In restriction digestion, BSA has been used to stabilize enzymes during DNA digestion. It is also widely used as a biomolecule to block active sites on surfaces. Formation of the anti-BSA/BSA immune complex is relevant to studies of the receptor site of the red blood cells . The five-channel BI-4500 SPR analysis system with integrated auto-sampler was used to study the binding interaction of the anti-BSA/BSA immune complex in real-time. The BI Autosampler can run two well-plates in any combination of 12, 96, and 384 well formats for a maximum of 768 different samples. The high throughput and full automation of this system greatly facilitates in optimizing the binding conditions, simplifying the experimental workflow, and analyzing the interaction kinetics.
On a CM Dextran sensor chip, different amounts of BSA were simultaneously immobilized in four channels via amine coupling. Immobilization was performed using an automated graduated injection feature of the BI SPR software package, which requires only one sample injection for the entire graduated immobilization process making immobilization simple, accurate, and sparing on sample consumption. Channels 1 through 4 were immobilized with different amounts of BSA (in the order of decreasing density) and channel 5 was left bare for reference subtraction. The ability to test interactions using multiple BSA densities strengthens the data quality by helping to identify and minimize non-ideal or secondary binding effects. Seven sets of three anti-BSA concentrations (2.5, 5.0, and 10 nM) were repeatedly exposed serially in all five channels. A regeneration injection of 20mM NaOH followed each binding interaction in order to release the anti-BSA from the immobilized BSA and expedite further testing (observed as upward spikes followed by a return to baseline).
Figure 1A shows the results of continuous BSA testing over a 13 hour period. The channels are shown overlapped so that the binding responses of each channel to the same anti-BSA injection can be more easily compared to each other. As expected, channels with greater amounts of BSA immobilized produced larger responses than that of channels immobilized with few amounts of BSA. Note that the consecutive injections are quite reproducible, demonstrating the extent of system stability, precision of sample delivery, and robustness of functionalize sensor surface.
Download a PDF of Application Note: 107 – Binding Kinetics Analysis with SPR: Interaction between Bovine Serum Albumin (BSA) and Anti-BSA
- Varga, L., Thiry, E., Fust, G. Immunology, 1988, 64, 381-384
- Sigal, G. B., Bamdad, C., Barberis, A., Strominger, J., Whitesides, G. M. Anal. Chem. 1995, 68, 490-497