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

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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 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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Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medicinal Chemistry

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
Original languageEnglish
Pages (from-to)767-772
Number of pages6
JournalThrombosis and Haemostasis
Volume79
Issue number4
StatePublished - 1998
Publication categoryResearch
Peer-reviewedYes