Binding Characteristics of the Antiendotoxin Antibody Xomen-E5 to Bacterial Whole-Cell Antigens

1993 ◽  
pp. 1211-1217
Author(s):  
K. J. Gorelick ◽  
D. M. Wood ◽  
J. B. Parent ◽  
E. Lim ◽  
K. D. Knebel ◽  
...  
Keyword(s):  
Whole Cell ◽  
Binding Characteristics ◽  
Antiendotoxin Antibody

scholarly journals Biotinylated Synthetic Chemokines: Their Use for the Development of Nonradioactive Whole-Cell Binding Assays

2003 ◽  
Vol 8 (3) ◽  
pp. 316-323 ◽  
Author(s):  
Anne-Christine Thierry ◽  
Geneviève Perrenoud ◽  
Stéphane Pinaud ◽  
Nicolas Bigler ◽  
Bérangère Denis ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Assay ◽  
Biologically Active ◽  
Binding Assays ◽  
Cell Binding ◽  
Chemical Route ◽  
Whole Cell ◽  
Migration Assay ◽  
Binding Characteristics ◽  
Whole Cells

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen. ( Journal of Biomolecular Screening 2003:316-323)

HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

1997 ◽  
Vol 77 (05) ◽  
pp. 0949-0954 ◽  
Author(s):  
J Prins ◽  
F R Lues ◽  
Y Y van der Hoek ◽  
J J.P Kastelein ◽  
B N Bouma ◽  
...  
Keyword(s):  
Case Control Study ◽  
Amino Acid Position ◽  
Case Control ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Apolipoprotein A ◽  
Significant Independent Risk Factor ◽  
And Gender ◽  
Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

Maternal and Fetal Platelet Responses and Adrenoceptor Binding Characteristics

1985 ◽  
Vol 53 (01) ◽  
pp. 095-098 ◽  
Author(s):  
C R Jones ◽  
R McCabe ◽  
C A Hamilton ◽  
J L Reid
Keyword(s):  
Adenylate Cyclase ◽  
Venous Blood ◽  
Adenosine Diphosphate ◽  
Binding Characteristics ◽  
Receptor Coupling ◽  
Threshold Response ◽  
Alpha Receptors ◽  
Maternal Responses ◽  
Epidural Anaesthetic

SummaryPaired blood samples were obtained from mothers (venous) and babies (cord venous blood) at the time of delivery by caesarean section under epidural anaesthetic. Fetal platelets failed to aggregate in response to adrenaline in vitro although adrenaline could potentiate the threshold response to adenosine diphosphate (1 μM). Fetal platelet responses to collagen and 8 Arg vasopressin did not differ significantly from maternal responses. Maternal and fetal platelets also showed similar inhibition of aggregation after activation of adenylate cyclase (PGE1 and parathormone), in contrast to the inhibition of adenylate cyclase by adrenaline.Alpha2 adrenoceptors were investigated using [3H] yohimbine binding receptor number and were reduced modestly but significantly on fetal compared to maternal platelets. The failure of fetal platelet aggregation in response to adrenaline appears to be related to a failure of receptor coupling and may represent a delayed maturation of fetal platelet alpha receptors or a response- to increased circulating catecholamines during birth.

Foetal steroid binding protein in British and Japanese women

1987 ◽  
Vol 114 (4) ◽  
pp. 584-588 ◽  
Author(s):  
M. Jawed Iqbal ◽  
Alastair Forbes ◽  
Mark L. Wilkinson ◽  
John W. Moore ◽  
Roger Williams ◽  
...  
Keyword(s):  
Ovarian Function ◽  
Binding Protein ◽  
Premenopausal Women ◽  
Japanese Women ◽  
Sex Hormone Binding Globulin ◽  
Partial Purification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Characteristics ◽  
Steroid Binding ◽  
Steroid Binding Protein

Abstract. In order to examine the newly-discovered sex-steroid binding protein, foetal steroid binding protein (FSBP) in different populations, its binding characteristics and its level were studied by two-tier column ligand binding assay and enzyme-linked immunosorbent assay (ELISA) respectively. In 10 Japanese premenopausal women, analysis of 5α-dihydrotestosterone (DHT) binding in the Cibacron Blue 3GA-Sepharose 6B portion of the column showed a rising plateau pattern with a mean maximum binding of 31.1 ± 7.41%, whereas of 9 similar British women, 8 displayed unsaturable, non-cooperative binding of 11.6 ± 8.22% (P < 0.01). After partial purification of FSBP in these samples, the protein exhibited saturable binding kinetics, median binding 25 (interquartiles 23–34) and 19 (13–25) nmol DHT/l in Japanese and British women, respectively (P < 0.05). By analyzing FSBP by ELISA in 56 Japanese (45 premenopausal) and 59 British (25 premenopausal) women, higher levels were obtained in the whole Japanese group (P = 0.0016) and in the premenopausal Japanese women (P = 0.018) than in their British counterparts. In both nationalities, FSBP levels were higher in premenopausal women, and there was a significant negative correlation of FSBP with age in both populations, particularly in postmenopausal women. FSBP levels did not correlate with weight, parity, sex hormone binding globulin or albumin levels. The influence of FSBP on free steroid levels remains unclear, but some relationship with ovarian function seems a possibility.

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