MONOCLONAL ANTIBODIES AGAINST THROHBIN-ANTITHROMBIN III COMPLEX: EPITOPE SPECIFICITY AND EFFECT ON THROMBIN-ANTITHROMBIN III INTERACTION

1987 ◽  
Author(s):  
S Asakura ◽  
N Yoshida ◽  
M Matsuda
Keyword(s):  
Monoclonal Antibodies ◽  
Antithrombin Iii ◽  
Normal Serum ◽  
Reactive Site ◽  
High Affinity ◽  
Epitope Specificity ◽  
New Information ◽  
Sds Page ◽  
Human Thrombin ◽  
Immunoglobulin Subclasses

Among monoclonal antibodies (MCA´s) raised against human thrombin (T)-antithrombin m (AT) complex (TAT), two MCA´s designated as JITAT-16 and 17 with high affinity, Kd = 4.6nMand 4.1 nfi, respectively, were selected and characterized for specificity and functions. Their respective immunoglobulin subclasses are IgGi and IgG2a, and epitopes were found to be different from each Dther as shown by crisscross inhibition experiments. Immuno-alotting of normal plasma and serum electrophoresed on non-SDS aolyacrylamide gel showed that these antibodies reacted with normal serum but not with plasma. This was verified by an anzyme-linked differential antibody immunosorbent assay using aither one of the MCA´s as the first antibody and the other MCA labeled with peroxidase as the second one. By immunoblotting after SDS-PAGE, we found that both antibodies reacted with TAT, nut not with its respective nascent constituent, AT or T. However, they reacted with reactive site-cleaved AT (or thrombin-nodified AT, ATM) and also a complex of AT with activated factor K (Xa-AT). These results indicate that both of these antibodies recognize enzyme-treated forms of AT, including AT molecules :omplexed with enzymes reversibly or irreversibly as well as ATM. Jpon incubation of T with AT in the presence of JITAT-16, T activity remained nearly unchanged and formation of irreversible rAT did not proceed as expected. Moreover, AT was preferentially :onverted to ATM. When JITAT-16 was added after completion of FAT formation, however, neither recovery of T activity nor generation of ATM was observed. These findings were not obtained vhen JITAT-17 had been substituted for JITAT-16. These data suggest that JITAT-16 may have converted AT from an inhibitor to a substrate for T after having recognized a possible intermediate reversible complex of AT with T. Undoubtedly, in the presence of a polyclonal antibody against AT, neither TAT formation nor ATM neneration was observed at all. The mechanism of the unique Function of JITAT-16 has not been fully clarified as yet, but this antibody seems to give us new information on the kinetic study of TAT formation and ATM generation when AT was allowed to react with enzymes.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals Effect of heparin on the glia-derived-nexin-thrombin interaction

1989 ◽  
Vol 257 (1) ◽  
pp. 191-196 ◽  
Author(s):  
A Wallace ◽  
G Rovelli ◽  
J Hofsteenge ◽  
S R Stone
Keyword(s):  
Rate Constants ◽  
Antithrombin Iii ◽  
Kinetic Properties ◽  
Association Rate ◽  
High Affinity ◽  
Diffusion Controlled ◽  
Different Types ◽  
Human Thrombin ◽  
Alpha Beta ◽  
Association Rate Constants

In order to determine the specificity of the interaction between thrombin and glia-derived nexin (GdN), the inactivation of proteolytically modified human thrombin species by GdN has been studied. The second-order rate constants for the inactivation of alpha-, beta T-, gamma T- and epsilon-thrombin by GdN were 1.41, 0.63, 0.33 and 1.91 microM-1.s-1 respectively. The kinetic properties of gdN were also investigated in the presence of different types of heparin, fractionated according to antithrombin III-binding affinity. Association rate constants of both gdN and antithrombin III with alpha-thrombin were obtained using unfractionated, low- and high-affinity heparin types. The different heparin types gave optimal rates of inhibition at similar heparin concentrations for both inhibitors. At optimal heparin concentrations, the rate of inactivation of alpha-thrombin by GdN was 0.5-1.2 nM-1.s-1, which suggests that, under these conditions, the interaction is diffusion-controlled.

scholarly journals O-Polysaccharide Epitopic Heterogeneity at the Surface of Brucella spp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry

1998 ◽  
Vol 5 (6) ◽  
pp. 862-870 ◽  
Author(s):  
Axel Cloeckaert ◽  
Vincent Weynants ◽  
Jacques Godfroid ◽  
Jean-Michel Verger ◽  
Maggy Grayon ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
The Other ◽  
Antigenic Structure ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Goal ◽  
Passive Protection ◽  
Epitope Specificity ◽  
New Information ◽  
High Degree

ABSTRACT Smooth Brucella strains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface ofBrucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucellaspecies and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees byBrucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

scholarly journals Effect of oversulphated chondroitin and dermatan sulphate upon thrombin and factor Xa inactivation by antithrombin III or heparin cofactor II

1988 ◽  
Vol 254 (2) ◽  
pp. 547-551 ◽  
Author(s):  
M F Scully ◽  
V Ellis ◽  
N Seno ◽  
V V Kakkar
Keyword(s):  
Antithrombin Iii ◽  
Chondroitin Sulphate ◽  
Factor Xa ◽  
Dermatan Sulphate ◽  
Physiological Control ◽  
Heparin Cofactor Ii ◽  
High Affinity ◽  
Human Thrombin ◽  
Sulphated Glycosaminoglycans ◽  
Inhibition Of Thrombin

The kinetics of inhibition of human thrombin and Factor Xa by antithrombin III or heparin cofactor II were examined under pseudo-first-order conditions as a function of the concentration of naturally occurring oversulphated chondroitin and dermatan sulphates. The sulphated glycosaminoglycans (GAGs) studied were chondroitin sulphate D (CSD) (GlcA-2-SO4-GalNAc-6-SO4), chondroitin sulphate K (CSK) (GlcA-3-SO4-GalNAc-4-SO4), chondroitin sulphate H (CSH) (IdA-GalNAc-4,6-diSO4) and polysulphated dermatan sulphate (DPS) (IdA-2-SO4 or -3-SO4-GalNAc-4,6-diSO4). The data for the antithrombin III inhibition of thrombin showed a low degree of maximal potentiation of this interaction (congruent to 10-fold), which would appear to be characteristic of GAGs devoid of the high-affinity antithrombin III binding site. In contrast there was a greater potentiation of the inhibition of thrombin by heparin cofactor II with DPS showing an activity comparable to heparin in this interaction at a concentration two orders of magnitude lower than dermatan sulphate. DPS potentiated antithrombin III-Factor Xa interaction by 1200-fold, similar to that shown by high-affinity heparin of 6 kDa. The antithrombin III-Factor Xa interaction was potentiated by all other GAGs studied to a degree similar to that of heparin pentasaccharide with high affinity for antithrombin III. The findings suggest more stringent structural requirements for GAG stimulation of antithrombin-thrombin interaction than for antithrombin-Factor Xa or heparin cofactor-thrombin interaction, which may also be of significance in physiological control of haemostasis.

Monoclonal antibodies to human plasma Protein X alias complement S-protein

1985 ◽  
Vol 5 (4) ◽  
pp. 343-352 ◽  
Author(s):  
Dieter Jenne ◽  
Ferdinand Hugo ◽  
Sucharit Bhakdi
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Protein ◽  
Antithrombin Iii ◽  
Plasma Concentrations ◽  
Fluid Phase ◽  
Gel Chromatography ◽  
S Protein ◽  
Sds Page ◽  
Protein X ◽  
Terminal Complement

Protein X alias complement S-protein was isolated by dissociation from purified XCSb-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immuno-precipitate of α2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of Oh-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monocJonal anti – Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80–90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500–700 pg/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.

Isolation and Partial Characterization of Equine Antithrombin III

1979 ◽  
Author(s):  
D. W. Estry ◽  
T. G. Bell ◽  
G. H. Tishkoff ◽  
J. C. Mattson ◽  
S. C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. in order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichiometry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals Fc-Dependent and Fc-Independent Opsonization of Cryptococcus neoformans by Anticapsular Monoclonal Antibodies: Importance of Epitope Specificity

2002 ◽  
Vol 70 (6) ◽  
pp. 2812-2819 ◽  
Author(s):  
Dale Netski ◽  
Thomas R. Kozel
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Peritoneal Macrophages ◽  
Normal Serum ◽  
Differential Interference Contrast Microscopy ◽  
Independent Manner ◽  
Opsonic Activity ◽  
Epitope Specificity ◽  
Interference Contrast Microscopy

ABSTRACT Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab′)2 fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab′)2 fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab′)2 fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab′)2 fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.

Characterization Of Rabbit Antithrombin III

1981 ◽  
Author(s):  
D Estry ◽  
J C Mattson ◽  
T G Bell ◽  
G H Tishkoff
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Heparin Binding ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Immunologic Assays

The rabbit is a well established model for studying the disseminated intravascular coagulation (DIC) associated with endotoxic syndromes. In order to establish the role of antithrombin III (AT III) in the modulation of DIC in the rabbit, characterization of rabbit AT III was undertaken. Rabbit antithrombin III, isolated according to modifications of the method of Thaler and Schmer, has a molecular weight comparable to that of human AT III (62,000 daltons) as measured by mobility on SDS-PAGE gels. Mixtures of rabbit and human AT III co-migrate as a single band on 7.5% SDS-PAGE gels. Rabbit AT III possesses both progressive and heparin activated (immediate) antithrombin activity in assays using human thrombin. Antisera raised against rabbit AT III demonstrates no cross reactivity with human AT III suggesting that despite physiologic and molecular weight similarities, antigenic differences are present. Incubation of rabbit antithrombin III with specific antisera, either prior to or after addition of heparin, did not alter the ability of antithrombin III to inhibit thrombin in either the immediate or progressive assays indicating that the antigenic determinants are not found in either the heparin binding or active thrombin binding sites. Crossed immunoelectrophoresis (IEP) demonstrates that antisera to rabbit AT III reacts with both free rabbit antithrombin III and AT III-thrombin complexes and can therefore be used in immunologic assays to quantitate total rabbit AT III (bound and free) and in crossed IEP to demonstrate the mobility of both free and complexed AT III.

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

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