Method of determination of pivka

#The present invention relates to The immunochemical assay of a PIVKA of every kind present in an organic specimen and a reagent therefor, which are utilized in the medical field.

#Vitamin K-dependent plasma proteins present in organisms include prothrombin, factor VII, factor IX, factor X, protein C, protein S, and protein Z. According to biosynthesis mechanisms of these proteins, a precursor protein is carboxylated at the .gamma.-positions of about 10 glutamic acid residues thereof in the presence of vitamin K and a carboxylase to be converted into an active-form normal protein, which manifests the physiological action thereof. These glutamic acid residues are present in the N-terminal region, which is referred to as the "Glaregion." In a state of physiological or clinical deficiency of vitamin K or in a state of suppressed action of vitamin K through administration of a vitamin K antagonist, proteins with incomplete carboxylation of those glutamic acid residues are formed.

#"PIVKA" is a generic term for those vitamin K-dependent plasma proteins with such incomplete carboxylation, and is an abbreviation of "Protein Induced by Vitamin K Absence or Antagonist." The number of noncarboxylated residues among the about 10 glutamic acid residues present in the amino-terminal region is not fixed. Thus, one kind of protein is composed of several kinds of PIVKA compounds present in mixture.

#FIG. 1 shows standard curves for PIVKA-II and PIVKA-X determined according to the method of the present invention.

#FIG. 2 shows determined values of PIVKA-II and PIVKA-X present in specimens obtained from patients with hepatopathy.

#The following Examples will now specifically illustrate the present invention, but should not be construed as limiting the scope of the invention.

#The blood plasma of the subject to which a vitamin K antagonist had been administered was admixed with the same amount of a 4M saturated solution of ammonium sulfate. The resulting mixture was stirred for 2 hours and centrifuged. The precipitate was dissolved in a 0.1M phosphate buffer solution, followed by dialysis. 0-0.6M NaCl fractionation was effected with DEAE-Sephacel to collect a PIVKA-II fraction, which was dialyzed and then passed through Heparin-Sepharose and Blue-Sepharose. PIVKA-II was further purified by gel filtration chromatography with Ultrogel AcA44 and prothrombin affinity column chromatography.

#Establishment of Anti-PIVKA-II monoclonal antibody-producing hybridoma cell strain