Multivalent immunoprobes can improve the sensitivity of biosensors because increased valency can strengthen the binding affinity between the receptor and target biomolecules. Here, we report surface-enhanced Raman scattering (SERS)-based immunoassays using multivalent antibody-conjugated nanoparticles (NPs) for the first time. Multivalent antibodies were generated through the ligation of Fab fragments fused with Fc-binding peptides to immunoglobulin G. This fabrication method is easy and fast because of the elimination of heterologous protein expression, high degrees of antibody modifications, and covalent chemical ligation steps. We constructed multivalent antibody−NP conjugates (MANCs) and employed them as SERS immunoprobes. MANCs improved the sensitivity of SERS-based immunoassays by 100 times compared to standard antibody−NP conjugates. MANCs will increase the feasibility of practical SERS-based immunoassays.
We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.
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