One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1 mm i.d. × 5 mm and contained 8-9 nmol of immobilized HSA. These microcolumns were used from 0.10-1.0 mL/min and gave results within 35 s to 2.8 min of sample injection. Limits of detection down to 0.10-0.28 ng/mL (1.5-4.2 pM) or 25-30 pg/mL (0.38-0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ± 0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

Two types of resin-conjugated Tamiflu analogs were synthesized by modifying our original synthetic route of oseltamivir phosphate (Tamiflu). The prepared resins bound to influenza virus neuraminidase, the main target of Tamiflu. The resins will be useful for isolating and identifying presumed endogenous vertebrate proteins that interact with Tamiflu, which might relate to the rarely observed abnormal behavior exhibited by some influenza patients treated with Tamiflu.

Protein G can be a valuable binding agent for antibodies and immunoglobulins in methods such as immunosensors, chromatographic-based immunoassays, and immunoaffinity chromatography. This report used the method of peak decay analysis along with frontal analysis and zonal elution studies to characterize the binding, elution and regeneration properties of affinity microcolumns that contained immobilized protein G. Frontal analysis was employed with rabbit immunoglobulin G (IgG) to characterize the binding capacity of these affinity microcolumns. Zonal elution experiments looking at the retained peaks for small injections of labeled rabbit IgG were used to optimize the column regeneration conditions. Peak decay analysis was then used to look at the effects of flow rate and elution pH on the release of several types of IgG from the protein G microcolumns. This approach made it possible to obtain detailed information on the use and behavior of such columns, as could be used in future work to optimize the capture or analysis of IgG and antibodies by such devices. The same approach and tools that were used in this report could also be adapted for work with affinity columns that make use of other supports, binding agents or targets.

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limits of detection, and analysis time. All these methods gave detection limits in the range of 8–19 ng/mL and precisions ranging from ± 5% to ± 10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.