Bioaffinity binding assays such as the immunoassay are widely used in life science research. In an immunoassay, specific antibodies are used to bind target molecules in the sample, and quantification of the binding reaction reveals the amount of the target molecules. Here we present a method to measure bioaffinity assays using the two-photon excitation of fluorescence. In this method, microparticles are used as solid phase in binding the target molecules. The degree of binding is then quantified from individual microparticles by use of two photon excitation of fluorescence. We demonstrated the effectiveness of the method using the human alpha-fetoprotein (AFP) immunoassay, which is used to detect fetal disorders. The sensitivity and dynamic range we obtained with this assay indicate that this method can provide a cost-effective and simple way to measure various biomolecules in solution for research and clinical applications.

Antigen uptake and the following maturation of dendritic cells (DCs) are pivotal to the initiation of specific antimicrobial immune responses. DCs also play an important role in the recruitment and activation of the cells of the innate immune system. We have examined the interactions of DCs with Borrelia burgdorferi to find explanations for the difficulties the human immune system has in dealing with the bacterium. Phagocytosis of B. burgdorferi by immature DCs and the effect of the bacterium on the maturation and interleukin-8 (IL-8) secretion of DCs were studied. Borreliae were phagocytized and processed into fragments by DCs; narrow tube-like pseudopods and broad pseudopods were used for the engulfment. The immature DC population gained a heterogeneous appearance within 2 h of incubation with the borreliae. A 24 h coculture with borreliae induced maturation and IL-8 secretion in the DCs in a manner comparable with the effect of lipopolysaccharides. All strains studied, including a mutant strain lacking outer surface proteins A and B, were capable of inducing these responses. Thus, our results did not show any clear inadequacy concerning the way DCs are dealing with B. burgdorferi. However, further studies on the subject are required.

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.

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A new type of instrumentation for single-step bioaffinity assays and microvolume fluorometry is presented. The concept is based on the use of two-photon excitation by a low-cost near-infrared laser and individual observation of bioactive fluorescent microparticles. The applicability of the instrument is demonstrated by a microparticle based multiplexed bioaffinity assay where several fluorescent markers are simultaneously excited. This instrument can be applied in the growing fields of drug discovery, in life science research, and in routine laboratory diagnostics.