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.

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.

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.

Prothrombin Evaluation as Obtained by Kinetics Studies of Antigen-Antibody Reaction in a Laser Nephelometer

1984 ◽  
Vol 52 (01) ◽  
pp. 015-018 ◽  
Author(s):  
A Girolami ◽  
A Sticchi ◽  
R Melizzi ◽  
L Saggin ◽  
G Ruzza
Keyword(s):  
Liver Cirrhosis ◽  
Cirrhotic Patient ◽  
Maximum Rate ◽  
Serum Proteins ◽  
Clotting Factors ◽  
Kinetics Studies ◽  
Maximum Value ◽  
Time Required ◽  
Antigen Antibody ◽  
Kinetics Of

SummaryLaser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard’s equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes

1992 ◽  
Vol 262 (2) ◽  
pp. G244-G248 ◽  
Author(s):  
C. Asher ◽  
D. Singer ◽  
R. Eren ◽  
O. Yeger ◽  
N. Dascal ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
The Other ◽  
Na Channel ◽  
Channel Activity ◽  
High Affinity ◽  
Other Hand ◽  
Exogenous Rna ◽  
Lower Intestine ◽  
Regulation Of Transport

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

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

Rapid detection of Australia/SH antigen and antibody by a simple and sensitive technique of immunoelectronmicroscopy

1971 ◽  
Vol 17 (7) ◽  
pp. 993-1000 ◽  
Author(s):  
A. E. Kelen ◽  
A. E. Hathaway ◽  
D. A. McLeod
Keyword(s):  
Complement Fixation Test ◽  
Complement Fixation ◽  
Practical Method ◽  
Rapid Screening ◽  
Blood Products ◽  
Agar Gel ◽  
Virus Particles ◽  
Antigen Antibody ◽  
Filtration Technique ◽  
Electronmicroscopic Examination

A simple and practical method is presented for demonstrating the presence of the Australia/SH antigen and its corresponding antibody in serum specimens, both qualitatively and quantitatively. The method is based on the electronmicroscopic visualization of characteristic aggregates of antigen–antibody complexes formed in the mixture of a serum specimen and the appropriate Australia/SH detector reagent. It involves the use of a microtechnique requiring minute amounts of reagents and provides, as a result of diffusion and filtration through agar gel, partially purified and concentrated preparations, ready for electronmicroscopic examination in less than an hour. The method is highly specific and yields reproducible results. Its sensitivity was found to be greater than that of the crossover electrophoresis test and closely approximates that of the complement fixation test, with the added advantage of not being affected by the "prozone phenomenon." The method can be recommended for use in laboratories equipped with electronmicroscopic facilities to establish a differential diagnosis of viral hepatitis cases, perform rapid screening of blood samples (blood products) for the presence of Australia/SH antigen, and clarify equivocal results obtained by other methods. It is expected that the agar–diffusion–filtration technique will also prove useful, in general, for enhancing the chances of detecting virus particles in suspensions of relatively low virus concentrations.

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