The development of microcolumns containing support material in high-performance affinity chromatography (HPAC) has led to improvements in the performance and efficiency of many types of separations.  One type of support material that can be used in the microcolumns is silica particles. Silica particles have the advantage of easy modification for stationary phase attachment. One important application of microcolumns containing modified silica is in the development of chromatographic immunoassays, which is a technique used to study relevant protein biomarkers. One important protein of interest is human serum albumin (HSA), which can also be used as a model analyte to optimize a chromatographic immunoassay or to target other biomarkers. The purpose of this project was to implement microcolumns containing modified silica particles for the development of HPAC-based competitive immunoassays. This work was conducted using a technique known as HPAC in which a chromatographic-based one-site immunometric assay was performed. Initially fluorescein-labeled anti-HSA antibodies were used to bind to HSA in solution. The limit of detection (LOD) was determined to be ~0.6 ng/mL HSA when the amount of labeled antibodies was 1 ng. In order to lower the LOD, a near-infrared fluorescent dye was employed. The next phase of the experiment involved conjugating a NIR fluorescent dye to the anti-HSA antibodies. This conjugate was then used in the assay and gave an LOD of 0.05 ng/mL when either 150 pg or 300 pg of NIR-fluorescent labeled HSA was used.        The objective of this study was to optimize a one-site chromatographic immunoassay using HSA as a model.  A LOD of less than 0.1 ng/mL was achieved by using a NIR fluorescent dye conjugated to anti-HSA antibodies. This research was supported by the MARC/U*STAR Program (Grant No. 5T34GM007672-33) and in part by the National Science Foundation (Grant No.CHE-1156560) and the National Institute of Health.

Implementation of novel miccrocolumns in high performance affinity chromatography and related applications

247th National Meeting (2014)

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