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A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen

Giri, Tusar K LU ; Hillarp, Andreas LU ; Härdig, Ylva; Zöller, Bengt LU and Dahlbäck, Björn LU (1998) In Thrombosis and Haemostasis 79(4). p.767-772
Abstract

A new method to determine the concentration of the free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of... (More)

A new method to determine the concentration of the free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specifity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.

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keywords
Antibodies, Monoclonal, Anticoagulants, Binding Sites, Calcium, Carrier Proteins, Dose-Response Relationship, Immunologic, Enzyme-Linked Immunosorbent Assay, Evaluation Studies as Topic, Humans, Immunoenzyme Techniques, Integrin alphaXbeta2, Ligands, Macromolecular Substances, Point Mutation, Protein S, Protein S Deficiency, Radioimmunoassay, Reproducibility of Results, Sensitivity and Specificity, Comparative Study, Journal Article, Research Support, Non-U.S. Gov't
in
Thrombosis and Haemostasis
volume
79
issue
4
pages
6 pages
publisher
F K Schattauer Verlag Gmbh
external identifiers
  • scopus:0031956214
ISSN
0340-6245
language
English
LU publication?
yes
id
79ec77a0-a2ab-4fdd-8014-2a61e472cbce
date added to LUP
2017-10-19 15:11:58
date last changed
2017-11-07 14:03:10
@article{79ec77a0-a2ab-4fdd-8014-2a61e472cbce,
  abstract     = {<p>A new method to determine the concentration of the free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca<sup>2+</sup> dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specifity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.</p>},
  author       = {Giri, Tusar K and Hillarp, Andreas and Härdig, Ylva and Zöller, Bengt and Dahlbäck, Björn},
  issn         = {0340-6245},
  keyword      = {Antibodies, Monoclonal,Anticoagulants,Binding Sites,Calcium,Carrier Proteins,Dose-Response Relationship, Immunologic,Enzyme-Linked Immunosorbent Assay,Evaluation Studies as Topic,Humans,Immunoenzyme Techniques,Integrin alphaXbeta2,Ligands,Macromolecular Substances,Point Mutation,Protein S,Protein S Deficiency,Radioimmunoassay,Reproducibility of Results,Sensitivity and Specificity,Comparative Study,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {4},
  pages        = {767--772},
  publisher    = {F K Schattauer Verlag Gmbh},
  series       = {Thrombosis and Haemostasis},
  title        = {A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen},
  volume       = {79},
  year         = {1998},
}