Antibody microarrays for native toxin detection

Rucker, Victor C.; Havenstrite, Karen; Herr, Amy E.

doi:10.1016/j.ab.2005.01.030

Cited by 96 publications

(52 citation statements)

References 32 publications

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“…The remaining replicates can be used to assess the apparent affinity of each antibody captured on the microarrays for a particular antigen. A binding curve for the antibodies produced by each cell can be constructed by applying a series of concentrations of antigen (e.g., 10 pM-100 nM) to the set of replicate microarrays and then measuring the fluorescent intensities of captured antigen as a function of concentration (13). Variations in the rates of secretion among cells or in the efficiency of capture among glass slides can affect the amount of antibody captured from each cell on each replicate.…”

Section: Resultsmentioning

confidence: 99%

Profiling antibody responses by multiparametric analysis of primary B cells

Story

Papa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (Ϸ10 3 -10 4 ). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigenspecific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.affinity ͉ immunomics ͉ microengraving ͉ soft lithography ͉ vaccines

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Section: Resultsmentioning

confidence: 99%

Profiling antibody responses by multiparametric analysis of primary B cells

Story

Papa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…PA has been previously detected in the serum of infected guinea pigs and rabbits after signs of toxemia are observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western methods (15,37), yet without any detection limits reported. More recently, a microarray was engineered to detect unlabeled native toxin (40). Although the reported limit of detection for PA was 1.3 g/ml, there exists potential in expanding upon immunoassays for toxin detection for infected hosts.…”

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confidence: 99%

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Mabry

Brasky

Geiger

et al. 2006

Clin Vaccine Immunol

114

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Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.The gram-positive Bacillus anthracis bacterium is the causative agent of anthrax. During inhalational anthrax, spores are inhaled into the lungs, leading to germination followed by the emergence of vegetative anthrax bacteria in circulation. Key anthrax virulence factors include the three components of the anthrax exotoxin, protective antigen (PA), lethal factor (LF), and edema factor (EF), which are secreted into the host's system (11). The PA and LF toxins combined are often referred to as anthrax lethal toxin (LeTx) and operate analogous to other A-B toxin systems (18).The PA component is secreted as an 83-kDa protein that is cleaved by a host furin-like protease to yield a 63-kDa fragment (5, 24) that readily heptamerizes. The PA 63 heptamer is bound to the host cell surface receptor ANTXR1 (ATR/ TEM8) (45) or ANTXR2 (CMG2) (7) with relatively high affinity (170 pM) (47). The LF component binds to the PA heptamer and gains entry into the cell through clathrin-dependent endocytosis (1). A drop in endosomal pH leads to a conformational change in PA and transport of LF into the host cell cytosol (17, 26). In the cytosol, LF toxin acts as a zinc metalloprotease, cleaving the N termini of mitogen-activated protein kinase kinases (9, 13), leading to a number of signs associated with toxemia and ultimately death (2, 32).In the rodent model, signs of anthrax infection are rapidly followed by death within a few hours, resulting in a very small, if existent, window for postexposure therapy after observed toxemia. However, in the nonhuman primate model and in human disease, the infection is characterized by delayed death up to 10 days following initial symptom onset (8, 10). It is theref...

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“…In addition, classic CT determinations require the use of either animal methods (5,6) or tissue culture methods (7,8), which are also time-consuming and are subjective in the interpretation of results. Efforts have been made recently to develop more-sensitive methods, including enzyme-linked immunosorbent assays (9), latex agglutination assays (10), coagglutination assays (5), liposome-based assays (9,11), radioimmunoassays (12), hydrogel-based immunoassays (13), monosaccharide-and antibody array-based assays (14)(15)(16)(17), PCR-based molecular assays (10,(18)(19)(20)(21), and biosensor-based assays (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). A reliable laboratory tool is desirable for clinical services and epidemiological investigation, to characterize CT activities rapidly and quantitatively.…”

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confidence: 99%

Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring

et al. 2013

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eWe report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml, the inoculation-to-detection time was 1.12 ؎ 0.38 h, and the values were inversely correlated with CT concentrations ( ‫؍‬ ؊1; P ‫؍‬ 0.01). The results indicate that the CT-RTCA assay with the Y-1 cell line provides a rapid and sensitive tool for the quantitative detection of CT activities in clinical specimens. Vibrio cholerae is a Gram-negative, comma-shaped, bacterial pathogen causing cholera, an acute secretory diarrheal disease. Epidemic cholera is common in developing countries and affects about 100,000 people annually (1). Cholera toxin (CT) is a major virulence determinant of V. cholerae, leading to rapidly progressing dehydration, shock, metabolic acidosis, and even death within hours without adequate and appropriate therapy (2). CT is a key biomarker of V. cholerae that is used for forecasting and assessing epidemic disease, monitoring and controlling cholera outbreaks, preventing epidemics, and guiding medical personnel in providing timely treatment of patients. There has been urgent demand for the development of novel sensitive assays for the detection and identification of CT proteins.Conventional biochemical and immunological methods for CT detection and identification can be completed only after V. cholerae is isolated. The bacterial isolation and subsequent CT detection and identification are laborious and usually require several days, resulting in significant delays in patient care and disease control (3, 4). In addition, classic CT determinations require the use of either animal methods (5, 6) or tissue culture methods (7,8), which are also time-consuming and are subjective in the interpretation of results. Efforts have been made recently to develop more-sensitive methods, including enzyme-linked immunosorbent...

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Antibody microarrays for native toxin detection

Cited by 96 publications

References 32 publications

Profiling antibody responses by multiparametric analysis of primary B cells

Profiling antibody responses by multiparametric analysis of primary B cells

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring

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