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.