Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations

1992 ◽  
Vol 67 (06) ◽  
pp. 631-638 ◽  
Author(s):  
Silvana Vigano D’Angelo ◽  
Sandra Tombesi ◽  
Santica Marcovina ◽  
Alberto Albertini ◽  
Patrizia Della Valle ◽  
...  
Keyword(s):  
Protein S ◽  
Plasma Samples ◽  
Potential Influence ◽  
Polyclonal Igg ◽  
Dependent Protein ◽  
Capture Antibody ◽  
Antigen Determination ◽  
Sample Dilution ◽  
Selection Of

SummaryTwo monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

1991 ◽  
Vol 66 (01) ◽  
pp. 049-061 ◽  
Author(s):  
Björn Dahlbäck
Keyword(s):  
Vitamin K ◽  
Complement System ◽  
Protein C ◽  
Protein S ◽  
High Affinity ◽  
Anticoagulant System ◽  
Dependent Protein ◽  
The Complement System ◽  
Β Chain ◽  
Dependent Domain

SummaryThe protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF)epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 γ-carboxy glutamic acid residues in the vitamin K-dependent domain, a β-hydroxylated aspartic acid in the first EGF-like domain and a β-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical α-chains and one β-chain. The α-and β-chains are linked by disulphide bridges. The cDNA cloning of the β-chain showed the α- and β-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the β-chain to contain the single protein S binding site on C4BP, whereas each of the α-chains contains a binding site for the complement protein, C4b. As C4BP lacking the β-chain is unable to bind protein S, the β-chain is required for protein S binding, but not for the assembly of the α-chains during biosynthesis. Protein S has a high affinity for negatively charged phospholipid membranes, and is instrumental in binding C4BP to negatively charged phospholipid. This constitutes a novel mechanism for control of the complement system on phospholipid surfaces. Recent findings have shown circulating C4BP to be involved in yet another calcium-dependent protein-protein interaction with a protein known as the serum amyloid P-component (SAP). The binding sites on C4BP for protein S and SAP are independent. SAP, which is a normal constituent in plasma and in tissue, is a so-called pentraxin being composed of 5 non-covalently bound 25 kDa subunits. It is homologous to C reactive protein (CRP) but its function is not yet known. The specific high affinity interactions between protein S, C4BP and SAP suggest the regulation of blood coagulation and that of the complement system to be closely linked.

DETERMINING THE DESALINATION UNIT COMPOSITION FOR COSTAL AREAS AND SMALL ISLANDS USE ANALYTIC HIERARCHY PROCESS

2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Imam Setiadi ◽  
Dinda Rita K. Hartaja
Keyword(s):  
Analytic Hierarchy Process ◽  
Energy Demand ◽  
Pairwise Comparison ◽  
Small Islands ◽  
Ahp Method ◽  
Analytic Hierarchy ◽  
Comparison Research ◽  
Hierarchy Process ◽  
Selection Of

Selection of the appropriate composition desalination units can be done with a variety of method approaches, one of the method is the Analytic Hierarchy Process. In determining the desalination unit with AHP method to consider is setting a goal, an alternative criteria and pairwise comparison. Research for the determination of the exact composition of the desalination unit in order to achieve sustainable drinking water suppy in coastal areas and small islands has been conducted. The results of the study are as follows, the energy demand of 50.83%, operator costs of 26.64%, maintenance costs of 14.13% and chemical requirement 8.4%. For an alternative composition desalination unit of RO 10 m3 / day is the best alternative composition with value of 59.61%, the composition of the next alternative is RO 20 m3/ day of 30.40% and the last alternative of the desalination unit composition is RO 120 m3/ day of 09.99%.Key words : Desalination, Mukti Stage Flash Composition, AHP

Optimization of the selection of analysis methods for the determination of naturally occurring radionuclides

Kerntechnik ◽  
2008 ◽  
Vol 73 (3) ◽  
pp. 118-121
Author(s):  
T. Heinrich ◽  
L. Funke ◽  
M. Köhler ◽  
U.-K. Schkade ◽  
F. Ullrich ◽  
...  
Keyword(s):  
Naturally Occurring ◽  
Analysis Methods ◽  
Naturally Occurring Radionuclides ◽  
Selection Of

Determination of the minimum number of technological bases of a part

2020 ◽  
pp. 73-75
Author(s):  
B.M. Bazrov ◽  
T.M. Gaynutdinov
Keyword(s):  
Cost Price ◽  
Labor Input ◽  
Part Surface ◽  
Technological Base ◽  
Size Accuracy ◽  
Minimum Number ◽  
The Cost ◽  
Input Cost ◽  
Selection Of

The selection of technological bases is considered before the choice of the type of billet and the development of the route of the technological process. A technique is proposed for selecting the minimum number of sets of technological bases according to the criterion of equality in the cost price of manufacturing the part according to the principle of unity and combination of bases at this stage. Keywords: part, surface, coordinating size, accuracy, design and technological base, labor input, cost price. [email protected]

Is Hydraulics Over-Used or Under-Used in Storm Drainage?

1994 ◽  
Vol 29 (1-2) ◽  
pp. 53-61
Author(s):  
Ben Chie Yen
Keyword(s):  
Unsteady Flow ◽  
Computational Time ◽  
Time Step ◽  
Flow Routing ◽  
Kinematic Wave Equation ◽  
Storm Drainage ◽  
Saint Venant Equations ◽  
Unsteady Flow Routing ◽  
Selection Of

Urban drainage models utilize hydraulics of different levels. Developing or selecting a model appropriate to a particular project is not an easy task. Not knowing the hydraulic principles and numerical techniques used in an existing model, users often misuse and abuse the model. Hydraulically, the use of the Saint-Venant equations is not always necessary. In many cases the kinematic wave equation is inadequate because of the backwater effect, whereas in designing sewers, often Manning's formula is adequate. The flow travel time provides a guide in selecting the computational time step At, which in turn, together with flow unsteadiness, helps in the selection of steady or unsteady flow routing. Often the noninertia model is the appropriate model for unsteady flow routing, whereas delivery curves are very useful for stepwise steady nonuniform flow routing and for determination of channel capacity.

Sign in / Sign up

Export Citation Format

Share Document