Reagent and method for measuring thrombin-antithrombin complex
Provided is a reagent for assaying a thrombin-antithrombin complex (TAT) in a blood sample from a subject by latex agglutination assay. The reagent includes a polycation. As a result, TAT complexes can be precisely assayed while circumventing the effect of heparin. The polycation is, for example, hexadimethrine bromide, chitosans, modified dextran, aminodextran, hydroxymethyl cellulose trimethylamine, lysozyme, spermine, spermidine, polylysine, polyarginine, polyornithine, protamine sulfate, hydroxyethyl cellulose trimethylamine, heparin-binding protein, polyallylamine, polyallylamine hydrochloride, poly(diallyl dialkyl amine), polyamideamine, polyamine, polyvinylbenzyltrimethylammonium chloride, polydiallyldimethylammonium chloride, polyethyleneimine, polypropyleneimine, polypropylethyleneimine, polyimidazoline, polyvinylamine, polyvinyl pyridine, poly(acrylamide/methacryloxypropyltrimethylammonium bromide), poly(diaryldimethylammonium chloride/N-isopropylacrylamide), poly(dimethylaminoethyl acrylate/acrylamide), poly(dimethylaminoethyl methacrylate), polydimethylaminoepichlorohydrin, polyethyleneiminoepichlorohydrin, polymethacryloxyethyl-trimethylammonium bromide, hydroxypropylmethacryloyloxyethyl dimethyl ammonium chloride, poly(methyldiethylaminoethylmethacrylate/acrylamide), poly(methyl/guanidine), polymethylvinylpyridinium bromide, poly(vinylpyrrolidone-dimethylaminoethyl methacrylate), or polyvinylmethylpyridinium bromide.
1. A method for assaying a TAT complex existing in a blood plasma sample from a patient receiving heparin, said blood sample comprising antithrombin multimers resulting from formation of heparin/antithrombin complexes the method comprising:
assaying a TAT complex in the blood plasma sample from the patient by performing a latex agglutination assay comprising contacting the sample with a latex agglutination assay reagent mixture,
wherein said reagent mixture comprises: (i) a first anti-TAT antibody which recognizes a TAT complex by binding to an antithrombin part of the TAT complex bound to latex, said first anti-TAT antibody has a reactivity to the TAT complex that is 100 times or higher-than a reactivity to free antithrombin; (ii) a second anti-TAT antibody which recognizes a TAT complex by binding to a thrombin part of the TAT complex bound to latex; and (iii) a polycation selected from the group consisting of hexadimethrine bromide and protamine sulfate,
wherein the final concentration of hexadimethrine bromide in the assay is 0.0002 to 0.002% (w/v), wherein the final concentration of protamine sulfate in the assay is 0.0005% to 0.005% (w/v), wherein the antithrombin multimers are capable to agglutinate in the presence of the first anti-TAT antibody of (i) and second anti-TAT antibody of (ii) bound to latex, thereby causing nonspecific agglutination; and wherein the polycation dissociates the antithrombin multimers, thereby reducing nonspecific agglutination.
2. The method according to claim 1 , wherein the polycation is hexadimethrine bromide.
3. The method according to claim 1 , wherein the polycation is protamine sulfate.