The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

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On the Role of Transferrin in 67Ga Uptake by Tumor Cells

Nuklearmedizin ◽

10.1055/s-0038-1629447 ◽

1988 ◽

Vol 27 (04) ◽

pp. 151-153

Author(s):

P. Thouvenot ◽

F. Brunotte ◽

J. Robert ◽

L. J. Anghileri

Keyword(s):

Specific Binding ◽

Subcellular Fractionation ◽

Animal Blood ◽

Tumor Bearing ◽

Cell Structures ◽

The Difference ◽

Sarcoma Cells ◽

In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

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Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647851 ◽

1975 ◽

Vol 33 (03) ◽

pp. 573-585 ◽

Cited By ~ 20

Author(s):

Masahiro Iwamoto

Keyword(s):

Tranexamic Acid ◽

Affinity Chromatography ◽

Dissociation Constant ◽

Factor Xiii ◽

Specific Binding ◽

The Other ◽

Inhibitory Mechanism ◽

Binding Studies ◽

Similar Affinity ◽

Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

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Concanavalin A-induced Aggregation of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647887 ◽

1975 ◽

Vol 33 (02) ◽

pp. 354-360 ◽

Cited By ~ 4

Author(s):

Heinrich Patscheke ◽

Reinhard Brossmer

Keyword(s):

Concanavalin A ◽

Binding Capacity ◽

Specific Binding ◽

Blood Platelets ◽

Residual Effect ◽

Independent Effect ◽

Con A ◽

Experimental Conditions ◽

Main Effect ◽

Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

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Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648973 ◽

1994 ◽

Vol 72 (06) ◽

pp. 848-855 ◽

Cited By ~ 34

Author(s):

Dzung The Le ◽

Samuel I Rapaport ◽

L Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Factor Viia ◽

Cell Damage ◽

Specific Binding ◽

Surface Membrane ◽

Annexin V ◽

Factor X ◽

Binding Studies ◽

Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648482 ◽

1992 ◽

Vol 67 (05) ◽

pp. 507-509 ◽

Cited By ~ 16

Author(s):

John Gibson ◽

Margaret Nelson ◽

Ross Brown ◽

Hatem Salem ◽

Harry Kronenberg

Keyword(s):

Enzyme Immunoassay ◽

Lupus Anticoagulant ◽

Specific Binding ◽

Technical Problem ◽

Serum Samples ◽

Citrate Buffer ◽

The Mean ◽

Sample Dilution ◽

Negative Population ◽

Serum Components

SummaryIn order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (± SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 ± 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.

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Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649639 ◽

1993 ◽

Vol 70 (04) ◽

pp. 625-630 ◽

Cited By ~ 71

Author(s):

Edward Young ◽

Benilde Cosmi ◽

Jeffrey Weitz ◽

Jack Hirsh

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Plasma Proteins ◽

Low Molecular Weight Heparin ◽

Specific Binding ◽

Anticoagulant Activity ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

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Calcium and the Fc Receptor on Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651120 ◽

1987 ◽

Vol 57 (03) ◽

pp. 298-301

Author(s):

William F Clark ◽

Gerald J M Tevaarwerk ◽

Bruce D Reid ◽

Suzanne Hall ◽

Anita Caveney ◽

...

Keyword(s):

Specific Binding ◽

Normal Subjects ◽

Human Platelets ◽

Fc Receptor ◽

Normal Male ◽

Scatchard Analysis ◽

Normal Female ◽

Apparent Difference ◽

Binding Data ◽

Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

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Interactions of Staphylokinase with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653799 ◽

1995 ◽

Vol 73 (03) ◽

pp. 472-477 ◽

Cited By ~ 5

Author(s):

H R Lijnen ◽

B Van Hoef ◽

D Collen

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Specific Binding ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Normal Plasma ◽

Atp Secretion ◽

Platelet Disaggregation ◽

Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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Specific Binding of Platelet Proteins to Immobilized Fibrin Monomers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657078 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1686-1689

Author(s):

Czeslaw S Cierniewski ◽

Tadeusz Krajewski ◽

Jolanta Waczyńska-Niewiarowska

Keyword(s):

Specific Binding ◽

Fibrin Monomers ◽

Platelet Proteins

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