunoreactivity in some neurons, glial cells (including astrocytes), gliomesenchymal cell nodules, vascular endothelial cells, inflammatory cells or microvascular walls. Cases with the most necrosis tended to have the least immunostaining. Immunoreactivity for IgG and fixed complement in anatomically intact or morphologically altered neural cells may indicate cellular lesions and potential cellular necrosis beyond the damage indicated by routine studies alone. Part of this immunoreactivity might represent a humoral autoimmune response.

Immunoglobulin G, complement factor C3 and lymphocytes in proliferative intraocular disorders

This study examines a possible immunological contribution to the development of proliferative intraocular disorders (PID) with traction retinal detachment. We analysed 24 periretinal membranes and 35 vitreous aspirates from patients with idiopathic proliferative vitreoretinopathy (PVR), traumatic PVR, and proliferative diabetic retinopathy (PDR). Lymphocytes and complement factor C3 deposits could not be detected in any of the membrane specimens. IgG was present in all but one of the PVR membranes but in less than half of the PDR specimens and there to a lesser extent. The IgG immunoreactivity was not collocalized with macrophages but instead located to the extracellular matrix. The intravitreal levels of IgG (ELISA) and protein were elevated in PID but the range of these biochemical changes was so wide that there were no significant differences of the IgG levels between the single types of PID. Using electrophoresis and Western blotting, C3 was detected in normal and pathologic vitreous but smaller C3 fragments indicative of C3 breakdown were only found in PID.

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta

Endogenous immunoglobulin-G was localised in ultrathin frozen sections of human term placenta by use of an indirect immuno electron-histochemical methodology. Immunoreactivity of endogenous IgG to rabbit anti-human immunoglobulin-G antibody was visualised by use of protein-A--colloidal gold complex. Gold marked the syncytiotrophoblast in both coated and uncoated regions of the apical plasmalemma, in vesicles and multivesicular bodies, and in vesicles near the basal plasmalemma. Immunoreactivity was also seen in the interstitial space between the trophoblast and the fetal endothelial layer as well as in various types of vesicles within the endothelial cells. No immunoreactivity was seen in the intercellular clefts of the endothelium. The pattern of localisation observed is consistent with receptor-mediated uptake of immunoglobulin-G into the syncytiotrophoblast of the human placenta followed by release into the interstitial space and then vesicular transport through the endothelium.

Immunoelectron microscopic observation of membranous nephropathy in children using protein A-gold complex method

The ultrastructural localization of immunoglobulins (IgG, IgA, IgM), complement component (C3c), or fibrinogen-related antigen (FRA) was investigated on 5 biopsy samples from 5 children with idiopathic MN or lupus nephritis using protein A-gold (PAG) complex method. The immunoreactivity of IgG was essentially confined to the mesangial and subepithelial electron-dense deposit (EDD) in all of 5 children, and more intense staining of IgG was observed in 3 children with stage II of MN than those with stage I and stage III of MN. Double immunocytochemical staining showed the same distribution of both IgG and IgM or both IgG and FRA in subepithelial EDD in 2 of 5 children. These findings suggest that IgG deposits are associated with the formation of subepithelial EDD in MN, and raise the possibility that IgM and FRA deposits may result from entrapment and/or immunological reaction.