Performance evaluation of the new rapid fertility assays in whole blood and plasma on PATHFAST.

BACKGROUND: The purpose of this study was to evaluate the validity of the PATHFAST fertility marker assays for the rapid measurement of female hormones including: LH, FSH, Estradiol (E2), Progesterone (P4), Prolactin (PRL), and HCG. As for the PATHFAST fertility marker assays, female hormones can be measured by whole blood, plasma, and serum. METHODS: The correlation of the heparin whole blood and the plasma samples in the PATHFAST was examined. The method comparison study of PATHFAST fertility marker assays was performed with the Elecsys 2010, AIA-360, IMMULITE 2000, miniVIDAS, and ARCHITECT i2000. Determination of the reference range values of the PATHFAST fertility marker assays was performed with serum samples which were obtained during the follicular phase, mid-cycle, luteal phase, and postmenopausal phase. RESULTS: The results of plasma samples of female hormones measured by the PATHFAST correlated highly with those of whole blood samples (r > 0.9). The results of LH, FSH, E2, P4, PRL, and HCG as measured by the PATHFAST correlated well with other commercial fertility assays (r > 0.9). Reference values of PATHFAST fertility marker assays were equivalent to those of other commercial methods. CONCLUSIONS: The PATHFAST system is an accurate diagnostic tool for the rapid assay of female hormones. The PATHFAST fertility marker assays can be useful in a physician's office laboratory (POL) as well as various clinical sites during infertility treatment.

A new time-resolved fluorometric microarray detection system using core-shell-type fluorescent nanosphere and its application to allergen microarray.

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab' fragment-conjugated fluorescent nanosphere (Fab' nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab' nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 mul of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r > 0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n = 5).

A core-shell-type fluorescent nanosphere possessing reactive poly(ethylene glycol) tethered chains on the surface for zeptomole detection of protein in time-resolved fluorometric immunoassay.

To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymerization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH(2) nanosphere). The fluorescent NH(2) nanosphere was constructed by incorporating fluorescent europium chelates with beta-diketonate ligands in the core of the NH(2) nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH(2) nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for alpha-fetoprotein (AFP) determination. Anti-human AFP Fab' fragment was covalently conjugated onto the maleimide nanosphere (Fab' nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 x 10(-)(21) mol, M(w,AFP) = 70000) for AFP. The imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when approximately 1.9 x 10(9) particles of the Fab' nanosphere were applied to the microtitration well.