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Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response-LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as "oligo-clonal" reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

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Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

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Defects in mitochondrial oxidative phosphorylation are a feature of many human diseases. To date, determination of oxidative phosphorylation has required fresh and live sample material and therefore also access to specialized equipment and trained personnel. Cryopreservation of samples is an attractive alternative, where samples can be collected and stored in an economic and practical fashion for later bulk assays. Here, we present an accurate, reliable method for estimating mitochondrial oxidative phosphorylation capacity of cryopreserved human cells. Broad adoption of this method will allow uncomplicated collection of samples and measurements of oxidative phosphorylation.

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Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

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Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

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Bacterial surface appendages of the type 4 pilus superfamily play diverse roles in adherence, aggregation, motility, signaling, and macromolecular transport. Here we describe two analytical approaches to study assembly of type 4 pili and of pseudopili produced by type 2 protein secretion systems: the shearing assay and immunofluorescence microscopy. These complementary antibody-based methods allow for semiquantitative analysis of fiber assembly. The shearing assay can be scaled up to yield crude extracts of pili that can be further analyzed by electron and atomic force microscopy or by mass spectrometry.

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OCTN1 was immuno-detected in the cervical cancer cell HeLa, in which the complete pattern of acetylcholine metabolizing enzymes is expressed. Comparison of immuno-staining intensity of HeLa OCTN1 with the purified recombinant human OCTN1 allowed measuring the specific OCTN1 concentration in the HeLa cell extract and, hence calculating the HeLa OCTN1 specific transport activity that was about 10 nmol×min(-1)×mg protein(-1), measured as uptake of [(3)H]acetylcholine in proteoliposomes reconstituted with HeLa extract. This value was very similar to the specific activity of the recombinant protein. Acetylcholine transport was suppressed by incubation of the protein or proteoliposomes with the anti-OCTN1 antibody and was strongly inhibited by PLP and MTSEA, known inhibitors of OCTN1. The absence of ATP in the internal side of proteoliposomes strongly impaired transport function of both the HeLa and, as expected, the recombinant OCTN1. HeLa OCTN1 was inhibited by spermine, NaCl (Na(+)), TEA, γ-butyrobetaine, choline, acetylcarnitine and ipratropium but not by neostigmine. Besides acetylcholine, choline was taken up by HeLa OCTN1 proteoliposomes. The transporter catalyzed also acetylcholine and choline efflux which, differently from uptake, was not inhibited by MTSEA. Time course of [(3)H]acetylcholine uptake in intact HeLa cells was measured. As in proteoliposomes, acetylcholine transport in intact cells was inhibited by TEA and NaCl. Efflux of [(3)H]acetylcholine occurred in intact cells, as well. The experimental data concur in demonstrating a role of OCTN1 in transporting acetylcholine and choline in HeLa cells.

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This article provides a novel method of constructing an optical fiber localized surface plasmon resonance (LSPR) biosensor. A gold nanoparticle (NP) assembled film as the sensing layer was built on the polyelectrolyte (PE) multilayer modified sidewall of an unclad optical fiber. By using a trilayer PE structure, we obtained a monodisperse gold NP assembled film. The preparation procedure for this LSPR sensor is simple and time saving. The optical fiber LSPR sensor has higher sensitivity and outstanding reproducibility. The higher anti-interference ability for response to an antibody makes it a promising method in application as a portable immuno-sensor.

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An immuno-recognition assay using a monoclonal antibody was developed to detect Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne). In vitro assays with isolates of P. grisea from perennial ryegrass, tall fescue (Festuca arundinacea), St. Augustinegrass (Stenotaphrum secundatum), crabgrass (Digitaria sanguinalis), finger millet (Eleusine coracana), wheat (Triticum aestivum), triticale (× Triticosecale rimpaui), and rice (Oryza sativa) showed positive reactions; however, the strength of the reactions differed among isolates. Reactions were more intense with isolates from perennial ryegrass, wheat, and triticale. All P. grisea isolates from perennial ryegrass collected from various regions of the United States showed positive reactions. P. grisea was detected at antigen dilution rates of 0.5×, 0.25×, 0.13×, 0.06×, and 0.03×. Dot-blot assays with Bipolaris sorokiniana, Colletotrichum graminicola, Curvularia lunata, Microdochium nivale, Pythium aphanidermatum, Rhizoctonia solani, or Sclerotinia homoeocarpa isolated from turfgrasses were negative. In vivo assays of symptomatic leaves of perennial ryegrass plants inoculated with P. grisea also showed positive reactions, and those inoculated with B. sorokiniana, P. aphanidermatum, R. solani, or S. homoeocarpa were negative. Intensity of reaction between the monoclonal antibody and P. grisea was proportional to disease severity in perennial ryegrass inoculated with P. grisea; however, there was no reaction in dot blots of leaf tissue collected during the latent period. P. grisea was detected in perennial ryegrass samples from golf course fairways affected by gray leaf spot in Connecticut, Massachusetts, Maine, New Jersey, Pennsylvania, and Rhode Island using this procedure. The monoclonal antibody recognition system is highly sensitive to P. grisea and can be used effectively for the rapid diagnosis of gray leaf spot of perennial ryegrass turf.