scholarly journals High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Loretta Yang ◽  
Shengcheng Wu ◽  
J. Christopher Cooper ◽  
Mallory K. Paul ◽  
Amanda L. Cummings ◽  
...  
Keyword(s):  
High Specificity ◽  
Affinity Reagents

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals Phage Display: A Powerful Technology for the Generation of High-Specificity Affinity Reagents from Alternative Immune Sources

2016 ◽  
pp. 85-99 ◽  
Author(s):  
William J. J. Finlay ◽  
Laird Bloom ◽  
Joanne Grant ◽  
Edward Franklin ◽  
Deirdre Ní Shúilleabháin ◽  
...  
Keyword(s):  
Phage Display ◽  
High Specificity ◽  
Affinity Reagents

scholarly journals DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Claudia Kolm ◽  
Isabella Cervenka ◽  
Ulrich J. Aschl ◽  
Niklas Baumann ◽  
Stefan Jakwerth ◽  
...  
Keyword(s):  
State Of The Art ◽  
Selection Process ◽  
High Specificity ◽  
Cost Effective ◽  
Cell Labelling ◽  
Dna Aptamers ◽  
Bacterial Cells ◽  
Combined Approach ◽  
Affinity Reagents ◽  
Assessment Techniques

AbstractDNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.

Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Loretta Yang ◽  
Sheng-Cheng Wu ◽  
Lu Meng ◽  
Christian Gerner-Smidt ◽  
Robert J. Woods
Keyword(s):  
High Specificity ◽  
Affinity Reagents

scholarly journals Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Robert J. Woods ◽  
Shengcheng Wu ◽  
Lu Meng ◽  
Christian Gerner‐Smidt ◽  
Matthew J. Sanders ◽  
...  
Keyword(s):  
High Specificity ◽  
Affinity Reagents

Structural and Chemical Studies of Pathological Fibers in Human Neurofibrillary Degeneration

1985 ◽  
Vol 43 ◽  
pp. 726-729
Author(s):  
K.S. Kosik ◽  
L.K. Duffy ◽  
S. Bakalis ◽  
C. Abraham ◽  
D.J. Selkoe
Keyword(s):  
Monoclonal Antibodies ◽  
Alzheimer Disease ◽  
Human Brain ◽  
Neurofibrillary Tangles ◽  
Morphological Analysis ◽  
High Specificity ◽  
Cytoskeletal Proteins ◽  
Neuritic Plaque ◽  
Protein Chemistry ◽  
Chemical Studies

The major structural lesions of the human brain during aging and in Alzheimer disease (AD) are the neurofibrillary tangles (NFT) and the senile (neuritic) plaque. Although these fibrous alterations have been recognized by light microscopists for almost a century, detailed biochemical and morphological analysis of the lesions has been undertaken only recently. Because the intraneuronal deposits in the NFT and the plaque neurites and the extraneuronal amyloid cores of the plaques have a filamentous ultrastructure, the neuronal cytoskeleton has played a prominent role in most pathogenetic hypotheses.The approach of our laboratory toward elucidating the origin of plaques and tangles in AD has been two-fold: the use of analytical protein chemistry to purify and then characterize the pathological fibers comprising the tangles and plaques, and the use of certain monoclonal antibodies to neuronal cytoskeletal proteins that, despite high specificity, cross-react with NFT and thus implicate epitopes of these proteins as constituents of the tangles.

A comparison of three cryotechniques (freeze-drying, cryosubstitution, cryosectioning) of specimen preparation for immunolabelling of bacterial antigens of Coxiella burnetii

1994 ◽  
Vol 52 ◽  
pp. 140-141
Author(s):  
T. F. McCaul ◽  
R. J. Gould
Keyword(s):  
Coxiella Burnetii ◽  
Freeze Drying ◽  
Stress Proteins ◽  
Cultured Cells ◽  
High Specificity ◽  
Specimen Preparation ◽  
Bacterial Antigens ◽  
Structural Preservation ◽  
Nuclear Ribonucleoprotein ◽  
Cellular Components

Immuno-electron microscopy has allowed the selective localisation of molecules with high resolution and high specificity. Cryopreparatory methods have provided better retention of antigenicity suitable for precise immunolabelling together with optimal structural preservation of cellular components. Cryosubstitution and cryoultramicrotomy have widely been exploited for immunolabelling. Molecular Distillation Dryer (MDD), a form of freeze-drying technique, has recently been used for immunolabelling of Plasmodium falciparum stress proteins and nuclear ribonucleoprotein particles in cultured cells. In the present study, we report the comparison of all three cryotechniques in the immunolabelling of bacterial antigens of Coxiella burnetii.The highly infectious C. burnetii was prefixed in 3% glutaraldehyde (66 mM cacodylate buffer, pH 6.8 ). The cells were then pre-embedded in 2% low-temperature agarose on Durapore hydrophilic membrane prior to cryofixation using a LifeCell CF100 metal-mirror system. A 1% glutaraldehyde in 100% methanol was used as a medium for cryosubstitution in a Reichert CS Auto Cryosubstitution apparatus.

Electronmicroscopic localization of ribonucleic acids in ciliate Bursaria truncatella during cell division

1990 ◽  
Vol 48 (3) ◽  
pp. 132-133
Author(s):  
Vladimir Popenko ◽  
Natalya Cherny ◽  
Maria Yakovleva
Keyword(s):  
Cell Division ◽  
High Specificity ◽  
Longitudinal Axis ◽  
Gold Complexes ◽  
Somatic Nucleus ◽  
Ribonucleic Acids ◽  
Biological Meaning ◽  
Bivalent Cations ◽  
Lowicryl K4m ◽  
Short Time

Highly polyploid somatic nucleus (macronucleus) of ciliate Bursaria truncatella under goes severe changes in morphology during cell division. At first, macronucleus (Ma) condences, diminishes in size and turns perpendicular to longitudinal axis of the cell. After short time, Ma turns again, elongates and only afterwards the process of division itself occurs. The biological meaning of these phenomena is not clear.Localization of RNA in the cells was performed on sections of ciliates B. truncatella, embedded in “Lowicryl K4M” at various stages: (1) before cell division (Figs. 2,3); (11) at the stage of macronucleus condensation; (111) during elongation of Ma (Fig.4); (1111) in young cells (0-5min. after division). For cytochemical labelling we used RNaseAcolloidal gold complexes (RNase-Au), which are known to bind to RNA containing cell ularstructures with high specificity. The influence of different parameters on the reliability and reproducibility of labelling was studied. In addition to the factors, discussed elsewhere, we found that the balance of mono- and bivalent cations is of great significance.

Hit and False-Alarm Rates of Selected ABR Indices in Differentiating Cochlear Disorders From Acoustic Tumors

1996 ◽  
Vol 5 (1) ◽  
pp. 90-96 ◽  
Author(s):  
Frank E. Musiek ◽  
Cynthia A. McCormick ◽  
Raymond M. Hurley
Keyword(s):  
False Alarm ◽  
Amplitude Ratio ◽  
High Specificity ◽  
Auditory Brainstem ◽  
Latency Difference ◽  
Cochlear Pathology ◽  
Brainstem Response ◽  
Absolute Latency ◽  
Two Indices ◽  
Low Sensitivity

We performed a retrospective study of 26 patients with acoustic tumors and 26 patients with otologically diagnosed cochlear pathology to determine the sensitivity (hit rate), specificity (false-alarm rate), and efficiency of six auditory brainstem response indices. In addition, a utility value was determined for each of these six indices. The I–V interwave interval, the interaural latency difference, and the absolute latency of wave V provided the highest hit rates, the best A’ values and good utility. The V/I amplitude ratio index provided high specificity but low sensitivity scores. In regard to sensitivity and specificity, using the combination of two indices provided little overall improvement over the best one-index measures.

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