HRP-conjugated plug-and-playable IgG-binding nanobodies as secondary antibody mimics in immunoassays

1988 ◽

Vol 46 ◽

pp. 382-383

Author(s):

Douglas R. Keene ◽

Robert W. Glanville ◽

Eva Engvall

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Human Skin ◽

Basement Membranes ◽

Primary Antibody ◽

Fat Cells ◽

Secondary Antibody ◽

Type Vi Collagen ◽

Dermal Matrix ◽

Immuno Electron Microscopy

A mouse monoclonal antibody (5C6) prepared against human type VI collagen (1) has been used in this study to immunolocalize type VI collagen in human skin. The enbloc method used involves exposing whole tissue pieces to primary antibody and 5 nm gold conjugated secondary antibody before fixation, and has been described in detail elsewhere (2).Biopsies were taken from individuals ranging in age from neonate to 65 years old. By immuno-electron microscopy, type VI collagen is found to be distributed as a fine branching network closely associated with (but not attached to) banded collagen fibrils containing types I and III collagen (Fig. 1). It appears to enwrap fibers, to weave between individual fibrils within a fiber, and to span the distance separating fibers, creating a “web-like network” which entraps fibers within deep papillary and reticular dermal layers (Fig. 2). Relative to that in the dermal matrix, the concentration of type VI collagen is higher around endothelial basement membranes limiting the outer boundaries of nerves, capillaries, and fat cells (Fig. 3).

Download Full-text

IgG-binding properties of novel bacterial Fc-receptor pH6 antigen (PsaA protein) Yersinia pestis

Immunology Letters ◽

10.1016/s0165-2478(97)88451-9 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 399

Author(s):

G Spirina

Keyword(s):

Yersinia Pestis ◽

Fc Receptor ◽

Binding Properties ◽

Igg Binding

Download Full-text

Differing Susceptibilities of Platelets from Normal Individuals and Patients with von Willebrand’s Disease to Attack by Quinine- and Quinidine-Dependent Antiplatelet Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646537 ◽

1989 ◽

Vol 61 (01) ◽

pp. 111-116

Author(s):

Sharron L Pfueller ◽

Robyn A Bilston ◽

Dana Logan ◽

Rosemary David ◽

Ian G Sloan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Serotonin Release ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease ◽

Antiplatelet Antibodies ◽

Normal Plasma ◽

Igg Binding ◽

Normal Individuals ◽

Drug Dependent

SummaryReactivity of quinine- and quinidine-dependent antiplatelet antibodies has been compared in platelet-rich-plasma (PRP) from normal donors and from patients with von Willebrand’s disease (vWd). One quinine-dependent antibody (Q. Ab) caused platelet aggregation and [14C] serotonin release with only 7 of 12 normal donors, while another Q. Ab and a quinidine-dependent antibody (Qd. Ab) caused aggregation and release with all 12. Drug- dependent IgG binding and PF 3 availability induced by the antibodies were, however, comparable in all donors. Differences in responsiveness were associated with platelets and not plasma. vWd platelets showed normal drug-dependent IgG binding, but decreased aggregation and serotonin release to most drug- dependent antibodies. Responsiveness was not restored by purified vWf:Ag, but, in one case, was corrected by normal plasma or cryoprecipitate. Drug-dependent binding of the Q. Ab which caused variable responsiveness in normals was to the same platelet antigens (GPIb and GPIIIa) in both normal and vWd platelets and did not require plasma components. Reduced PF 3 availability was seen with some antibodies in some vWd patients. Plasma from two of these patients inhibited aggregation of normal platelets to Q. Ab and one of these inhibited aggregation to ADP. Antiplatelet antibodies were detected in these two plasmas by ELISA. Thus some Q. Ab produce different responses with platelets from different donors. In vWd, reduced responsiveness to Q.Ab and Qd. Ab may result from production of inhibitory antiplatelet antibodies.

Download Full-text

Partial Inhibition of Platelet Aggregation and Fibrinogen Binding by a Murine Monoclonal Antibody to GPIIIa: Requirement for Antibody Bivalency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648991 ◽

1994 ◽

Vol 72 (06) ◽

pp. 964-972 ◽

Cited By ~ 16

Author(s):

Jeffery L Kutok ◽

Barry S Coller

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Murine Monoclonal Antibody ◽

Platelet Glycoprotein ◽

Igg Antibody ◽

Surface Receptors ◽

Partial Inhibition ◽

Fibrinogen Binding ◽

Igg Binding ◽

Agonist Concentration

SummaryWe produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) Ilia. 7H2-IgG and 7H2-F(ab’)2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, ∼40,000 7H2-IgG antibody molecules bind per platelet. In contrast, ∼80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s).

Download Full-text

Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650123 ◽

1981 ◽

Vol 45 (01) ◽

pp. 027-033 ◽

Cited By ~ 10

Author(s):

K Sugiura ◽

M Steiner ◽

M Baldini

Keyword(s):

Shape Change ◽

Fluorescein Isothiocyanate ◽

Recovery Phase ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Volume ◽

Igg Antibody ◽

Heterologous Antiserum ◽

Igg Binding ◽

Series Of Experiments

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

Download Full-text