A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen.
A new method to determine the concentration of 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 specificity 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.
Giri
T K
TK
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Hillarp
A
A
Härdig
Y
Y
Zöller
B
B
Dahlbäck
B
B
D000911:Antibodies, Monoclonal / Q000276:immunology; D000925:Anticoagulants / Q000494:pharmacology / Q000627:therapeutic use; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques; D016167:Integrin alphaXbeta2; D008024:Ligands; D046911:Macromolecular Substances; D017354:Point Mutation; D017293:Protein S / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D018455:Protein S Deficiency / Q000097:blood / Q000175:diagnosis* / Q000188:drug therapy; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D002352:Carrier Proteins; D016167:Integrin alphaXbeta2; D008024:Ligands; D046911:Macromolecular Substances; D017293:Protein S; D002118:Calcium