BIO-IMMUNOASSAY FOR STAPHYLOKINASE IN BLOOD

A bio-immunoassay (BIA) for the determination of staphylokinase (STA) activity in a plasma milieu has been developed. MA-7H11, a murine mono clonal antibody raised against STA, which has a high affinity for STA but does not interfere with the complex formation between plasmin(ogen ) and STA or with plasminogen activation by STA, was coated on microti ter plates at a concentration of 4 mug/ml. STA-containing samples were incubated overnight at 4-degrees-C and, after extensive washing, boun d STA was quantitated by incubation with plasminogen (final concentrat ion 0.5 muM) for 1 h at 37-degrees-C, followed by determination of gen erated plasmin from the absorbance at 405 nm 10 min after addition of the chromogenic substrate S-2403 (final concentration 0.3 mM). Calibra tion curves constructed with natural (STAN) or recombinant (STAR) STA were linear between approximately 1 and 10 nM, with a lower detection limit of less-than-or-equal-to 1 nM in buffer and in plasma of the hum an, baboon or hamster. Following bolus injection of STAR in hamsters, the disposition rate of STAR activity from plasma, determined with the BIA correlated very well (r = 0.98) with that of STAR-related antigen determined by ELISA, indicating that STAR is cleared in a functionall y active form. The initial half-life was about 2 min, as determined wi th both methods. Following continuous intravenous infusion over 1 h in baboons, the plasma clearance of STAR activity, determined from the i nfusion rate and the steady-state plasma level of STAR activity, range d between 45 and 62 ml/min for doses of STAR between 0.063 and 0.250 m g/kg.