A microwell plate-based multiplex immunoassay for simultaneous quantitation of antibodies to infectious viruses.

Antibodies (Abs) to disease-causing viruses in human blood are important indicators of infection status. While ELISA has been widely used to detect these Abs, a multiplex assay system for simultaneous detection of multiple Abs is still a desirable alternative method for a more efficient screening process because of the lack of multiplexing ability in ELISA. However, as all antibodies are based on immunoglobulin and recognized commonly by the same secondary antibody, it is impossible to multiplex the conventional indirect ELISA in a 96-microwell plate-based platform. To overcome this hurdle, we designed an assay consisting of two steps: capturing target Abs by specific antigens on DNA-encoded gold nanoparticles; and quantifying the target Abs by producing RNase H-mediated detection signals based on the DNA and additional RNA probes. With this newly designed method, we could simultaneously analyze three infectious disease-related Abs, anti-HIV Ab, anti-HCV Ab, and anti-HBV Ab, on the microwell-based platform. The assay performance was evaluated by comparison with ELISA. Furthermore, the accuracy and precision of the assay in a practical application was also estimated by determining the amount of target Abs in human serum solutions.

Orphanin FQ/nociceptin blocks methamphetamine place preference in rats.

Orphanin FQ/nociceptin (NOC) has been reported to regulate dopaminergic neurotransmission in rewarding pathway, and to suppress the development of conditioned place preference (CPP) induced by certain addictive drugs. In this study, we investigated the effect of NOC on CPP induced by repeated administration of methamphetamine (MAP) in rats. Repeated administration of MAP (1 mg/kg, i.p.) induced substantial CPP. MAP-induced CPP was completely suppressed by NOC (10 nmol, i.c.v.). Pretreatment with [Nphe1]nociceptin(1-13)NH2 (50 nmol, i.c.v.), an antagonist of the NOC receptor, antagonized the suppressive effect of NOC on MAP-induced CPP. These results suggest that NOC blocks MAP-induced CPP by activation of the NOC receptor.

Mechanism of nicotine-evoked release of 3H-noradrenaline in human cerebral cortex slices.

1. The mechanism of stimulation of noradrenaline (NA) release by nicotine (NIC) was investigated in human cerebral cortex slices preloaded with 3H-noradrenaline. 2 NIC (10-1000 micro M) increased 3H-NA release in a concentration-dependent manner. 3. NIC (100 micro M)-evoked 3H-NA release was largely dependent on external Ca2+, and was attenuated by omega-conotoxin GVIA (0.1 micro M) but not by nitrendipine (1 micro M). 4. Tetrodotoxin (1 micro M) and nisoxetine (0.1 micro M) attenuated the NIC (100 micro M)-evoked release of 3H-NA. 5. Mecamylamine (10 micro M), dihydro-beta-erythroidine (10 micro M) and d-tubocurarine (30 micro M), but not alpha-bungarotoxin (alpha-BTX, 0.1 micro M), attenuated the NIC (100 micro M)-evoked release of 3H-NA. 6. NIC (100 micro M)-evoked release of 3H-NA was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 micro M) and D(-)-2-amino-5-phosphonopentanoic acid (D-AP5, 100 micro M), but attenuated by MK-801 (10 micro M). MK-801 (0.1-1000 micro M) displaced the specific binding of 3H-nisoxetine with K(i) values of 91.2 micro M. NIC (100, 300 and 1000 micro M) did not induce 3H-D-aspartate release in human cerebral cortex slices. 7. NIC (100 micro M)-evoked release of 3H-NA was attenuated by 7-nitroindazole (10 micro M), N(G)-nitro-L-arginine methyl ester HCl (L-NAME, 30 micro M), N(G)-monomethyl-L-arginine acetate (L-NMMA, 300 micro M). [(3)H]-NA release induced by NIC (100 micro M) was attenuated by methylene blue (3 micro M) and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ, 10 micro M), and enhanced by zaprinast (30 micro M). 8. In conclusion, NIC stimulates the release of 3H-NA through activation of alpha-BTX-insensitive nicotinic acetylcholine receptors in the human cerebral cortex slices and this action of NIC is associated with modulation of the NO/cGMP pathway.