Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.
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The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo. (+info)
Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H.
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A unique monoclonal Ig lambda light chain dimer (protein LOI) was isolated from the serum and urine of a patient with hypocomplementemic membranoproliferative glomerulonephritis. In vitro the lambda light chain dimer efficiently activated the alternative pathway of complement (AP). When added to normal human serum, LOI temporarily enhanced AP hemolytic activity, but during a prolonged incubation the hemolytic activity was depleted. Protein LOI was found to bind to factor H, the main regulator molecule of AP. By binding to the short consensus repeat domain 3 of factor H, the dimer LOI blocked one of three interaction sites between H and C3b and thus inhibited the activity of H and induced an uncontrolled activation of the AP. Structural analysis showed that LOI belonged to the Vlambda3a subgroup of lambda light chains. The variable (V) region of LOI was most closely related to the predicted product of the Vlambda3 germline gene Iglv3s2, although it contained several unique residues that in a tertiary homology model structure form an unusual ring of charged residues around a hydrophobic groove in the putative Ag binding site. This site fitted considerably well with a putative binding site in the molecular model of domain 3 of factor H containing a reciprocal ring of charged amino acids around a hydrophobic area. Apparently, functional blocking of factor H by the Ab fragment-like lambda light chain dimer had initiated the development of a severe form of membranoproliferative glomerulonephritis. Thus, the lambda light chain dimer LOI represents the first described pathogenic miniautoantibody in human disease. (+info)
Isolation, characterization and sequence analysis of five IgG monoclonal anti-beta 2-glycoprotein-1 and anti-prothrombin antigen-binding fragments generated by phage display.
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We have isolated five monoclonal IgG anti-beta 2-glycoprotein-1 (anti-beta 2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta 2GP-1-enriched mAbs (B14, B22, B27) reacted with beta 2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with beta 2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for beta 2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between beta 2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to beta 2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta 2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta 2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients. (+info)
Peripheral CD4+ T cell maturation recognized by increased expression of Thy-1/CD90 bearing the 6C10 carbohydrate epitope.
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The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40-80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1- variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10- naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope. (+info)
Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.
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Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD. (+info)
Ly-49CB6 NK inhibitory receptor recognizes peptide-receptive H-2Kb.
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NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide. (+info)
Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen.
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Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects. (+info)
Concanavalin A receptors on the surface membrane of lymphocytes from patient's with Hodgkin's disease and other malignant lymphomas.
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Concanavalin A (Con A) induces movement of its receptors on the cell surface membrane. This induction results in a concentration of Con A site complexes on one pole of the cell to form a cap. A marked difference was found in the mobility of Con A receptor between lymphocytes from normal persons and lymphocytes from patients with Hodgkin's disease and other malignant lymphomas. Lymphocytes isolated from tonsils of patients undergoing tonsillectomy and from axillary lymph nodes of breast cancer patients exhibited approximately 30% of cells with caps, which is identical with the cap formation ability of normal lymphocytes. In biopsy material from patients with Hodgkin's disease and other malignant lymphomas, a significant decrease in the ability of the lymphocytes to form caps was observed. This difference in the mobility of Con A sites was even more pronounced in lymphocytes isolated from the peripheral blood. In 123 patients with Hodgkin's disease and other malignant lymphomas, cap formation ranged between 3 and 12%. The ability of cells, from a normal donor or a lymphoma patient, to form caps was independent of the source from which the lymphocytes were isolated, e.g., lymph node, spleen, or blood. Lymphocytes from patients with lymphoma were also agglutinated by Con A to a higher degree than normal lymphocytes. These findings are discussed in relation to the association of the lymphocytes with these malignancies and as a possible aid in their differential diagnosis. (+info)