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Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%o. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence -Asn-49-Arg-50-Phe-51-Gly-52-of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

A rapid and highly sensitive CE immunoassay method integrating mixing, reaction, separation, and detection on-chip is described for the measurement of alpha-fetoprotein (AFP), a liver cancer marker in blood. Antibody-binding reagents, consisting of 245-bp DNA coupled anti-AFP WA1 antibody (DNA-WA1) and HiLyte dye-labeled anti-AFP WA2 antibody (HiLyte-WA2), and AFP-containing sample were filled into adjacent zones of a chip channel defined by the laminar flow lines of the microfluidic device using pressure-driven flow. The channel geometry was thus used to quantitatively aliquot the reagents and sample into the chip. DNA-WA1 was electrokinetically concentrated in the channel and sequentially transported through the AFP-sample zone and HiLyte-WA2 zone by ITP in such a manner that the AFP sandwich immune complex formation took place in the sample and HiLyte-WA2 zones. The sandwich AFP immune complex was then detected by LIF after CGE in a separation channel that was arranged downstream of the reaction channel. AFP was detected within 136 s with a detection sensitivity of 5 pM. The on-chip immunoassay described here, applying ITP concentration, in-channel reaction, and CGE separation, has the potential of providing a rapid and sensitive method for both clinical and research applications.

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.

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