Human tissue plasminogen activator-human plasminogen activator inhibitor complex immunoassay and kit therefor

An immunological assay for active human plasminogen activator inhibitor which comprises:

• (1) providing two aliquots of a human specimen containing (i) a human tissue plasminogen activator-human plasminogen activator inhibitor complex and (ii) active human plasminogen activator inhibitor,
• (2) adding a sufficient amount of free human tissue plasminogen activator to one of the two aliquots to complex the active human plasminogen activator inhibitor,
• (3) contacting both the aliquots individually with a first antibody linked to an insoluble solid carrier, and then contacting a labeled second antibody therewith, or
• (4) simultaneously contacting the first antibody linked to the insoluble solid carrier and the labeled second antibody with the aliquots individually,
• steps (3) and (4) being performed in the presence of a surfactant,
• (5) measuring solid phase bound label to determine the amount of human plasminogen activator-human tissue plasminogen activator inhibitor complex in each of the aliquots, and
• (6) determining the amount of active human plasminogen activator inhibitor based on the difference between the amount of human tissue plasminogen activator-human plasminogen activator inhibitor complex in the two aliquots,
• and wherein
• (a) there is used as the first antibody a monoclonal antibody JTI-4 produced by the hybridoma JTI-4 (FERM BP-2318) which specifically binds a human plasminogen activator inhibitor linked to an insoluble solid carrier having a specular surface,
• (b) there is used as the second antibody a polyclonal antibody which specifically binds a human tissue plasminogen activator labeled with an enzyme, and
• (c) there is used as the surfactant a nonionic surfactant having an HLB (Hydrophilic-Lipophilic Balance) of at least 16 and a concentration of 0.001-0.1 w/w %, said nonionic surfactant being at least one member selected from the group consisting of polyoxyalkylene alkyl aryl ether series, polyoxyalkylene alkyl ether series, polyoxyalkylene polyhydric alcohol fatty acid ester series and polyoxyethylene polyoxypropylene polyol series.