Search: (AMNE:(MEDICAL AND HEALTH SCIENCES Basic Medicine Medicinal Chemistry)) pers:(Hillarp Andreas) pers:(Dahlbäck Björn) >
A new direct, fast ...
A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen
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- Giri, Tusar K (author)
- Lund University,Lunds universitet,Klinisk kemi, Malmö,Forskargrupper vid Lunds universitet,Clinical Chemistry, Malmö,Lund University Research Groups
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- Hillarp, Andreas (author)
- Lund University,Lunds universitet,Klinisk kemi, Malmö,Forskargrupper vid Lunds universitet,Clinical Chemistry, Malmö,Lund University Research Groups
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Härdig, Ylva (author)
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- Zöller, Bengt (author)
- Lund University,Lunds universitet,Klinisk kemi, Malmö,Forskargrupper vid Lunds universitet,Clinical Chemistry, Malmö,Lund University Research Groups
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- Dahlbäck, Björn (author)
- Lund University,Lunds universitet,Klinisk kemi, Malmö,Forskargrupper vid Lunds universitet,Clinical Chemistry, Malmö,Lund University Research Groups
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(creator_code:org_t)
- 1998
- 1998
- English 6 s.
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In: Thrombosis and Haemostasis. - 0340-6245. ; 79:4, s. 767-772
- Related links:
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https://lup.lub.lu.s...
Abstract
Subject headings
Close
- 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.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Läkemedelskemi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Medicinal Chemistry (hsv//eng)
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
Publication and Content Type
- art (subject category)
- ref (subject category)
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