antibody-DNA Conjugates Research Articles

Photothermal effect for localized desorption of primary lymphocytes arrayed on an antibody/DNA-based biochip.

This work proposes a miniaturized system able to perform multiple cell capture followed by cell-type selective release from a biochip surface. Unlabelled lymphocytes were first specifically captured onto a DNA array by antibody-DNA conjugates. The immobilized cells were subsequently released under spatiotemporal control within local heating generated by intense Surface Plasmon Resonance (SPR) produced by laser illumination.

Detection of Antigens Using a Protein–DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A*

The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2(+) cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2(+) cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.

DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging

Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms—such as lab-on-a-chip or microarrays—to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody–DNA conjugates. These protein–DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein–DNA microarray spots but also their fast and specific orthogonal release according to the antibody–DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features.

The fabrication of a piezoelectric immunosensor based on DNA–antibody conjugate layer

In this article, we report a method of antibody immobilization carried out by hybridizing DNA–antibody conjugates on a mixed self-assembled monolayer composed of DNA thiols and mercaptopropionic acid via sequence-specific hybridization. The proposed method was applied to fabricate an immunosensor for detecting human immunoglobulin G (IgG). Under the optimized experimental conditions, a wide linear range from 50.0 to 500 μg/ml was reached with a detection limit of 30.13 μg/ml. The developed immunosensor possesses advantages such as simple fabrication, wide linear range, easy regeneration, and excellent reproducibility.

Citations

BioTechniquesVol. 45, No. 2 CitationsOpen AccessCitationsNijsje Dorman†Nijsje Dorman††Selected and written by Nijsje Dorman, a freelance writer in Boston, MA.Search for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000112921AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail MatchmakerThe relative ease of amplifying nucleic acids has encouraged protein detection schemes that have a DNA readout. One example of this is immunoPCR, which uses antibody-DNA conjugates as templates for amplification and detection. Though immunoPCR uses physical coupling to bridge the protein-DNA divide, the link may instead be accomplished by noncovalent binding of nucleic acid aptamers to protein substrates. One of the most attractive of such strategies is the proximity ligation assay, in which two aptamers bind to the target, are ligated together, and then this newly formed template is detected by real-time PCR. However, because identifying two noninterfering aptamers introduces some complexity, a single conformation-switching aptamer is an attractive alternative. In this case, upon binding its target, the aptamer undergoes a conformational change that is transduced into an assayable signal. In a report in Analytical Biochemistry, Yang and Ellington propose harnessing the conformational change so that it exposes an annealing site for an oligo that will be ligated to the aptamer. Ligation adds a second binding site for a PCR primer (another was already in the unligated aptamer), thereby making the aptamer a viable substrate for real-time PCR. The authors explored this premise using aptamers previously developed for binding thrombin and platelet-derived growth factor (PDGF). As the original aptamer sequences were not designed for conformation switching, the authors had to add sequences that adopt a ligation-unfavorable structure in the absence of analyte binding, but that provide a platform for annealing of the intended ligation partner once the aptamer-protein interaction has occurred. In addition, a primer site for PCR amplification and an annealing site for a TaqMan probe were required. All of this had to be done with adequate spacing to provide room to accommodate both the target protein and the ligase, and of course the sensitivity and specificity of the aptamer could not be compromised. The authors were pleased to discover that only minimal optimization was needed to convert the original aptamers into ligatable conformation-switching probes, and that limits of detection were compatible with physiologically relevant concentrations of the two analytes. Although clinical samples were not measured, the simplicity and robustness of this approach appears to be an attractive fit for biosensors to detect any of the many clinically relevant factors for which aptamers have been derived.Image reprinted with permission. ©2008 Elsevier.Yang and Ellington. Real-time PCR detection of protein analytes with conformation-switching aptamers. Anal. Biochem. [Epub ahead of print, May 20, 2008].Behind the ScreenIn a recent issue of Cell Stem Cell, Desbordes et al. offer a glimpse behind the curtains of a new method for high-throughput screens in hESC. The technical challenge posed by screening the effects of compound libraries on stem cell renewal and differentiation has been how to accommodate the specialized culture conditions needed for these cells within the context of a highly parallel automated analysis. The authors found that Accutase treatment created a single-cell suspension that could be dispensed at 6000 cells per well into a 384-well plate. Two days after plating, FGF2 supplementation was discontinued, placing the cells on the cusp of two possible fates: ongoing self-renewal, characterized by high levels of the stemness marker Oct4 in relation to DAPI stain; or differentiation, which is detectable by a low Oct4-DAPI ratio. At this same time point, the researchers added members of a 2880-compound library consisting of drugs, natural products, and other bioactive agents. After 5 days of treatment, automated image analysis was used to detect compounds that consistently demonstrated high or low Oct4 levels; potential hits were then analyzed in dose-response experiments. Further validation of apparent activators of Oct4 expression required lower-throughput methods, and included measurement of Nanog expression, enumeration of undifferentiated colonies, and cell cycle analysis. In-depth analysis of compounds that reduced Oct4 levels were performed in the 384-well plate environment, with staining for trophoblast, mesendoderm/endoderm, and neuroectoderm markers. Transcriptional profiling of activator and inhibitor compounds provided preliminary insights into pathways explaining the disparate fates. This detailed methodological description will significantly facilitate deciphering and directing the behavior of hESCs, and should spur future improvements to diminish post-plating cell death (reducing the number of cells required) and to better standardize growth conditions (minimizing the number of assays in which positive and negative controls failed quality checks).Desbordes et al. 2008. High-throughput screening assay for the identification of compounds regulating self-renewal and differentiation in human embryonic stem cells. Cell Stem Cell 2:602-612.

Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.

Detection of femtogram amounts of biogenic amines using self-assembled DNA-protein nanostructures

Chimera Biotec, founded in 2000 as a spin-off from academic research in Germany, has implemented its proprietary DNA-antibody conjugates within the highly innovative Imperacer product line, enabling the ultra-sensitive detection of proteins and other antigens. The ready-to-use, commercially available Imperacer kits offer a performance-enhancing tool to improve the limit of detection of conventional ELISA protocols by more than 1,000-fold. Hence, the detection and quantification of trace amounts of basically any antigen, ranging from large proteins to small molecules such as hormones or biogenic amines, can be accomplished with an Imperacer-boosted immunoassay.

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR.

An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.

Analyte detection with DNA-labeled antibodies and polymerase chain reaction

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.

Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates.

An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 x 10(-22) moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately x 10(5)) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.