Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia.

Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

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.