This review focuses on the application of novel technologies for generating biocompatible surfaces for high-throughput screening (HTS) of proteins. Various methods of coupling and spotting proteins on self-assembled monolayer (SAM) surfaces will be described along with the protein chip challenges pertaining to spot homogeneity, morphology, biocompatibility and reproducibility.
We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format. The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions. All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 L total volume). The detection limits for the immobilized probes on the microarray surface were 0.5 g/mL (200 fmol/spot) for the peptide antigen and 0.1 g/mL (3 fmol/spot) for the recombinant antibodies. Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 g/mL. Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface. For recombinant antibody fragments with a kDa of 10100 nM, we have established an easy and direct immunoassay. In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications. Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria. Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.
We report a novel high-throughput (HTP) protein chip platform, constructed on gold using self-assembly techniques, for conducting high quality antigen-antibody interactions. Biotinylated monolayers were used to immobilize a streptavidin surface with high packing density. This biocompatible platform was then used for detection of serum IgM antibodies. Serum samples of patients suspected to suffer from Lyme borreliosis were used to validate the protein chip platform using biotinylated peptide AAOspC8 molecules as the test probes. Various experimental parameters such as the effect of concentration of probes, targets, temperature of incubation, and their effect on the resulting signal-to-noise ratio are described in detail. Highly specific protein interaction data with a high signal-to-noise ratio were obtained with serum sample solutions as low as 1 microL/spot (1/10 diluted).
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