A specific, highly sensitive radioimmunoassay has been developed for the determination of Serratia protease. The radioimmunoassay (RIA) was based upon competition of the protease with 1Z51-labeled protease for antiprotease, followed by a second antibody to separate bound enzyme from free enzyme. The RIA provided a range of 1 to 10 ng for determining the enzymes under conditions in which the enzymatic activity could not be measured. The assay was completely inhibited in the presence of human plasma. The inhibition resulted from a complex formation of the enzyme with plasma a,macroglobulin. By treatment of the complex with acetone, however, the RIA could be achieved.
1 Abbreviations used: RIA, radioimmunoassay; TSP. homogeneously purified Serratia protease; lz51-TSP, Serratia protease labeled with V; anti-TSP, anti-.Serratia protease serum prepared in rabbit; c+M, a,macroglobulin; B,, no binding.
Methods
Preparation of TSP. TSP was purified from cultured filtrate of Serratia sp. E,, by successive column chromatographies of DEAE-cellulose and Sephadex G-75 (2). The TSP was confirmed to be a homogenous protein with M, 6.0 x lo4 (2).
Radioiodination of TSP. TSP was labeled with lz51 according to the method of Yagi (5) with a slight modification. To the mixture of TSP (30 pg) and Na 1z51(500 PCi) in 0.3 ml of 0.1 M phosphate buffer, pH 7.0, was added 0.1 ml of 2.5 mM chloramine-T in the same buffer. After standing at 23Β°C for 5 min, 0.1 ml of 5 mM NaHSO, in the same buffer was added to the mixture. lz51-TSP was separated from free lz51 by a gel filtration of a column (1.4 x 23 cm) of Sephadex G-25 (fine). The specific activity of 1251-TSP was about 5.4 pCiIpg* The 12'I-TSP was kept at -20Β°C.
Preparation of antiserum. The antiserum against TSP (anti-TSP) was obtained by three subcutaneous injections of 5 mg of TSP at 3-week intervals to a rabbit (male, 4 kg). The TSP in 1 ml of 0.9% NaCl was emulsified with 1 ml of Freund incomplete adjuvant (Difco) for the first injection and suspended in 1 ml of 0.5% aluminium