Immunoradiometric assays for the Ca(II)-dependent and Ca(II)-independent conformation of human protein C

Abstract

From a rabbit antiserum against human protein C, two subpopulations of antibodies recognizing Ca(II)-dependent and NonCa(II)-dependent epitopes of human protein C respectively were affinity purified for use in solid-phase immunoradiometric assays. The two assays were specific for PC:Ca(II)Ag and PC:NonCa(II)Ag and highly sensitive with a lower limit of detection of 2 ng/ml. The concentration of PC:Ca(II)Ag and PC:NonCa(II)Ag and their ratio in healthy volunteers was 0.91 ± 0.16 U/ml, 0.95 ± 0.14 U/ml and 0.97 ± 0.18. In a group of patients treated with oral anticoagulants the ratio PC:Ca(II)Ag/PC:NonCa(II)Ag was significantly decreased (to about 0.6) as compared with normal controls suggesting the presence of sub- and noncarboxylated protein C molecules (PIVKA-PC) in plasma of patients on oral anticoagulants. Analysis of plasmas of patients with a hereditary (n-8) or isolated (n-4) type II protein C deficiency and a history of thrombo-embolic disease did not reveal patients with a PC:Ca(II)Ag level and a PC:Ca(II)Ag/PC:NonCa(II)Ag ratio below the lower limit of the normal range suggesting that the frequency of genetic protein C variants with defects in the Ca(II)-dependent conformation is low.