Optimized Small Molecule Antibody Labeling Using the Amicon Pro Device
Protein immuno-detection encompasses a broad range of analytical methodologies, including Western blotting, flow cytometry and microscope-based applications. These assays, which detect, quantify and/or localize expression for one or more proteins in biological samples, are reliant upon fluorescent or enzyme-tagged, target-specific antibodies. While indirect detection strategies are sufficient for single targets, multiplexing requires uniquely labeled reagents. This is further underscored by the growing use of clinical samples where increasing information content per test, through multiplex assays, is paramount to improved understanding of biological processes. However, while small molecule labeling kits are commercially available with a range of detection moieties, the workflow is hampered by a requirement for multiple buffer exchange steps that are both time-consuming and subject to sample loss. In addition to the basic workflow, optimization of reaction conditions is often required to ensure labeling of the resulting conjugate is commensurate with specifications outlined by the assay platform in which it will be used.
Many small molecule labeling kits are N-hydroxysuccinimide (NHS) chemistry-based, targeting lysine residues for conjugation; samples must be devoid of ammonium ions and primary amines necessitating buffer exchange. Dialysis, gel filtration and diafiltration represent the most common methods for buffer exchange. By combining buffer exchange with concentration, diafiltration potentially offers the faster, more efficient option. The Amicon™ Pro centrifugal device was specifically engineered to maximize buffer exchange via continuous flow diafiltration. The base was designed to exactly match contours of the 0.5mL Amicon™ Ultrafilter facilitating optimal exposure of the membrane’s surface area, while reducing volume between the plastic sidewalls (Figure 1). The tip also narrows promoting sample diafiltration within the exchange chamber; under continuous centrifugal pressure, buffer is metered in, mixed with the sample and forced across the membrane boundaries. While many smaller devices have volume constraints (≤0.5mL) and thus require multiple cycles for effective desalting, this upper reservoir holds up to 10mL of buffer permitting continuous single spin exchange.
To assess the relative capacity for buffer exchange, testing was carried out against representative devices for dialysis, gravity gel filtration and centrifugal diafiltration. Four parameters were measured: (1) processing time, (2) buffer exchange efficiency, (3) protein retention and (4) specific activity (functional integrity). The results are summarized in Table 1. The Amicon™ Pro device offered roughly equivalent or superior performance for all four attributes, suggesting it is well suited for Ab labeling. The device demonstrated the highest protein recovery; on average, <3% of starting BSA was lost. In fact, centrifugal-based methods, where concentration and buffer exchange are combined, had greater protein retention than either dialysis or gravity columns. The device also delivered a protein sample with similar specific activity as the dialysis method; the smaller diafiltration device demonstrated a relative loss in specific activity most likely due to sample instability arising from repeated concentration/dilution cycling. The poor performance of gravity columns may reflect stability issues arising from sample dilution and subsequent concentration. Lastly, the Amicon Pro device was relatively fast and required little hands-on effort (1 spin). Other methods were much longer; required multiple repetitive processing steps and/or use of a separate device for concentration to complete.
The degree of labeling is influenced by the molar ratio of reactants, activity of labeling reagents and nature of the probe. Optimization of the dye/protein molar ratio (F/P ratio) is often required to ensure a probe’s signal strength is consistent with signal-to-noise requirements of the assay. In the Amicon Pro device, reactions are initiated by centrifugal buffer exchange with the labeling mix replacing the Ab storage buffer in the 0.5mL attachment. Thus, a range of labeling conditions can be quickly assessed through diafiltration-based reaction termination. F/P ratios were determined following labeling in the presence of increasing amounts of NHS-fluorescein dye. Not surprising, the probes’ F/P values increased with increasing dye; however, this response appeared to plateau at 30 µg, a quantity far greater than standard kit recommendations (Figure 2). More significantly, labeling was almost instantaneous (≥ 70% following exchange spin) and reached a maximum at 30 minutes post spin independent of dye input (Figure 3). By contrast, following a standard two-hour tube format, F/P values continued to increase with dye concentration. Consistent with kinetics of NHS-based labeling, the tube format also demonstrated a slower, more linear response with signal leveling off at 60 minutes. The observed differences in F/P values between the two methods is best explained by the fact that the centrifugal process is continuous; dye concentration remains high and constant, but unbound is rapidly forced out of the Amicon Pro’s reaction chamber thus minimizing exposure to excess label.
Translating the outlined benefits to the overall workflow (upfront cleanup, labeling, unbound dye removal), the Amicon Pro device reduced processing time to 30 minutes (as compared to two days for dialysis-based approaches). The device also demonstrated a significant improvement in Ab recovery (on average, 50% greater yield); this most likely reflects single device containment for the entire process. For commercial Abs, sample loss can be cost-prohibitive as well as limiting the number of tests performed with a given labeled batch. Functional performance of a number of differentially-labeled conjugates was further evaluated by Western blotting using the SNAP i.d.™ detection system. From the dilution series depicted in Figure 4, for a similar amount of primary Ab (1 µg/mL), signal intensity was greater with user-labeled Abs than a commercially available conjugate. Further, there were no adverse effects on the probe’s performance (signal intensity, specificity or background) incurred by using significantly greater amounts of dye as was done with the Amicon Pro device.
0.5 mL Device
Amicon Pro Device
|Process Time||16 hours||40 minutes||45 minutes||15 minutes|
|Percentage of Salt Removal A||99.9 ± 0.04||99.6 ± 0.24||100 ± 0.004||100 ± 0.001|
|Percentage of Protein Retention B||82 ± 7.2||97 ± 4.2||92 ± 2.7||97 ± 0.7|
|Specific Activity C|
|4.1 ± 0.35||3.3 ± 0.22||3.9 ± 0.4||4.3 ± 0.07|
|Concentration Step Required||YES||YES||NO||NO|
A Desalting efficiency – removal of 1 M NaCl as measured by a change in sample conductivity
B Recovery – 500 µg BSA was buffer exchanged; recovery quantified using the Direct Detect™ IR- spectrometer
C Enzymatic activity – a chemiluminescent phosphatase assay was performed following buffer exchange of GST-LPP. Specific activity is expressed as A405 signal per µg GST-LPP.