2.1.4 Results and discussion
2.1.4.1 Detection antibody
In the chosen format of a competitive quantification, the detection antibody represents the signal-giving component. Several parameters such as labeling ratio, incubation time and detection antibody concentration were optimized to enhance the signal intensity and thereby tune the limit of detection.
Selection of antibody with highest affinity
As described in the theoretical part earlier, the affinity of the used detection antibody as well as its concentration, had to be considered when optimizing the competitive quantifica-tion method. Amongst several detecquantifica-tion antibodies, the mouse anti-human PSA antibody with the clone number CHYH2 exhibited the highest affinity with a KD of 8.2 nM and was therefore chosen for all subsequent experiments.
Labeling of detection antibody
A directly labeled detection antibody was preferred rather than the utilization of a sec-ondary antibody to keep the number of incubation steps as low as possible. As the fluores-cent properties of the labeled antibody influence the overall assay sensitivity, it was tested which labeling strategy provided the highest signal intensities. As depicted in figure 2.3 A, a fluorescent dye was conjugated to the detection antibody in different dye:protein ratios.
The gained fluorescent intensities of the dye labeled antibody were then compared with a quantum dot (QD) labeling as seen in figure 2.3 B. Quantum dots are known to exhibit a fluorescent yield well above the ones of dyes and therefore might be an alternative option to increase signal intensities.
An increased dye:protein ratio resulted in higher signal intensity (figure 2.3 A). An overla-beling, which lead to a reduced affinity and binding, was, however, observed at a dye:protein ratio of 8:1. Consequently, a dye:protein ratio of 4:1 was found to be the most suitable strategy because the highest signal intensities were achieved here. Moreover, concentration changes resulted in a more pronounced signal intensity difference and smaller concentration changes could better be detected.
A site-click quantum dot labeling (quantum dot was conjugated to glycosylation of anti-body) did not show any superior fluorescent properties in comparison with a dye labeling.
An explanation for this might be the increased size of a quantum dot (26 nm in diam-eter). During the incubation process, the quantum dot-coupled antibody might behave
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Dye vs. QD
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CHYH2 = anti-PSA detection antibody, DY649P1 = Cy5-like fluorescent dye, QD = quantum dots, FU = fluorescent units
Figure 2.3: Labeling of detection antibody using the Cy5-like fluorescent dye DY-649P1 (A) or fluorescent quantum dots (B).
Experimental setup: Different concentrations of labeled detection antibody were incubated on 2D epoxysilanized microarrays with immobilized PSA.
more like a rolling nanoparticle with a size of approximately 36 nm, rather than a free moving molecule. In this regard, a reduced diffusion rate due to sterical hindrances and mass transportation limitations might prevent the antibody from binding. Similar obser-vations are also described and discussed in literature [14].
Other signal increasing methods such as rolling circle amplification or reporter enzymes for the generation of fluorescent dyes would not have met the requirements of a fast and reproducible one-step solution for the desired application.
Incubation time
Investigating the incubation time as the most time-consuming step, a compromise between an adequate signal intensity and a short duration had to be made. The gained results, represented in figure 2.4, indicate that the steady-state antibody binding capacity was at-tained after one hour. In contrast to the quickly rising signal intensity, only a moderate background noise increase was observed. As the signal-to-noise ratio (S/N) did not fur-ther improve at incubation times beyond fifteen minutes, this incubation time was chosen for our setup. As discussed by Kusnezow et al. [15], the optimal design of a microarray assay to compensate kinetic limitations would span an incubation time of several hours.
Yet, it is also mentioned that prolonged incubation times lead to a pronounced dissocia-tion. Consequently, a longer incubation time would not necessarily result in a better assay performance as dissociation processes can be dependent on additional and probably more uncontrollable parameters. This also applies for washing steps, which in our setup were reduced to a minimum of two PBST washing steps followed by three rinsing steps with water at the end of each incubation.
Moreover, choosing a time point not in the plateau level but in the dynamic range, required an exact incubation time control to enable a precise quantification.
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Figure 2.4: Influence of incubation time on signal intensity
Experimental setup: Labeled detection antibody in a concentration of 3 ng/ml was incubated for different amounts of time on a 2D epoxysilanized microarray with immobilized PSA.
Concentration of detection antibody
According to the equation transformation made in the introduction, the concentration of the detection antibody turned out to be the most influential parameter regarding the quan-tification range. In theory, an equilibrium between the detection antibody in solution, the antigen in solution and the immobilized antigen has to be reached. As demonstrated in figure 2.5, the dynamic range of the sigmoidal competitive quantification curve could be adjusted by the concentration of detection antibody. Hereby, the inflexion point is
de-termined by the antibody affinity and defined as the point at which the ratio of antigen bound and antigen unbound in solution equals 1:1. Given a target concentration of PSA of 0.035 - 0.35 nM (1 -10 ng/ml) in solution, the most suitable concentration of the detection antibody was at 0.02 nM (3 ng/ml). Lower concentrations would have been thinkable too, but taking into account that lower concentrations of detection antibody would also result in a decreased absolute fluorescent signal, a compromise had to be made here.
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Figure 2.5: Effect on the dynamic range using different detection antibody concentrations
Experimental setup: Each sample of a PSA serial dilution series was incubated with a defined concentration of labeled detection antibody. Sample mixture with detection antibody was then given on a 2D epoxysilanized microarray with immobilized PSA. After washing, fluorescence intensity of each PSA spot was measured and analyzed. A semilog curve fit was performed for data points within the dynamic range.