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Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System.

Henseleit A, Pohl C, Kaltenbach HM, Hettwer K, Simon K, Uhlig S, Haustein N, Bley T, Boschke E - Biosensors (Basel) (2015)

Bottom Line: The bivalence and heterogeneity of the antibody caused a complex binding mechanism.This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA).The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Dresden 01062, Germany. anja.henseleit@tu-dresden.de.

ABSTRACT
We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

No MeSH data available.


Related in: MedlinePlus

Kinetics of HSA-specific antibodies binding to immobilized HSA. Plots generated by applying the bivalent analyte model (red lines) overlay the plots associated with each antibody concentration (black lines; from top to bottom: 3.4 µM, 0.9 µM, 0.2 µM, 53.9 nM, 13.5 nM, 3.4 nM). Values resulting from the average of two sensing spots and the subtraction of reference surface signals from raw signal measurements are presented on the graph. The graph below depicts the residuals of the fits.
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biosensors-05-00027-f004: Kinetics of HSA-specific antibodies binding to immobilized HSA. Plots generated by applying the bivalent analyte model (red lines) overlay the plots associated with each antibody concentration (black lines; from top to bottom: 3.4 µM, 0.9 µM, 0.2 µM, 53.9 nM, 13.5 nM, 3.4 nM). Values resulting from the average of two sensing spots and the subtraction of reference surface signals from raw signal measurements are presented on the graph. The graph below depicts the residuals of the fits.

Mentions: For kinetic measurements, antibody solutions ranging in concentration from 3.4 nM to 3.4 µM were sequentially injected. Data representing binding of the antibodies to HSA were evaluated using three different models. Figure 2, Figure 3 and Figure 4 show overlaid fits of different binding models, and the parameters are presented in Table 1.


Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System.

Henseleit A, Pohl C, Kaltenbach HM, Hettwer K, Simon K, Uhlig S, Haustein N, Bley T, Boschke E - Biosensors (Basel) (2015)

Kinetics of HSA-specific antibodies binding to immobilized HSA. Plots generated by applying the bivalent analyte model (red lines) overlay the plots associated with each antibody concentration (black lines; from top to bottom: 3.4 µM, 0.9 µM, 0.2 µM, 53.9 nM, 13.5 nM, 3.4 nM). Values resulting from the average of two sensing spots and the subtraction of reference surface signals from raw signal measurements are presented on the graph. The graph below depicts the residuals of the fits.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384080&req=5

biosensors-05-00027-f004: Kinetics of HSA-specific antibodies binding to immobilized HSA. Plots generated by applying the bivalent analyte model (red lines) overlay the plots associated with each antibody concentration (black lines; from top to bottom: 3.4 µM, 0.9 µM, 0.2 µM, 53.9 nM, 13.5 nM, 3.4 nM). Values resulting from the average of two sensing spots and the subtraction of reference surface signals from raw signal measurements are presented on the graph. The graph below depicts the residuals of the fits.
Mentions: For kinetic measurements, antibody solutions ranging in concentration from 3.4 nM to 3.4 µM were sequentially injected. Data representing binding of the antibodies to HSA were evaluated using three different models. Figure 2, Figure 3 and Figure 4 show overlaid fits of different binding models, and the parameters are presented in Table 1.

Bottom Line: The bivalence and heterogeneity of the antibody caused a complex binding mechanism.This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA).The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Dresden 01062, Germany. anja.henseleit@tu-dresden.de.

ABSTRACT
We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

No MeSH data available.


Related in: MedlinePlus