To facilitate the DDI, the covalent conjugate HA24 was synthesized from a 24mer ssDNA oligonucleotide and recombinant STV. Synthesis and purification of HA24 was carried out from the corresponding thiolated oligonucleotide, tA24 (for sequences, see Materials and Methods) using a heterobispecific cross-linker, as previously described (11
). The covalent DNA–STV conjugate can be used as a versatile molecular adapter, allowing for convenient tagging of biotinylated proteins with a single-stranded oligonucleotide. To employ the DDI method for the functionalization of a microplate with capture antibodies, preconjugates are produced from HA24 and one molar equivalent of the biotinylated antibody leading to the formation of a preconjugate, capable of hybridizing to surface-bound capture oligonucleotides (Fig. ). STV-coated microplates were used as a solid support for capturing, and to this end, were functionalized with biotinylated oligonucleotides, complementary to the DNA sequence in HA24. In a first set of experiments, the functionalization of the capture plate was investigated using biotinylated antibodies from goat directed against human IgG (GAH). To estimate the immobilization efficiency of the DDI, similar amounts of the biotinylated GAH were immobilized either directly in wells of microplates by physisorption, by means of the STV–biotin interaction in wells of the STV-coated microplate or by DDI, using the oligonucleotide-functionalized STV microplates (Fig. ).
Figure 3 DDI of capture antibodies from goat directed against human IgG (GAH). Immobilization efficiencies of three alternative techniques are compared: biotinylated GAH was immobilized by either direct physisorption (triangles), biotin–STV interaction (more ...)
Subsequent to immobilization of the GAH, serial dilutions of human IgG as a target antigen were incubated in the wells containing fixed amounts of the GAH capture antibody. Signal detection was first carried out by regular ELISA. As shown in Figure A, the capture plates produced by DDI led to the best detection limit of <30 amol/µl hIgG, corresponding to 4.5 ng/ml. Immobilization by physisorption and STV–biotin interaction led to slightly worse results, with a detection limit of ~100 amol/µl hIgG (15 ng/ml). This result is in agreement with prior studies of DDI (10
), in which the high efficiency of protein immobilization had been attributed to: (i) the reversibility of DNA hybridization, enabling a denser packing on formation of the protein layer; (ii) the lean structure of the rigid double-helical DNA spacer between the surface and the protein which may also contribute to a larger effective surface area; and (iii) to a higher biological activity, which may result from the larger distance between surface and antibody, enabling a more homogeneous type of reaction during the formation of the immuno-complex.
In a second set of experiments, the capture plates produced by the methods described above were used in an IPCR assay (Fig. B), employing pre-synthesized oligomeric conjugates comprised of biotinylated GAH, recombinant STV and bis-biotinylated dsDNA marker fragments (13
). Detection of PCR amplicons was achieved using TaqMan real-time PCR quantification (21
). Similar to the ELISA case, the performance of capture plates prepared by DDI exceeded those prepared by physisorption or STV–biotin interaction. In particular, DDI allowed a detection limit of ~0.3 amol/µl hIgG, corresponding to 45 pg/ml, while physisorption and STV–biotin interaction were slightly worse (~3 amol/µl, 450 pg/ml). Owing to the high sensitivity of IPCR, the detection limit of all three IPCR assays was found to be ~100-fold better than in the case of the ELISA analysis.
To further demonstrate the applicability of DDI-based immunoassays, the tumor marker human CEA (22
) was chosen as a target. In a model study, serial dilution samples were prepared by spiking normalized human plasma (BISEKO) with purified CEA. Commercially available polyclonal anti-CEA antibody (ACA) from rabbit was biotinylated, coupled to the HA24 conjugate, and the resulting preconjugate was used for preparing ACA-functionalized microplates by DDI. Serial dilutions of the CEA in blood serum were incubated in the wells containing the ACA, and signal detection was carried out either by regular ELISA using an ACA–STV–alkaline phosphatase conjugate and fluorogenic AttoPhos or by IPCR, using an oligomeric ACA–STV–bis-biotinylated dsDNA conjugate (13
) and real-time TaqMan PCR. As shown in Figure , the limit of detection for CEA was ~50 amol/µl (10 ng/ml) CEA for DDI–ELISA, while with the DDI–IPCR method, an ~1000-fold increase in sensitivity was obtained, allowing the detection of ~0.05 amol/µl (10 pg/ml) of CEA. In comparison, a regular ELISA assay, based on physisorbed ACA, only allowed the detection of ~500 amol/µl (100 ng/ml) of CEA (data not shown). These results clearly confirm the robustness and sensitivity of the DDI-based immunoassays, making them applicable to the detection of antigens in highly complex matrices, such as blood serum.
Figure 4 Application of the DDI–IPCR combined assay for the detection of CEA. The signals obtained for serial dilutions of CEA in human blood serum are compared for two different assays, i.e. the DDI–ELISA (light gray) and DDI–IPCR (black). (more ...)
As an additional test of applicability, IgG from rabbit (rIgG) was chosen as a target antigen. For this, biotinylated goat anti-rabbit IgG (GAR) antibody was coupled with HA24 and the resulting preconjugate was used in the DDI-based functionalization of a microplate. Serial dilutions of the rIgG antigen in buffer were allowed to bind and the target adsorbed to the surface was quantified by real-time IPCR, employing a pre-formed conjugate comprised of biotinylated GAR, STV and bis-biotinylated dsDNA (13
). In a parallel assay, the detection of IgG from humans (hIgG) was conducted for comparison. As indicated in Figure , the detection of rIgG led to much better results, as compared with those obtained for hIgG. As little as 0.03 amol/µl (4.5 pg/ml) of rIgG was detectable, while the analysis of hIgG only allowed the detection of 1 amol/µl (150 pg/ml) of antigen. The alterations in sensitivity reflect individual differences in the kinetic and thermodynamic binding properties of the two antibodies, GAR and GAH, employed in the immunoassays.
Figure 5 Detection of IgG from human (hIgG, black rectangles) and rabbit (rIgG, light-gray circles) by means of the DDI–IPCR assay. The standard deviation of duplicate measurements was <8%, e.g. 1.31 ± 0.093 for detection of 1 (more ...)
A general advantage of DDI-based immunoassays relies on the high-specificity immobilization mediated by the base pairing of complementary nucleic acids. This even allows for simultaneously immobilizing many different DNA-tagged complexes site-specifically on a DNA microarray (9
). Thus, DDI-based immunoassays, in principle, allow that the binding of the antigen target by DNA-tagged antibodies is carried out in homogeneous solution, and subsequently, the immuno-complexes formed are captured at the DNA-functionalized substrate by nucleic acid hybridization. This approach not only would reduce the number of incubation steps, but it also might lead to increased sensitivity, since the antibody–antigen interaction occurs faster in homogeneous solution than it does at the solid/liquid interphase in regular immunoassays.
To investigate these types of in-solution capture assays, we carried out a series of experiments using the rIgG/GAR system (Fig. ). Initially, a two-step assay was investigated. To this end, serial dilutions of rIgG in buffer were mixed with fixed amounts of the HA24–GAR preconjugate. To study how capture reagent concentration influences the sensitivity of the immunoassay, three different concentrations of the HA24–GAR were used, ranging from 2, 10 to 50 nM (Fig. A). The mixtures of the HA24–GAR with the rIgG were immediately applied to the wells of a microplate containing capture oligonucleotides, thereby allowing for the DNA-directed adsorption of the immuno-complexes formed in solution. Subsequent to incubation for 30 min, the plate was washed and the rIgG immobilized was quantified by real-time IPCR using a 500 pM solution of a conjugate, comprised of biotinylated GAR, STV and bis-biotinylated dsDNA (13
). As expected, both the signal intensities and sensitivity of the assay clearly depended on the concentration of capture reagent (Fig. A). A best sensitivity of ~0.1 amol/µl (15 pg/ml) was obtained for 10 nM of capture conjugate, while both an increase to 50 nM and a decrease to 2 nM of capture conjugate led to a reduced sensitivity of 300 amol/µl (45 ng/ml) and 2 amol/µl (300 pg/ml), respectively. However, the signal-to-noise ratio was not as good as that obtained in the analogous three-step assay (Fig. ). To explain the HA24–GAR concentration-dependent assay sensitivity in Figure A, one needs to consider the relative stoichiometric amount of HA24–GAR conjugate complexes and rIgG molecules. At high ratios of HA24–GAR:rIgG, binding sites of rIgG are likely to be blocked, and consequently, binding of the IPCR detection reagent is sterically hindered. Thus, lower IPCR signals result. At low ratios of HA24–GAR:IgG, incomplete oligonucleotide tagging of the antigen occurs, and thus, the sensitivity is decreased.
Figure 6 Performance of DDI-based in-solution capture assays for the detection of rIgG. (A) Two-step immunoassay: serial dilutions of the rIgG analyte were mixed with varying amounts of capture reagent HA24-GAR, ranging from 2 (rectangles), 10 (circles) to 50 (more ...)
To study the feasibility of a one-step immunoassay, serial dilutions of the rIgG antigen were mixed with a fixed amount of HA24–GAR capture reagent, either 4 or 10 nM, respectively, and 25 pM of the IPCR detection conjugate, comprised of the biotinylated GAR, recombinant STV and bis-biotinylated dsDNA. The mixtures were immediately applied to oligonucleotide-coated microplate wells to allow for the DNA-directed adsorption of the immuno-complexes formed in solution. Subsequently, quantification of signals was carried out by PCR and PCR–ELISA. As indicated in Figure B, signal intensities and the assay’s overall sensitivity strongly depended on the amount of capure reagent. The lower concentration of capture reagent, 4 nM HA24–GAR, led to an increased detection limit of 0.1 amol/µl (15 pg/ml) of rIgG, as compared with 2 amol (300 pg/ml), respectively, obtained in the case of 10 nM HA24–GAR. In both cases, an unusually shaped dose–response curve was evident, indicating a significant decrease in signal intensity when high amounts of rIgG antigen were present in the sample (see curves in Fig. B). This shape was highly reproducible, and most likely, is caused by limited amounts of the detection conjugate. This leads to an incomplete labeling of antigen molecules, and thus, decreased signal intensities. Unfortunately, this effect cannot be compensated for by increasing the detection conjugate concentration. Since IPCR is highly sensitive to the concentration of the detection conjugate, i.e. the amount of marker DNA incubated in the reaction vessel (12
), an increase in detection conjugate concentration was inevitably associated with high background signals, and thus, a dramatic decrease in sensitivity (data not shown).
Figure C allows one to directly compare the conventional three-step assay, exclusively based on successive reagent incubation steps, with the corresponding two- and one-step assays described above. It is clearly evident that the decrease in incubation steps is associated with a decrease in the overall sensitivity of the immunoassay. As discussed above, signal intensities depend on the relative amounts of target, capture and detection reagents. Consequently, best results are obtained with the conventional protocol, taking advantage of the repeated reagent incubation and washing steps. The in-solution capture assays, however, not only allow for a significant reduction in assay time but also still offer a significant increase in sensitivity, as compared with conventional ELISA detection. Additional enhancement in sensitivity might well be achieved by systematic optimization of assay conditions, in particular, the concentration of reagents.