On-Chip Binding Assay: MC-CE Device Integrated with Multisegment Circular-Ferrofluid-Driven Micromixing Injection for Assay of Free Bilirubin and Albumin Residual Binding Capacity for Bilirubin

2015 ◽  
pp. 262-279
Keyword(s):  
Binding Assay ◽  
Binding Capacity ◽  
On Chip

scholarly journals Bordetella bronchiseptica Adherence to Cilia Is Mediated by Multiple Adhesin Factors and Blocked by Surfactant Protein A

2005 ◽  
Vol 73 (6) ◽  
pp. 3618-3626 ◽  
Author(s):  
Jessica A. Edwards ◽  
Nathan A. Groathouse ◽  
Scott Boitano
Keyword(s):  
Binding Assay ◽  
Binding Capacity ◽  
Protein A ◽  
Upper Airway ◽  
Surfactant Protein ◽  
Bordetella Bronchiseptica ◽  
Surfactant Protein A ◽  
Dermonecrotic Toxin ◽  
Bacterial Adhesins ◽  
Tracheal Epithelial

ABSTRACT In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg− phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg− strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.

scholarly journals Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2020 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2019 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

Oestrogen and progesterone receptor binding capacity and oestrogen receptor alpha expression (ERα mRNA) along the cervix of cycling ewes

2008 ◽  
Vol 20 (3) ◽  
pp. 350 ◽  
Author(s):  
M. Rodríguez-Piñón ◽  
C. Tasende ◽  
P. Puime ◽  
E. G. Garófalo
Keyword(s):  
Progesterone Receptor ◽  
Oestrogen Receptor ◽  
Binding Assay ◽  
Binding Capacity ◽  
Longitudinal Axis ◽  
Oestrous Cycle ◽  
Receptor Alpha ◽  
Oestrogen Receptor Alpha ◽  
Functional Differences ◽  
Mrna Concentration

The aim of the present work was to study the oestrogen receptor (ER) and progesterone receptor (PR) binding capacity and the oestrogen receptor alpha (ERα) mRNA concentration in cranial and caudal cervix during the ovine oestrous cycle. Cervical samples of synchronised Corriedale ewes were obtained on Day 1 (n = 7), 6 (n = 6) or 13 (n = 7) after oestrus detection (Day 0). The ER and PR binding capacity by ligand-binding assay and the ERα mRNA concentration by solution hybridisation in both cranial and caudal zones of the cervix were determined. The ER and PR binding capacity were higher (P < 0.005) on Day 1 than on Days 6 and 13 in both cranial and caudal zones. The ERα mRNA concentrations were higher (P < 0.0001) on Day 1 than on Days 6 and 13 only in the caudal zone. The PR binding capacity and ERα mRNA concentration were higher (P < 0.005) in the caudal than in the cranial zone on Day 1. The ER and PR expression in the ovine cervix varied during the oestrous cycle in agreement with the known upregulation exerted by oestrogen and downregulation exerted by progesterone. Differences in ER and PR expression along the longitudinal axis of the ovine cervix were found, reflecting histological and functional differences between the cranial and caudal zones.

Properties and localization of endothelin-1-specific receptors in rat testicles

1992 ◽  
Vol 263 (1) ◽  
pp. R15-R18
Author(s):  
H. Sakaguchi ◽  
M. Kozuka ◽  
S. Hirose ◽  
T. Ito ◽  
H. Hagiwara
Keyword(s):  
Binding Assay ◽  
Binding Capacity ◽  
Physiological Role ◽  
Spermatogenic Cells ◽  
Myoid Cells ◽  
Rat Testis ◽  
Endothelin 1 ◽  
Specific Receptors ◽  
Membrane Bound ◽  
Endothelin 3

The characterization and localization of rat testicular endothelin receptors were studied. Receptor binding assay with radiolabeled members of the endothelin family revealed that endothelin-1-specific receptors were present in rat testes; a maximal binding capacity of 250 +/- 62 fmol/mg protein and a dissociation constant value of 0.35 +/- 0.06 nM were calculated from the Scatchard plot. The affinities for endothelin analogues were endothelin-1 = endothelin-2 much greater than endothelin-3 much greater big endothelin-1 for the membrane-bound receptors. Receptors of endothelin-1 were localized in peritubular myoid cells and interstitial cells of rat testis by 125I-labeled endothelin-1 autoradiography; the receptors were undetectable in spermatogenic cells. The presence of endothelin-1 receptors in the rat testis raises the possibility that one or more of the endothelins may play a physiological role in the regulation of testicular function.

Immunoassay for estrogen receptor does not detect inactivated receptor.

1989 ◽  
Vol 35 (5) ◽  
pp. 824-828 ◽  
Author(s):  
L A Molnar ◽  
N Dhirani ◽  
K Tran ◽  
W A McBlain
Keyword(s):  
Estrogen Receptor ◽  
Binding Assay ◽  
Binding Capacity ◽  
Antigenic Site ◽  
Breast Tumors ◽  
Assay Methods ◽  
Steroid Binding ◽  
Commercial Enzyme Immunoassay ◽  
Accurate Quantification

Abstract Accurate quantification of estrogen receptor (ER) is essential for optimal clinical characterization of individual cases of breast cancer. If breast tumors are mishandled, the relatively labile ER protein may lose its steroid-binding capacity (become inactivated) and not be measurable by the routine steroid-binding assay. We tested whether the commercial enzyme immunoassay of Abbott Laboratories could quantify inactivated ER. Samples of powdered breast tumors from humans were exposed to various temperature and homogenization conditions known to inactivate ER, and any remaining ER was quantified by both the immunoassay and the steroid-binding assay. For all inactivation conditions tested, the two assay methods detected the same proportions of remaining ER. We conclude that the inactivation reaction for ER also alters one or both of the antigenic site(s) necessary for the immunoassay. Hence, for breast tumors mishandled to the extent of inactivating ER, the immunoassay offers no advantage over the more conventional steroid-binding assay for quantifying any remaining ER.

scholarly journals Synthesis and Characterization of Molecular Imprinting Polymer Microspheres of Piperine: Extraction of Piperine from Spiked Urine

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Rachel Marcella Roland ◽  
Showkat Ahmad Bhawani
Keyword(s):  
Extraction Efficiency ◽  
Binding Assay ◽  
Binding Capacity ◽  
Molar Ratio ◽  
Polymer Particles ◽  
Imprinted Polymer ◽  
Molecularly Imprinted ◽  
Ft Ir ◽  
Cross Linker

Molecularly imprinted polymer (MIP) microspheres for Piperine were synthesized by precipitation polymerization with a noncovalent approach. In this research Piperine was used as a template, acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, and 2,2′-azobisisobutyronitrile (AIBN) as an initiator and acetonitrile as a solvent. The imprinted and nonimprinted polymer particles were characterized by using Fourier transform infrared spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). The synthesized polymer particles were further evaluated for their rebinding efficiency by batch binding assay. The highly selected imprinted polymer for Piperine was MIP 3 with a composition (molar ratio) of 0.5 : 3 : 8, template : monomer : cross-linker, respectively. The MIP 3 exhibits highest binding capacity (84.94%) as compared to other imprinted and nonimprinted polymers. The extraction efficiency of highly selected imprinted polymer of Piperine from spiked urine was above 80%.

scholarly journals Identification of a heparin-releasable hepatic lipase binding protein from rat liver

1998 ◽  
Vol 330 (2) ◽  
pp. 785-789 ◽  
Author(s):  
Belinda BREEDVELD ◽  
Kees SCHOONDERWOERD ◽  
Hans JANSEN
Keyword(s):  
Rat Liver ◽  
Binding Assay ◽  
Solid Phase ◽  
Binding Capacity ◽  
Specific Binding ◽  
Hepatic Lipase ◽  
Binding Activity ◽  
Cross Linking ◽  
Major Protein ◽  
Slot Blot

Hepatic lipase (HL) plays a key role in the metabolism of several lipoproteins. Metabolically active HL is bound in liver parenchymal cells to specific binding sites. We studied the nature of the HL binding in rat liver. Rat livers were perfused with heparin, which lead to a loss of 80% of the HL binding capacity of the liver. The heparin-containing perfusates possessed HL binding capacity, determined by slot-blot assay. The perfusates were loaded on to a heparin-Sepharose column and eluted with a linear salt gradient (0.2-1 M). HL binding activity, assessed by a slot-blot binding assay, eluted both at 0.3 M and at 0.8 M NaCl. A 0.5 M NaCl eluate was used to further characterize the HL binding activity. In this fraction the major protein had a molecular mass of 70 kDa. The fraction showed saturable HL binding in a solid-phase binding assay. Cross-linking of the 0.5 M NaCl fraction to 125I-labelled HL yielded a complex of 130 kDa, suggesting the cross-linking of the 57 kDa 125I-labelled HL to a protein of about 73 kDa. We concluded that heparin releases a protein of about 73 kDa from rat liver, which associates with HL. This protein may represent the HL binding site in liver.

scholarly journals Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2019 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips. In particular, we took advantage of bradykinin-derived peptidic baits to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

scholarly journals Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Reproduction ◽  
2000 ◽  
pp. 77-83 ◽  
Author(s):  
B Strom Holst ◽  
B Larsson ◽  
C Linde-Forsberg ◽  
H Rodriguez-Martinez
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Zona Pellucida ◽  
Binding Assay ◽  
Binding Capacity ◽  
Storage Conditions ◽  
Deep Freezing ◽  
Sperm Binding ◽  
Significant Difference ◽  
Scanning Electron

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.

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