Eliminating Nonspecific Binding Sites for Highly Reliable Immunoassay via Super-resolution Multicolor Fluorescence Colocalization

Nanoscale ◽  
2021 ◽  
Author(s):  
Shenfei Zong ◽  
Yun Liu ◽  
Kuo Yang ◽  
Zhaoyan Yang ◽  
Zhuyuan Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Super Resolution ◽  
Specific Adsorption ◽  
Nonspecific Binding ◽  
Nonspecific Adsorption ◽  
Multicolor Fluorescence

Non-specific adsorption in immunoassays has always been a major problem that affects the reliability of assay results. Despite the emergence of various methods which can reduce nonspecific adsorption, a universal...

A comparison of cobalamin binding by liver and kidney in rat and man

1985 ◽  
Vol 68 (3) ◽  
pp. 357-364 ◽  
Author(s):  
J. S. D. Scott ◽  
E. P. W. Bowman ◽  
W. G. E. Cooksley
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Competitive Inhibition ◽  
Rat Kidney ◽  
Binding Constants ◽  
Human Kidney ◽  
Specific Adsorption ◽  
Receptor Protein ◽  
Mean Values ◽  
Liver And Kidney

1. Binding of cobalamin (Cbl) was compared in liver and kidney plasma membranes prepared from rat and human tissues. 2. Single, high-affinity, saturable (200 pmol/l), binding sites for TC II-Cbl were found in all tissues; by contrast no receptors were present for free cobalamin, for which only non-specific adsorption occurred. 3. Binding constants for TC II-CNCbl determined for liver and kidney plasma membranes were of a similar magnitude. Mean values for Ka (litre/nmol) were 16.7 (rat liver), 18.8 (rat kidney), 8.0 (human liver) and 7.5 (human kidney). 4. Results for binding TC II-OHCbl instead of TC II-CNCbl showed no difference in Ka and Bmax. values, although the non-specific adsorption was decreased to a third. 5. Competitive inhibition results showed that the receptors are specific for the TC II molecule and that binding is unaffected either by the cobalamin moiety or by the presence of free cobalamin. Degradation of the receptor protein molecules by trypsin (10 μg/ml) resulted in 90% inhibition of binding. 6. It is concluded that differences between liver and kidney in cobalamin uptake and accumulation cannot be attributed to differences in their TC II receptors.

scholarly journals Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

1985 ◽  
Vol 33 (5) ◽  
pp. 474-476 ◽  
Author(s):  
V Muresan ◽  
M C Constantinescu
Keyword(s):  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Binding Sites ◽  
Sodium Periodate ◽  
Luminal Surface ◽  
Nonspecific Binding ◽  
Coated Pits ◽  
Neuraminidase Treatment ◽  
Plasmalemmal Vesicles ◽  
Endothelial Surface

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

scholarly journals Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4

2021 ◽  
Vol 118 (3) ◽  
pp. e2009493118
Author(s):  
Yufei Shan ◽  
Stephen Matthew Farmer ◽  
Susan Wray
Keyword(s):  
Neuronal Migration ◽  
Binding Sites ◽  
Cortical Neurons ◽  
Actin Polymerization ◽  
Granule Cells ◽  
System Development ◽  
Super Resolution ◽  
Direct Interaction ◽  
Nervous System Development ◽  
Gnrh Neurons

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein–protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

Receptor Analysis: Data Processing in the Presence of Nonspecific Binding Sites

1987 ◽  
pp. 107-113
Author(s):  
H. Moeller ◽  
Ch. Fusch ◽  
A. Kuch ◽  
N. Manolopoulos ◽  
G. Oettling
Keyword(s):  
Data Processing ◽  
Binding Sites ◽  
Analysis Data ◽  
Nonspecific Binding

scholarly journals R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues.

1991 ◽  
Vol 39 (7) ◽  
pp. 921-926 ◽  
Author(s):  
G Sohn ◽  
C Sautter
Keyword(s):  
Indirect Immunofluorescence ◽  
Binding Sites ◽  
Aquatic Macrophytes ◽  
Elodea Canadensis ◽  
Fluorescent Labeling ◽  
Fluorescent Label ◽  
Nonspecific Binding ◽  
Immunofluorescence Method ◽  
Rabbit Igg ◽  
Highly Sensitive

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.

Highly hydrophilic polyhedral oligomeric silsesquioxane (POSS)-containing aptamer-modified affinity hybrid monolith for efficient on-column discrimination with low nonspecific adsorption

The Analyst ◽  
2019 ◽  
Vol 144 (5) ◽  
pp. 1555-1564 ◽  
Author(s):  
Yiqiong Chen ◽  
Dandan Zhu ◽  
Xinyue Ding ◽  
Guomin Qi ◽  
Xucong Lin ◽  
...  
Keyword(s):  
Polyhedral Oligomeric Silsesquioxane ◽  
Specific Adsorption ◽  
Nonspecific Adsorption ◽  
Oligomeric Silsesquioxane

A highly hydrophilic aptamer-modified POSS-containing hybrid affinity monolith is presented for efficient on-column discrimination with low non-specific adsorption.

Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins

1974 ◽  
Vol 20 (9) ◽  
pp. 1150-1154 ◽  
Author(s):  
Victor S Fang ◽  
Samuel Refetoff
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Nonspecific Binding ◽  
High Concentration ◽  
Assay Sensitivity ◽  
Thyroxine Binding Globulin ◽  
Fixed Concentration

Abstract Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T3) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T3. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T3 were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T3 from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T3 from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T3 quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T3-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T3 binding.

scholarly journals Active Cation Transport and Ouabain Binding in High Potassium and Low Potassium Red Blood Cells of Sheep

1971 ◽  
Vol 58 (1) ◽  
pp. 94-116 ◽  
Author(s):  
Philip B. Dunham ◽  
Joseph F. Hoffman
Keyword(s):  
Binding Sites ◽  
High Specificity ◽  
Cation Transport ◽  
Nonspecific Binding ◽  
Ouabain Binding ◽  
High Potassium ◽  
High K ◽  
Low Potassium ◽  
The Difference ◽  
Specificity Of Binding

Red cells from high K sheep contained 82 mM K/liter cells and had a pump flux of 0.86 mM K/liter cells x hr; similarly, LK cells had 16.5 mM K/liter cells and a pump flux of 0.12 mM K/liter cells x hr. Using [3H]-ouabain, the relation between the number of ouabain molecules bound per cell and the concomitant per cent inhibition of the pump was found to be approximately linear for both HK and LK cells. The number of glycoside molecules necessary for 100 % inhibition of the pump was 42 for HK cells and 7.6 for LK cells, after correction for six nonspecific binding sites for each type of cell. The ratio of ouabain molecules/cell at 100 % inhibition was 5.5, HK to LK, and the ratio of the normal K pump fluxes was 7.2, HK to LK. The similarity of these ratios suggests that an important difference between HK and LK cells, determining the difference in pump fluxes, is the number of pump sites. The turnover times (ions/site x min) are 6000 and 4800 for HK and LK cells, respectively. The results also indicate a high specificity of binding of ouabain to pump sites.

The Effect of Temperature Changes on Tachyphylaxis to Angiotensin In Vitro

1972 ◽  
Vol 50 (6) ◽  
pp. 498-502 ◽  
Author(s):  
Patrick LeMorvan ◽  
Djuro Palaic
Keyword(s):  
Cell Membrane ◽  
Guinea Pig ◽  
Dose Response ◽  
Binding Sites ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Maximal Response ◽  
Nonspecific Binding ◽  
Temperature Changes

The effect of heat on the contractility and binding of 14C-angiotensin to guinea pig aortic strips was studied. It was found that heating strips to 47 °C for 20 min causes an increase in maximal response as well as a shift to the left of the dose–response curve for angiotensin while responses to noradrenaline (NA) remain unchanged. This procedure also increases the onset of tachyphylaxis to 10−5 M angiotensin. Heating (47 °C) for varying periods of time decreases the binding of 14C-angiotensin by about 60%. This effect does not seem to be related to the time of heating. It is postulated that this reduction in binding reflects denaturation of nonspecific binding sites on the cell membrane. Heating (47 °C, 20 min) enhanced the binding of 14C-angiotensin to aortic strips which had been rendered tachyphylactic to 10−5 M angiotensin. It is suggested that this is due to the induction of new binding sites.

Sign in / Sign up

Export Citation Format

Share Document