Antibody Probes Research Articles

Recovery and identification of Pseudo-nitzschia (Bacillariophyceae) frustules from natural samples acquired using the environmental sample processor.

Many species within the diatom genus Pseudo-nitzschia are difficult to distinguish without applying molecular analytical or microscopy-based methods. DNA, antibody and lectin probes have previously been used to provide rapid and specific detection of species and strains in complex field assemblages. Recently, however, well-documented cryptic genetic diversity within the group has confounded results of DNA probe tests in particular. Moreover, the number of species descriptions within the genus continues to increase, as do insights into toxin production by both new and previously described species. Therefore, a combination of classical morphological techniques and modern molecular methodologies is needed to resolve ecophysiological traits of Pseudo-nitzschia species. Here, we present an approach to recover and identify frustules from sample collection filters used for toxin analysis onboard the Environmental Sample Processor (ESP), an in situ sample collection and analytical platform. This approach provides a new and powerful tool for correlating species presence with toxin detected remotely and in situ by the ESP, and has the potential to be applied broadly to other sampling configurations. This new technique will contribute to a better understanding of naturally occurring Pseudo-nitzschia community structure with respect to observed domoic acid outbreaks.

Facile and sensitive detection of influenza viruses using SERS antibody probes

We report facile and sensitive influenza virus detection method using surface-enhanced Raman scattering antibody probes.

Colloidal Gold Probe-Based Immunochromatographic Strip Assay for the Rapid Detection of Microbial Transglutaminase in Frozen Surimi

Adding microbial transglutaminase (MTGase) to frozen surimi to enable the surimi to be sold as a higher-grade product at a higher price defrauds surimi product manufacturers and undercuts legitimate industry prices. Therefore, it is important to develop an accurate method of detecting the presence of MTGase in surimi. In this study, an immunochromatographic strip assay with a colloidal gold antibody probe was successfully developed and used to rapidly and qualitatively detect MTGase in surimi samples. The results were obtained in less than 10 min. The limit for the qualitative detection of MTGase using the immunochromatographic strip assay was identified as 1.0 μg/mL. The results of the immunochromatographic strip analysis of frozen surimi samples were verified by comparison with the results of a sandwich enzyme-linked immunosorbent assay. The colloidal gold probe-based immunochromatographic strip assay was thus found to be a rapid, economical, and user friendly method of detecting MTGase in surimi.

Differentiation of individual cell populations in a touch mixture using antibody probes and flow cytometry

Differentiation of individual cell populations in a touch mixture using antibody probes and flow cytometry

Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles.

Bright signal outputs are needed for fluorescence detection of biomolecules at their native expression levels. Increasing the number of labels on a probe often results in crowding-induced self-quenching of chromophores, and maintaining the function of the targeting moiety (e.g., an antibody) is a concern. Here we demonstrate a simple method to accommodate thousands of fluorescent dye molecules on a single antibody probe while avoiding the negative effects of self-quenching. We use a bottlebrush polymer from which extend hundreds of duplex DNA strands that can accommodate hundreds of covalently attached and/or thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA assembly sequesters the intercalated fluorophores against dissociation and can be tethered through DNA hybridization to an IgG antibody. The resulting fluorescent nanotag can detect protein targets in flow cytometry, confocal fluorescence microscopy, and dot blots with an exceptionally bright signal that compares favorably to commercially available antibodies labeled with organic dyes or quantum dots.

Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

Confirmatory detection of sperm and semen Via proximity ligation real-time PCR

Semen and sperm identification play a critical role in forensic investigation for understanding the circumstances surrounding a crime and determining whether a sexual act occurred or not. Yet current methods for sperm detection vary widely in speed, sensitivity, and specificity. Also, the only undisputable confirmatory test for the presence of semen is the microscopic observation of spermatozoa. This process can be extremely time consuming and labor intensive, as each sample must be processed and examined individually. Particularly in samples with low levels or no spermatozoa, analysts may spend hours searching a slide. Also, failure to identify sperms by microscopic examination is not conclusive for their absence. Here, we demonstrate the development of a confirmatory method employing Proximity Ligation Real Time PCR (PLiRT-PCR) for the identification of semen and sperm from sexual assault evidence. PLiRT-PCR is designed to detect and quantitate the expression of protein markers through an antibody-protein binding reaction followed by qPCR. Two protein targets have been chosen as candidates for the assay: cysteine-rich secretor protein 2 (CRISP-2) and prostate-specific antigen (PSA). CRISP-2 is specifically expressed in the male reproductive tract and localized inside the acrosome of spermatozoa. The acrosomal location protects CRISP-2 from environmental damage until lysis, allowing for successful detection even on aged forensic samples. Also, its location on the surface of the acrosome makes it likely for epitopes to be accessible to antibody probes. PSA was selected as a second candidate due to its high concentration in semen and its useful role when processing samples from azoospermic or vasectomized perpetrators. This study discusses the identification, specificity, and the limit of detection of Crisp2 and PSA markers in samples containing body fluid mixtures. Data demonstrate the potential for PLiRT-PCR as a confirmatory test for semen. The approach allows high throughput semen and sperm detection to be processed in parallel on a 96 well plate, enabling analysts to make the most informed decisions on the best analytical path forward in each case, preserve valuable evidence, overcome the drawbacks associated with the microscopic observation of spermatozoa, and save analyst time.

Effects of polyvalent immunoglobulins in patients with recurrent pregnancy loss and antibodies to the choriocarcinoma cell line JEG-3

ObjectiveThe benefit of polyvalent immunoglobulins (IVIG) for patients with recurrent pregnancy loss (RPL) is controversially discussed. Anti-trophoblast antibodies as an expression of immune pathology have been associated with RPL. We investigated whether the antibody activity against the choriocarcinoma cell line JEG-3 in RPL patients is influenced in vitro by IVIG. Study designSera of 110 unexplained RPL patients with positive anti-JEG-3 antibodies were coincubated with IVIG in different concentrations (10mg/ml, 20mg/ml, 40mg/ml). Coincubation with human albumin in identical concentrations served as control. Anti-JEG-3 reactivity was measured by using flow cytometry in comparisons with two in-house standards antibody probes of low and high reactivity as described before. Anti-JEG-3 reactivity above the 95% confidence interval of controls was defined as positive. ResultsIncubating RPL sera with 10mg/ml IVIG significantly decreased anti-JEG-3 activity (p<0.001). Increasing IVIG concentration to 40mg/ml resulted in a slightly additionally reduction (p=0.42). In contrast, coincubation with albumin in identically concentrations did not affect anti-JEG-3 activity (p>0.40). ConclusionCoincubation with IVIG in vitro leads to a significant suppression of anti-JEG-3 activity in the sera of RPL patients.

PEA-CLARITY: 3D molecular imaging of whole plant organs.

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

A carcinoembryonic antigen optoelectronic immunosensor based on thiol-derivative-nanogold labeled anti-CEA antibody nanomaterial and gold modified ITO

A dual carcinoembryonic antigen (CEA) biosensing alternative optoelectric platforms has been developed based on gold modified ITO electrode. The new platform is constructed by assembly of a thiol-derivative-nanogold (TDN) labeled anti-CEA (TDN-anti-CEA) antibody on a gold/indium tin oxide (ITO) electrode to create a high CEA sensitive surface. The gold film deposited on ITO glass provides a desirable substrate for the immobilization of the TDN-anti-CEA antibody and improves the compatibility between nanogold and electrode which results in a significant amplification. By using the same TDN-anti-CEA antibody probe, the CEA (analyte) was analyzed using two different methods; electrochemical impedance spectroscopy (EIS) and ultraviolet (UV) spectroscopy, the results showed that the limit of detection was 1 and 2pg/ml by EIS and UV methods, respectively. Given their promising advantages, it is likely that dual platforms will evolve to be an attractive tool for use in many different fields.

AdamTS‐A Developmental Function in Neuronal Development and Migration in Drosophila Melanogaster

The Drosophila central nervous system (CNS) is an excellent model system in which to dissect the genetic and molecular processes that control the development of the nervous system. Through a forward genetic screen, we previously identified, a larval phenotype that leads to the formation of ectopic neural masses. These masses are composed of dozens to thousands of neuronal cell bodies at the junctures between the brain and the eyes, the ventral nerve cord and the leg imaginial discs, as well as within nerves. We determined that this mutant phenotype arises due to a transposable element insert within the 5' untranslated region of a disintegrin and metalloprotease with thrombospondin type 1 motif A (AdamTS‐A) gene. The AdamTS family of proteases are secreted proteins thought to play critical roles in extracullular matrix (ECM) formation and function, and their regulation has been linked to cancer progression. We have recently generated RNA and antibody probes to the AdamTS‐A and will report the RNA and protein expression of Adam TS‐A during larval CNS development.Support from the NINDS/NIH grant number NS0365570 and the Opportunities in Genomics Research through the Genome Institute at Washington University in St. Louis, grant number R25H600668703.

Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS)

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor’s cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)—a high throughput technique for separating cell populations based on their optical properties—and then subjected to STR analysis.We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele’s protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.

Streptococcal Antibody Probe Crosses the Blood Brain Barrier and Interacts within the Basal Ganglia

Within the brain, the basal ganglia (basal nuclei) regulates wanted movement and inhibits unwanted movement. This area of the brain is intertwined with capillary beds that bring nutrients to the brain and form the blood brain barrier. During disease state, antibodies are increased in circulation and movement of these antibodies into the basal ganglia can occur. Streptococcal infection can lead to the generation of antibodies that have autoimmune activity within the brain. These antibodies have been implicated in neurological disorders. In our laboratory, an in vitro study of a monoclonal mouse antibody generated against the class 1 epitope of the M6 protein has demonstrated binding within the basal ganglia of Lewis rat brains. Here we present an in vivo study using Lewis rats injected with either the streptococcal antibody or an anti-myosin positive control. The interaction and movement of the antibody from blood vessels into the tissues of the basal ganglia was determined through the use of immunofluorescence and fluorescent microscopy and is contrasted with IgG injected and uninjected controls. Our data demonstrates that the streptococcal antibody penetrates the blood brain barrier within 24 hours (as determined by the presence of immunofluorescence outside of blood vessels) and remains significantly elevated above control values even 72 hours after injection (p < 0.05). In contrast, the anti-myosin positive control was not visualized in the interstitial fluid until 48 hours post injection and was no longer significantly above control levels by 72 hours. IgG injected controls did not display movement of antibody into the brain. Therefore, the streptococcal antibody is capable of crossing the blood brain barrier and interacting with tissues of the basal ganglia.

Single-Cell Western Blotting.

Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

Radioiodinated Peptidic Imaging Probes for in Vivo Detection of Membrane Type-1 Matrix Metalloproteinase in Cancers.

Membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis, and holds great promise as an early biomarker for malignant tumors. Therefore, the ability to evaluate MT1-MMP expression could be valuable for molecular biological and clinical studies. For this purpose, we aimed to develop short peptide-based nuclear probes because of their facile radiosynthesis, chemically uniform structures, and high specific activity, as compared to antibody-based probes, which could allow them to be more effective for in vivo MT1-MMP imaging. To the best of our knowledge, there have been no reports of radiolabeled peptide probes for the detection of MT1-MMP in cancer tissues. In this study, we designed and prepared four probes which consist of a MT1-MMP-specific binding peptide sequence (consisting of L or D amino acid isomers) and an additional cysteine (at the N or C-terminus) for conjugation with N-(m-[(123/125)I]iodophenyl) maleimide. We investigated probe affinity, probe stability in mice plasma, and probe biodistribution in tumor-bearing mice. Finally, in vivo micro single photon emission computed tomography (SPECT) imaging and ex vivo autoradiography were performed. Consequently, [(123)I]I-DC, a D-form peptide probe radioiodinated at the C-terminus, demonstrated greater than 1000-fold higher specific activity than previously reported antibody probes, and revealed comparably moderate binding affinity. [(125)I]I-DC showed higher stability as expected, and [(123)I]I-DC successfully identified MT1-MMP expressing tumor tissue by SPECT imaging. Furthermore, ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles corresponded to MT1-MMP-positive areas. These findings suggest that [(123)I]I-DC is a promising peptide probe for the in vivo detection of MT1-MMP in cancers.

A scanometric antibody probe for facile and sensitive immunoassays.

We have developed a novel scanometric antibody probe for rapid, sensitive, and naked-eye-visible immunoassays. Using this probe, we clearly demonstrated the successful scanometric detection and identification of influenza A viruses on a microarray. In addition, the sensitivity of the scanometric immunoassay was comparable to that of the fluorescence-based method.

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae.

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the "pellicle") overlaid by a 15-45 nm thick layer of CPS (the "capsule"). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Microfluidic Western blotting of low-molecular-mass proteins.

We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.

Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

Design, synthesis, and validation of Axl-targeted monoclonal antibody probe for microPET imaging in human lung cancer xenograft.

Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with 64Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe 64Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, 64Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher 64Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, 64Cu-DOTA-h173 uptake in A549 is significantly higher than that of 64Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, 64Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.