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Simultaneous assay of free and total protein S by ELISA using monoclonal and polyclonal antibodies
C Gardiner1, I J Mackie, S J Machin
1Department of Haematology, University College London, UK.
The measurement of protein S is essential in the investigation of thrombophilia. Protein S forms a complex with C4b binding protein which exists in equilibrium with the free form but only the free form has anticoagulant cofactor activity. The accurate measurement of protein S has previously proved problematical. We have investigated the use of an ELISA method for protein S, using monoclonal antibodies directed against the free form, which can be used in parallel with polyclonal antibodies for the simultaneous assay of total protein S. The total protein S measurement was based on a well established method while the monoclonal antibody free protein S assay (mAb PS) was less complex than previous assays with no requirement for prior PEG precipitation and showed improved precision (intra-assay CV = 4.8% and 7.1%, between assay CV = 7.1% and 8.6% respectively). The mAb assay compared well with the conventional PEG precipitation ELISA for free protein S and a functional protein S assay based on the prothrombin time, in normal subjects, patients with thrombophilia, congenital protein S deficient patients or out-patients stabilized on oral anticoagulants. This assay overcomes many of the problems associated with protein S measurement and is a useful tool in the investigation of thrombophilia.
The measurement of protein S is essential in the investigation of thrombophilia. Protein S forms a complex with C4b binding protein which exists in equilibrium with the free form but only the free form has anticoagulant cofactor activity. The accurate measurement of protein S has previously proved problematical. We have investigated the use of an ELISA method for protein S, using monoclonal antibodies directed against the free form, which can be used in parallel with polyclonal antibodies for the simultaneous assay of total protein S. The total protein S measurement was based on a well established method while the monoclonal antibody free protein S assay (mAb PS) was less complex than previous assays with no requirement for prior PEG precipitation and showed improved precision (intra-assay CV = 4.8% and 7.1%, between assay CV = 7.1% and 8.6% respectively). The mAb assay compared well with the conventional PEG precipitation ELISA for free protein S and a functional protein S assay based on the prothrombin time, in normal subjects, patients with thrombophilia, congenital protein S deficient patients or out-patients stabilized on oral anticoagulants. This assay overcomes many of the problems associated with protein S measurement and is a useful tool in the investigation of thrombophilia.