scholarly journals The SA/rABC technique: a new ABC procedure for detection of antigens at increased sensitivity.

1995 ◽  
Vol 43 (1) ◽  
pp. 31-37 ◽  
Author(s):  
I M Grumbach ◽  
R W Veh
Keyword(s):  
Binding Sites ◽  
Signal Intensity ◽  
Detection System ◽  
Molar Ratios ◽  
Tissue Sections ◽  
Biotin Binding ◽  
Increased Sensitivity ◽  
Bioanalytical Applications ◽  
Theoretical Considerations ◽  
Maximal Signal Intensity

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin-peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.

The Estimation of the Number of Binding Sites for Antibody on Antigen Molecules with Reference to Factor VIII and its Antibody

1976 ◽  
Vol 35 (02) ◽  
pp. 274-288 ◽  
Author(s):  
Judith Pool ◽  
Rosemary Biggs ◽  
R. G Miller
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Theoretical Basis ◽  
Human Antibody ◽  
Rabbit Antibody ◽  
Number Of Binding Sites ◽  
Theoretical Considerations

SummaryThe theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1–3 sites for human antibody. The data for rabbit antibody suggest 5–6 sites per factor VIII molecule.

scholarly journals Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

2014 ◽  
Vol 406 ◽  
pp. 143-147 ◽  
Author(s):  
Raju Ahmed ◽  
Emma Spikings ◽  
Shaobo Zhou ◽  
Andrew Thompsett ◽  
Tiantian Zhang
Keyword(s):  
Detection System ◽  
Binding Activity ◽  
Biotin Binding ◽  
Endogenous Biotin

scholarly journals Phosphorylated Alpha-Synuclein Within Cutaneous Autonomic Nerves of Patients With Parkinson’s Disease: The Implications of Sample Thickness on Results

2020 ◽  
Vol 68 (10) ◽  
pp. 669-678
Author(s):  
Ningshan Wang ◽  
Jennifer Garcia ◽  
Roy Freeman ◽  
Christopher H. Gibbons
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Alpha Synuclein ◽  
Sweat Glands ◽  
Autonomic Nerves ◽  
Pgp 9.5 ◽  
Protein Gene Product ◽  
Tissue Sections ◽  
Increased Sensitivity ◽  
Skin Biopsies

The detection of cutaneous phosphorylated alpha-synuclein (P-syn) in patients with Parkinson’s disease (PD) has ranged from 30% to 100% across different studies. We hypothesize that part of the variability in P-syn detection is due to methodological differences using sections of different tissue thickness. Three skin biopsies were obtained from 29 individuals with PD and 21 controls. Tissues were cut into 10-, 20-, and 50-µm-thick sections and double-stained with protein gene product (PGP) 9.5 and P-syn. We quantified the deposition of P-syn with and without PGP 9.5 in sweat glands, pilomotor muscle, and blood vessels using confocal digital images of autonomic structures. Overall, the P-syn-positive rates with PGP 9.5 colocalization in subjects with PD were 100% using 50 µm sections, 90% using 20 µm sections, and 73% using 10 µm sections with 100% specificity. (No P-syn was detected within control subjects.) Without PGP 9.5, colocalization of the P-syn-positive rates was 100% for all samples, but specificity dropped below 70%. In this study, double-immunostained 50 µm skin biopsy tissue sections are superior to 20 and 10 µm tissue sections at detecting P-syn in subjects with PD. The increased sensitivity is likely secondary to a combination of greater volume of tissue analyzed and improved visualization of nerve fiber architecture.

Relative Affinity of Plasmin for the α1-Macroglobulin, Cl Inactivator and α1-Antitrypsin Inhibitors

1975 ◽  
Author(s):  
Peter C. Harpel
Keyword(s):  
Complex Formation ◽  
Protease Inhibitors ◽  
Disulfide Bond ◽  
Light Chain ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Molar Ratio ◽  
Molar Ratios ◽  
Relative Affinity ◽  
Quantitative Contribution

The quantitative contribution of three major plasma protease inhibitors in binding plasmin has been studied. Mixtures of plasmin and each of the purified inhibitors were analyzed by SDS-acrylamide gel electrophoresis. Plasmin remained bound to its inhibitors in the presence of SDS and urea. A 1 : 1 molar ratio for complex formation was established, and treatment of the complexes with a disulfide bond reducing agent showed that the light chain of plasmin contained the binding sites for both CĪ inactivator and α-antitrysin. Limited degradation of all three inhibitors by plasmin was observed, and the altered inhibitor remained complexed to the enzyme. The competitive binding of 125I plasmin to mixtures of these inhibitors was followed by sucrose density ultracentrifugation and by SDS-gel electrophoresis. In mixtures containing physiologic molar ratios of enzyme and inhibitors, over 80% of the bound plasmin was complexed to the α2-macroglobufui (α2M). No evidence for an exchange of plasmin between the inhibitors was obtained.

Characterization of cholecystokinin binding sites in goldfish brain and pituitary

1996 ◽  
Vol 271 (1) ◽  
pp. R137-R143 ◽  
Author(s):  
B. A. Himick ◽  
S. R. Vigna ◽  
R. E. Peter
Keyword(s):  
Binding Sites ◽  
Hormone Secretion ◽  
Pituitary Hormone ◽  
Tissue Sections ◽  
High Affinity ◽  
Hypothalamic Nuclei ◽  
High Affinity Binding Site ◽  
Vagal Lobe ◽  
Feeding Center ◽  
Reversible Time

The characterization and distribution of cholecystokinin (CCK)/gastrin binding sites were determined in the goldfish central nervous system (CNS). Binding of 125I-sulfated CCK octapeptide (125I-CCK-8s) in tissue sections was found to be saturable, reversible, time dependent, and displaceable by CCK/gastrin-like peptides. Analysis of saturable equilibrium binding revealed a high-affinity binding site (dissociation constant of 0.706 +/- 0.188 nM), which also displayed high affinity for gastrin-17s and caerulein. Lower affinities were observed for the nonsulfated forms of CCK-8 and gastrin-17. These findings suggest that a single primitive CCK/gastrin receptor exists in the goldfish CNS. The distribution of CCK/gastrin binding sites in the goldfish brain and pituitary revealed high densities within the telencephalon and preoptic hypothalamus, as well as within hypothalamic nuclei associated with the brain feeding center. High densities of binding sites were also localized within the midbrain tegmentum and optic tectum of the midbrain, the facial lobe and vagal lobe of the hindbrain, and within the pituitary pars distalis. Overall, these findings support previous studies that indicate that CCK/gastrin-like peptides play a role in the central regulation of feeding behavior and pituitary hormone secretion in fish.

Biotinylated Polythiophene Copolymer – a Novel Electroactive Biomaterial Utilizing the Biotin-Streptavidin Interaction

1992 ◽  
Vol 292 ◽  
Author(s):  
Jeong-Ok Lim ◽  
Manjunath Kamath ◽  
Kenneth A. Marx ◽  
Sukant K. Tripathy ◽  
David L. Kaplan ◽  
...  
Keyword(s):  
Binding Sites ◽  
Solid Substrate ◽  
Emission Peak ◽  
One Dimensional ◽  
Langmuir Blodgett ◽  
Significant Area ◽  
Pressure Area ◽  
Biotin Binding ◽  
Monolayer Assembly ◽  
Area Expansion

AbstractA novel hierarchical biomaterial capable of incorporating any biotinylated biomolecule has been created. Our strategy is to biotinylate one-dimensional electroactive polymers and use a bridging streptavidin protein on Langmuir-Blodgett (LB) organized films. The following copolymeric system which enables functionalization of other molecules and formation of good monolayers was employed. Biotinylated poly(3-methanolthiophene-co-3-undecylthiophene) (B-PMUT) demonstrated a significantly better isotherm implying superior molecular packing compared to poly(3-methanolthiophene-co-3-undecylthiophene) (PMUT) on the LB airwater surface. The isotherm showed significant area expansion when streptavidin was injected below the B-PMUT monolayer in 0.1mM NaH2PO4/0.1 M NaCl buffer (pH 6.8) subphase. We then incorporated biotinylated phycoerythrin (B-PE) into this novel biomaterial by binding the unoccupied biotin binding sites on the bound streptavidin (4 sites total). The pressure-area isotherm of the protein injected monolayer showed area expansion. A characteristic fluorescent emission peak at 576nm was detected from the monolayer transferred onto a solid substrate. These observations demonstrated the function of B-PMUT in hierarchical monolayer assembly of molecules incorporating the biotin / streptavidin interaction.

scholarly journals Lectin-binding properties of human breast cancer cell lines and human milk with particular reference to Helix pomatia agglutinin.

1995 ◽  
Vol 43 (3) ◽  
pp. 275-281 ◽  
Author(s):  
U Schumacher ◽  
E Adam ◽  
S A Brooks ◽  
A J Leathem
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Binding Sites ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Breast Cancer Cell Lines ◽  
Human Breast ◽  
Membrane Glycoproteins ◽  
Tissue Sections ◽  
Helix Pomatia Agglutinin

Several studies have shown binding of a variety of lectins to breast cancer cells in tissue sections. In particular, binding of the lectin from the Roman snail, Helix pomatia agglutinin (HPA), to breast cancer cells is linked with a poor prognosis. The molecular basis for lectin binding to metastatic breast cancers is not known. To elucidate this in a model system, lectin-binding patterns of seven human breast cancer cell lines were investigated, their cell membranes were isolated, and HPA binding was assessed. In addition, the influence of fixation and processing on lectin-binding sites was also investigated. Binding of lectins to the tumor cells was very heterogeneous between and within the different cell lines and was influenced by fixation and processing. However, some cell lines showed HPA-binding sites both in vivo and in tissue sections. Analysis of the isolated cell membrane glycoproteins from these cell lines on Western blots revealed that HPA can bind to several membrane glycoproteins. In contrast, human milk shows only one major milk glycoprotein that is HPA-positive. Therefore, a switch in glycosylation appears to be taking place during the transformation to a metastatic phenotype.

scholarly journals Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin.

1989 ◽  
Vol 37 (2) ◽  
pp. 249-256 ◽  
Author(s):  
K Fujimoto ◽  
N Araki ◽  
K S Ogawa ◽  
S Kondo ◽  
T Kitaoka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Biologically Active ◽  
Myocardial Cells ◽  
Thin Sections ◽  
Gold Particles ◽  
Calmodulin Binding ◽  
Tissue Sections

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.

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