Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats

Abstract

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.

Peak decay analysis and biointeraction studies of immunoglobulin binding and dissociation on protein G affinity microcolumns

“…This has previously been done by making repeated measurements of the binding capacity for rabbit IgG over the course of up to 50 application and elution cycles. In work using a similar protein G support to that employed in this current study, it was found that a variation in the binding capacity of less than one 1% (e.g., 0.3-0.9%) occurred from one application/elution cycle to the next during the course of these experiments [6].…”

“…These microcolumns were stored in a neutral pH phosphate buffer at 4°C when not in use. The types of protein G microcolumns that were used in this study have been found to be stable for up to one year and over approximately 200 application and elution cycles [6].…”

Affinity chromatography: A review of trends and developments over the past 50 years

“…Immunoglobulin-binding proteins are a third class of biological binding agents that are often used in affinity chromatography [100,101,[157][158][159][160][161][162][163][164][165][166][167]. As indicated by Figure 6, this combination is the second most-reported form of bioaffinity chromatography.…”

“…Mixtures of immunoglobulin-binding proteins or recombinant forms such as protein A/G and protein G/L have also been used in affinity methods [100,101]. These immunoglobulin-binding proteins can be used to capture and purify antibodies, as well as to measure immunoglobulins in samples [100,101,[157][158][159][160][161][162][163][164][165][166][167].…”

High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents

“…70,71 Protein G columns have also been used to adsorb antibodies for use in competitive binding immunoassays. 72,73 This latter work has utilized such columns in a simultaneous injection competitive immunoassay to measure HSA 72 and in sequential injection-based methods to analyze HSA or transferrin. 72,73…”