We therefore added 5 μl of mineral oil into each well before
they were sealed. Mineral oil prevented evaporation and improved the performance of detection of various concentrations of rIL-3 (Fig. 4) and rSCF (not shown). To compare various immunoassays for detecting cytokines, we tested the performance of Nano-iPCR I and II, iPCR and ELISA for detection of rIL-3 and rSCF at various concentrations. For IL-3, polypropylene wells of the 96-well PCR plate (Eppendorf) were coated with extravidin, followed by anti-IL-3 polyclonal antibody (Nano-iPCR I). Alternatively, wells of TopYield strips (NUNC) were coated directly with anti-IL-3 antibody (Nano-iPCR II, iPCR and ELISA). Next, free binding sites were blocked with selleck TPBS-2% BSA and rIL-3 at various concentrations was added. After incubation, unbound IL-3 was removed by washing with TPBS. Further course of the procedures differed depending www.selleckchem.com/products/bmn-673.html on the method used (see Section 2 and Fig. 1). Analysis of data obtained showed that Nano-iPCR I (Fig. 5A) exhibited clear concentration-dependent differences in the range of 0.01–100 ng/ml of IL-3 with Cq values from ~ 35 (at 100 ng/ml) to ~ 46 (at 0.01 ng/ml). These relatively high values probably reflect low protein binding capacity of PCR polypropylene wells. Nano-iPCR
II (Fig. 5C) performed in polycarbonate TopYiled strips showed lower Cq values in the range from ~ 19 (at 100 ng/ml) to ~ 32 (at 0.01 ng/ml). With iPCR (Fig. 5E), the dose–response curve was similar to that of Nano-iPCR II assay, except for even lower Cq values, from ~ 15 (at 100 ng/ml) to ~ 24 (at 0.01 ng/ml). This was in part caused by lower Cq values in negative controls
(without IL-3) in iPCR compared to Nano-iPCR, and could be related to higher nonspecific binding of the biotinylated template used for iPCR. In contrast to Nano-iPCR and iPCR, ELISA assay (Fig. 5G) was less sensitive and the range of IL-3 concentrations detectable by the assay was narrower (between 0.1 and 10 ng/ml). Similar data were obtained when various assays were used for detection of rSCF. Thus, Nano-iPCR I (Fig. 5B), compared to Nano-iPCR II (Fig. 5D) and iPCR (Fig. 5F), was Tacrolimus (FK506) characterized by relatively high Cq values (including negative controls without rSCF) and higher sensitivity at low concentration of rSCF. ELISA assay (Fig. 5H) was again less sensitive, and also the range of rSCF concentrations detectable by the assay was reduced (0.1–10 ng/ml). The data indicate that Nano-iPCRs and iPCRs are superior in sensitivity and exhibit broader range of detectable concentrations than ELISA. To prove the convenience of Nano-iPCR we attempted to determine changes in the amount of rSCF during growth of BMMCs in RPMI-1640 medium supplemented with 10% FCS and SCF. Cell-free samples from cell cultures were collected at 24 h intervals for 5 days.