scholarly journals Studies on the antigenic determinants in the self-association of IgG rheumatoid factor

1981 ◽  
Vol 154 (1) ◽  
pp. 112-125 ◽  
Author(s):  
FA Nardella ◽  
DC Teller ◽  
M Mannik
Keyword(s):  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Igg Rheumatoid Factor ◽  
Normal Human ◽  
Self Association ◽  
Equilibrium Ultracentrifugation ◽  
Antigen Antibody

The number, location, and other characteristics of the antigenic determinants for self-association of IgG-rheumatoid factors (IgG-RF) were examined using the IgG-RF isolated from the plasma of one patient as a model system. Affinity chromatography was employed for isolation of the IgG-RF. Sedimentation equilibrium ultracentrifugation was used to study the various interactions. The antigenic valence of IgG-RF Fc, normal human Fc, and rabbit Fc fragments was two for the interaction with Fab fragments from IgG-RF, as might be expected from the molecular symmetry of IgG. The antigenic valence of intact normal IgG, however, was only one, indicating that when one of the available antigenic determinants interacted with the Fab fragment of IgG-RF, the other determinant becomes sterically inaccessible. Reduction and alkylation, known to increase the flexibility of the hinge region, did not alter the antigenic valence of IgG for Fab fragments of IgG-RF. The antigenic valence of IgG-RF in self-association could not be experimentally determined but must be two to permit the observed concentration-dependent further polymer formation of IgG-RF dimers. Unique antigenic determinants on the Fc fragments of IgG-RF were sought and not found, thus reaffirming the formation of two antigen-antibody bonds as the basis for dimerization of IgG-RF molecules. The pFc' and Fc' fragments, representing Cγ3 domains of IgG, failed to show significant interaction with Fab fragments of IgG-RF, indicating that the antigenic determinants were not expressed by the Cγ3 regions but are located either on Cγ2 region or require intact Cγ2 and Cγ3 regions for expression. These conclusions were corroborated by the antigenic valence of one for the Fc(i) fragment, a new papain-generated intermediate fragment of Fc, composed of two intact Cγ3 domains and one intact Cγ2 domain. Normal IgG, because of its valence of one for interaction with IgG-RF, would effectively terminate further polymerization of IgG-RF dimers. This may well in part explain the finding of smaller IgG-RF complexes in the serum than in synovial fluid of patients with rheumatoid arthritis.

Thrombocytopenia after second exposure to abciximab is caused by antibodies that recognize abciximab-coated platelets

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2054-2059 ◽  
Author(s):  
Brian R. Curtis ◽  
Julia Swyers ◽  
Ajit Divgi ◽  
Janice G. McFarland ◽  
Richard H. Aster
Keyword(s):  
Healthy Subjects ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Fibrinogen Receptor ◽  
Patients At Risk ◽  
Life Threatening ◽  
Normal Human ◽  
Normal Antibodies

Abstract Thrombocytopenia, often severe, occurs in 1% to 2% of patients given the fibrinogen receptor antagonist abciximab, a chimeric Fab fragment containing murine specificity-determining and human framework sequences. The cause of this complication has not yet been defined. Studies of 9 patients who developed profound thrombocytopenia (platelets <10 × 109/L [10 000/μL]) within a few hours of being given abciximab a second time showed that each had a strong immunoglobulin G (IgG) antibody that recognized platelets sensitized with abciximab. Five patients also had IgM antibodies. IgG antibodies reactive with abciximab-coated platelets were also found in 77 (74%) of 104 healthy subjects. However, the patient antibodies could be distinguished from “normal” ones in 2 ways: (1) only the patient antibodies reacted preferentially with platelets sensitized with the intact monoclonal antibody 7E3 from which the murine sequences in abciximab are derived; and (2) the “normal” antibodies could be inhibited by Fab fragments derived from normal human IgG, whereas the patient antibodies were relatively resistant to this treatment. The findings suggest that antibodies from the patients are specific for murine sequences in abciximab and are capable of causing life-threatening thrombocytopenia upon injection of this drug. The antibodies commonly found in healthy subjects are specific for the papain cleavage site of any Fab fragments and, although they react with abciximab-coated platelets, appear not to cause significant thrombocytopenia. It may be possible to identify patients at risk for developing thrombocytopenia if given abciximab by screening for antibodies that recognize 7E3-coated platelets.

scholarly journals Structure and interactions of cartilage proteoglycan binding region and link protein

1985 ◽  
Vol 228 (1) ◽  
pp. 77-85 ◽  
Author(s):  
F Bonnet ◽  
D G Dunham ◽  
T E Hardingham
Keyword(s):  
Polyclonal Antibodies ◽  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Gel Chromatography ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Lysine Residues ◽  
Proteoglycan Aggregates ◽  
Self Association

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.

Localization of Intracellular Antigens in thin Sections with Ferritin Labeled Antibody

1970 ◽  
Vol 28 ◽  
pp. 174-175
Author(s):  
M.S. Shahrabadi ◽  
T. Yamamoto
Keyword(s):  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Antigenic Determinants ◽  
Conjugate Prior ◽  
Thin Sections ◽  
Specific Attachment ◽  
Intracellular Antigen ◽  
Antigen Antibody ◽  
Ideal Method ◽  
The Ideal

The technique of labeling of macromolecules with ferritin conjugated antibody has been successfully used for extracellular antigen by means of staining the specimen with conjugate prior to fixation and embedding. However, the ideal method to determine the location of intracellular antigen would be to do the antigen-antibody reaction in thin sections. This technique contains inherent problems such as the destruction of antigenic determinants during fixation or embedding and the non-specific attachment of conjugate to the embedding media. Certain embedding media such as polyampholytes (2) or cross-linked bovine serum albumin (3) have been introduced to overcome some of these problems.

Immunochemical Studies on Antithrombin III

1979 ◽  
Author(s):  
E.J. McKay
Keyword(s):  
Antithrombin Iii ◽  
The Other ◽  
Antigenic Determinants ◽  
Immunochemical Analysis ◽  
Paradoxical Effect ◽  
Test Protocol ◽  
Agar Gel ◽  
High Affinity ◽  
Time Required ◽  
Antigen Antibody

Depressed Antithrombin III (AT) levels Increase thrombic tendency in man, therefore value in assaying this protein has been established. Immunochemical analysis of AT in clinical disease has however proved controversial, consequently systematic studies were undertaken to rationalize the requirements necessary to optimise these methods in particular electro-Immunoassay. The known binding affinity of AT for heparin has been exploited to differentiate high affinity AT from its inhibitor - protease complexes and has resulted in reports stating that heparin added to the agar gel prior to electrophoresis significantly reduces the time required for completion of antigen/antibody complexes. Our studies however have demonstrated that the antibody required for quantitative analysis must be capable of not only reacting with “native” antigenic determinants of AT but also with “neo” antigens that are exposed when inhibitor-protease complexes are formed. Heparin should not be used in the test protocol, for it has a paradoxical effect on Immunopreclpltation in gels, masking some antigenic determinants of unbound - high affinity AT on one hand, and appear to disrupt the Immunoprecipitin “rocket” formed with the inhibitor-protease complexes during electrophoresis on the other.

Demonstration of the Exposure of New Antigenic Determinants Following Antigen–antibody Combination

Nature ◽  
1965 ◽  
Vol 205 (4976) ◽  
pp. 1079-1081 ◽  
Author(s):  
C. S. HENNEY ◽  
D. R. STANWORTH ◽  
P. G. H. GELL
Keyword(s):  
Antigenic Determinants ◽  
Antigen Antibody

scholarly journals IMMUNOLOGIC STUDIES OF WATER-SOLUBLE HUMAN AMYLOID FIBRILS

1969 ◽  
Vol 130 (4) ◽  
pp. 797-808 ◽  
Author(s):  
Edward C. Franklin ◽  
Mordechai Pras
Keyword(s):  
Human Serum ◽  
Comparative Studies ◽  
Amyloid Fibrils ◽  
Complement Fixation ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Antigenic Determinants ◽  
Normal Tissues ◽  
Absorption Studies ◽  
Normal Human

Eight preparations of soluble amyloid and degraded amyloid (DAM) were compared immunologically. Unlike amyloid fibrils, six of eight preparations of DAM proved to be relatively strong immunogens. Antisera to DAM reacted weakly or not at all with normal human serum or extracts of normal tissues, but were specifically reactive with amyloid fibrils or DAM. Comparative studies of DAM'S from eight different subjects showed some degree of cross-reactivity among them, yet demonstrated that they were not identical. Similar conclusions were obtained by quantitative precipitin and complement fixation analyses. Comparison of the amyloid fibrils with the homologous DAM by complement fixation and absorption studies demonstrated the existence in DAM of antigenic determinants that were lacking or inaccessible in the native fibrils. A search for amyloid precursors and antibodies to amyloid in the sera of 12 patients proved unsuccessful.

Dissociation of mannan--serum complexes and detection of Candida albicans mannan by enzyme immunoassay variations.

1982 ◽  
Vol 28 (2) ◽  
pp. 306-310 ◽  
Author(s):  
E Reiss ◽  
L Stockman ◽  
R J Kuykendall ◽  
S J Smith
Keyword(s):  
Candida Albicans ◽  
Enzyme Immunoassay ◽  
Alkali Treatment ◽  
Human Sera ◽  
Normal Human ◽  
Sandwich Enzyme Immunoassay ◽  
Antigen Antibody ◽  
Bead Method

Abstract Candida albicans mannan was added to normal human sera and the resulting complexes were dissociated by boiling (boil) with EDTA or by alkali treatment (bead method). The mannan released was detected by "sandwich" enzyme immunoassay (EIA) or by EIA inhibition. Each EIA took 2.3 h to perform. The total time for the boil-EIA combination was 2.7 h and for the bead-EIA, 3.8 h. The temperature favorable for antigen--antibody incubation was 4 degrees C. The sandwich EIAs were preferable to EIA inhibition because absorbance was directly proportional to mannan concentration, within-run variation was decreased, and accuracy was increased. The boil-sandwich EIA had the highest sensitivity in the 12.5 to 200 micrograms/L range.

scholarly journals Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments

2000 ◽  
Vol 46 (11) ◽  
pp. 1755-1761 ◽  
Author(s):  
Harri Härmä ◽  
Piia Tarkkinen ◽  
Tero Soukka ◽  
Timo Lövgren
Keyword(s):  
Prostate Specific Antigen ◽  
Single Particle ◽  
Binding Capacity ◽  
Specific Antigen ◽  
Genetically Engineered ◽  
Covalent Attachment ◽  
Serum Samples ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Total Psa

Abstract Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode. Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R >0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

scholarly journals Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1226-1234 ◽  
Author(s):  
C Legrand ◽  
V Dubernard ◽  
AT Nurden
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Synthetic Peptides ◽  
Divalent Cations ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Plasma Fibrinogen ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Activated Platelets

Abstract Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400–411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.

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