MC-3 Extension of the sensitivity of time resolved immunofluorometry to the attomolar range after novel multiple labelling of streptavidin: R.C. Morton, E. Reichstein, M.J. Khosravi and E.P. Diamandis (CyberFluor Inc., 179 John Street, Toronto, Ontario, Canada M5T 1X4)

Abstract

Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A2 (PLA2), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein. Antibodies were raised in rabbits against recombinant human PLA2 proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 μg/L for the TR-FIA for GIB PLA2, 1 μg/L (GIIA), 35 μg/L (GIID), 3 μg/L (GIIE), 4 μg/L (GIIF), 14 μg/L (GIII), 11 μg/L (GV), 2 μg/L (GX), 92 μg/L (GXIIA) and 242 μg/L (GXIIB). All secreted PLA2s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA2 in serum (median 19.0 μg/L, range 13.1–33.7 μg/L, n=4) as compared to the healthy blood donors (median 1.8 μg/L, range 0.8–3.4 μg/L, n=28, P<0.0001). The concentration of GIIA PLA2 in the sera of septic patients (median 315.7 μg/L, range 15.9–979.6 μg/L, n=34) was highly elevated as compared to that of the blood donors (median 1.8 μg/L, range 0.8–5.8 μg/L, n=28, P<0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA2 in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA2s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA2s above the detection limits of the current TR-FIAs.