scholarly journals Isolation of Low Abundance Proteins and Cells Using Buoyant Glass Microbubble Chromatography

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Steingrimur Stefansson ◽  
Daniel L. Adams ◽  
Cha-Mei Tang
Keyword(s):  
Protein Binding ◽  
Biological Samples ◽  
Magnetic Beads ◽  
Solid Phase ◽  
Binding Capacity ◽  
Protein A ◽  
The Other ◽  
Neurofilament Protein ◽  
Surface Areas ◽  
Fast Flow

Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining them in situ.

The Effect Of Coagulation On The Association Of VIII:CAg With VIIIR:Ag

1981 ◽  
Author(s):  
J A van Mourik ◽  
P H G Lantinga ◽  
J A Hellings
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Calcium Concentration ◽  
Solid Phase ◽  
Binding Capacity ◽  
The Other ◽  
Void Volume ◽  
Concomitant Increase ◽  
Initial Value ◽  
Factor Viii Related Antigen

Solid-phase immunoradiometric assays specific for Factor VIII coagulant antigen (VIII:CAg) and Factor VIII related antigen (VIIIR:Ag) have been used to assay the immunoreactivity of these antigens in plasma and whole blood during coagulation at physiological calcium concentration. When non-anticoagulated plasma, prepared from blood immediately after venipuncture, was incubated at 37°C, the concentration of VIII:CAg and VIlIR:Ag did not change. However, when whole blood, collected withoutanticoagulant, was incubated, the concentration of VIII:CAg gradually decreased to 50% of the initial value whereas the concentration of VIIIR:Ag remained unchanged. Gelchromatographic analyses revealed that coagulation of plasma leads to progressive dissociation of VIII:CAg from the factor VIII:VWF complex. When plasma was chromatographed before the onset of coagulation, VIII:CAg was eluted at the void volume together with VIIIR:Ag whereas after coagulation of the plasma VIII:CAg devoid of VIIIR:Ag was eluted after the void volume. Similarly, when the supernatant plasma from blood was chromatographed before the onset of coagulation, VIII:CAg together with VIIIR:Ag was eluted at the void volume whereas during and after coagulation the amount of VIII:CAg associated with VIIIR:Ag gradually decreased. However, no concomitant increase of the concentration of dissociated VIII:CAg was noted under the latter conditions. It seems likely, therefore, that adherance of dissociated VIII:CAg to cellular constituents accounts for the loss of VIII:CAg during coagulation of blood. On the other hand, it can not be excluded that cellular enzymes, extruded during coagulation, affect the antibody-binding capacity of VIII:CAg.Further studies indicate that, at least in part, dissociation of the factor VIII:VWF complex during coagulation is mediated by thrombin.

EDTA-DEPENDENT MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S

1987 ◽  
Author(s):  
S Marcovina ◽  
R Coppola ◽  
M P Protti ◽  
C Gelfi ◽  
P M Mannucci
Keyword(s):  
Monoclonal Antibodies ◽  
Ascitic Fluid ◽  
Solid Phase ◽  
Protein A ◽  
Fluid Phase ◽  
Protein S ◽  
The Other ◽  
Human Protein ◽  
Sephadex Column ◽  
Plot Analysis

Splenocytes from a Balb/c mouse immunized with purified human protein S (PS) were fused with murine hybridoma cell line SP2/0-Agl4 and cultured in Iscove's medium without addition of fetal bovine serum. Hybrid supernatants were screened for the presence of specific antibodies by solid-phase ELISA and cloned by the limiting dilution technique. Pour clones, named S2, S3, S8, and S10, were selected, recloned several times, and injected intraperitoneally into Balb/c mice for the production of antibody-rich ascitic fluid. The monoclonal antibodies (Mabs), all of IgGl subclass with k light chain, were purified from ascitic fluid by Protein-A chromatography. The specificity of Mabs was controlled by the immunoblotting technique: the Mabs appeared to react only with two plasma proteins, one with a MW of about 70.000 dal tons comigrating with purified PS, and the other with a MW >300.000 da that is likely to be the C4b-binding protein-PS complex. No interaction has been observed with PS-depleted plasma. As tested by a fluid phase radio immunoassay, the binding of Mabs to PS was significantly higher in the presence of EDTA while was totally inhibited in the presence of Ca2+. Scatchard plot analysis of the binding between 125I-PS and Mabs showed that the binding affinity of the antibodies ranged from 108 to 109 1/mol. Each EDTA-dependent Mab was then immobilized on Sepharose 4B-CNBr and used to purify PS from barium precipitation of citrated plasma. The fraction eluted with 2 mmol of CaCl2 from the immunoadsorbent appeared to contain only two proteins when stained with Cocmassie Blue. By immuno blotting, all Mabs reacted with both proteins, one comigrating with purified PS and the other with a MW >300.000. Essentially homogeneous PS, free from the high MW component, was obtained when the barium citrate adsorbate was applied to a DEAE-Sephadex column and the protein peack containing the balk of PS was sussequently applied to the immunoadsorbent and eluted with 2 mmol CaCl2.In summary, we have described an unusual set of EDTA-dependent monoclonal antibodies to PS and their use for the purification of homogeneous protein S from human plasma.

scholarly journals Ig Glycosylation in Ulcerative Colitis: It’s Time for New Biomarkers

2021 ◽  
Vol 12 ◽  
Author(s):  
Riccardo Capecchi ◽  
Paola Migliorini ◽  
Federico Zanzi ◽  
Simona Maltinti ◽  
Ilaria Puxeddu ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Clinical Remission ◽  
Solid Phase ◽  
Binding Capacity ◽  
Protein A ◽  
Mucosal Healing ◽  
Therapeutic Outcome ◽  
Serum Samples ◽  
Biologic Drugs ◽  
Number Of Patients

Background: Ulcerative colitis (UC) is a chronic relapsing disease, which needs a continue monitoring, especially during biological therapies. An increasing number of patients is treated with anti-Tumor Necrosis factor (TNF) drugs, and current research is focalized to identify biomarkers able to monitor the disease and to predict therapeutic outcome.Methods: We enrolled consecutive UC patients treated with anti-TNF, naïve to biologic drugs. Therapeutic outcome was evaluated after 54 weeks of treatment in terms of clinical remission (Partial Mayo Score -PMS- <2) and mucosal healing (Mayo Endoscopic Score <2). On serum samples collected at baseline and after 54 weeks of treatment, a Lectin-based ELISA assay was performed, and specific glycosylation patterns were evaluated by biotin-labelled lectins. We have also collected 21 healthy controls (NHS) samples, age and sex-matched.Results: Out of 44 UC patients enrolled, 22 achieved clinical remission and mucosal healing after 54 weeks. At baseline, when Protein A was used as coating, UC patients non-responders showed a reduced reactivity to Jacalin (JAC) in comparison with NHS (p = 0.04). After one year of treatment, a decrease in JAC binding was seen only in responders, in comparison with baseline (p = 0.04). When JAC binding was tested selecting IgG by means of Fab anti-IgG Fab, UC patients displayed an increased reactivity after anti-TNF therapy (p < 0,0001 vs controls). At baseline, PMS inversely correlates with JAC binding when Fab anti-IgG Fab was used in solid phase (r2 = 0,2211; p = 0,0033). Patients with higher PMS at baseline (PMS ≥5) presented lower binding capacity for JAC in comparison with NHS and with lower PMS patients (p = 0,0135 and p = 0,0089, respectively).Conclusion: Ig glycosylation was correlated with clinical and endoscopic activity in patients with UC. JAC protein A-selected Ig showed a possible role in predicting therapeutic effectiveness. If these data would be confirmed, Ig glycosylation could be used as biomarker in UC.

scholarly journals P456 Ig glycosylation in ulcerative colitis: it’s time for new biomarkers

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S450-S451
Author(s):  
R Capecchi ◽  
P Migliorini ◽  
F Zanzi ◽  
S Maltinti ◽  
I Puxeddu ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Clinical Remission ◽  
Solid Phase ◽  
Binding Capacity ◽  
Protein A ◽  
Mucosal Healing ◽  
Therapeutic Outcome ◽  
Serum Samples ◽  
Biologic Drugs ◽  
Number Of Patients

Abstract Background Ulcerative colitis (UC) is a chronic relapsing disease, which needs a continue monitoring, especially during biological therapies. An increasing number of patients is treated with anti-Tumor Necrosis factor (TNF) drugs, and current research is focalized to identify biomarkers able to monitor the disease and to predict therapeutic outcome. Methods We enrolled consecutive UC patients treated with anti-TNF, naïve to biologic drugs. Therapeutic outcome was evaluated after 54 weeks of treatment in terms of clinical remission (Partial Mayo Score -PMS- <2) and mucosal healing (Mayo Endoscopic Score <2). On serum samples collected at baseline and after 54 weeks of treatment, a Lectin-based ELISA assay was performed, and specific glycosylation patterns were evaluated by biotin-labelled lectins. We have also collected 21 healthy controls (NHS) samples, age and sex-matched. Results Out of 44 UC patients enrolled, 22 achieved clinical remission and mucosal healing after 54 weeks. At baseline, when Protein A was used as coating, UC patients non-responders showed a reduced reactivity to Jacalin (JAC) in comparison with NHS (p=0,04). After one year of treatment, a decrease in JAC binding was seen only in responders, in comparison with baseline (p=0,04). When JAC binding was tested selecting IgG by means of Fab anti-IgG Fab, UC patients displayed an increased reactivity after anti-TNF therapy (p<0,0001 vs controls). At baseline, PMS inversely correlates with JAC binding when Fab anti-IgG Fab was used in solid phase (r2= 0,2211; p=0,0033). Patients with higher PMS at baseline (PMS ≥5) presented lower binding capacity for JAC in comparison with NHS and with lower PMS patients (p= 0,0135 and p=0,0089 , respectively). Conclusion Ig glycosylation was correlated with clinical and endoscopic activity in patients with UC. JAC protein A-selected Ig showed a possible role in predicting therapeutic effectiveness. If these data would be confirmed, Ig glycosylation could be used as biomarker in UC.

Solid-phase immunoelectron microscopy for rapid diagnosis of enteroviruses

1984 ◽  
Vol 42 ◽  
pp. 226-227 ◽  
Author(s):  
K. Pegg-Feige ◽  
F. W. Doane
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Immunoelectron Microscopy ◽  
Detection Method ◽  
Solid Phase ◽  
Protein A ◽  
Plant Viruses ◽  
Rapid Diagnosis ◽  
Virus Diagnosis ◽  
Antiviral Antibody

Immunoelectron microscopy (IEM) applied to rapid virus diagnosis offers a more sensitive detection method than direct electron microscopy (DEM), and can also be used to serotype viruses. One of several IEM techniques is that introduced by Derrick in 1972, in which antiviral antibody is attached to the support film of an EM specimen grid. Originally developed for plant viruses, it has recently been applied to several animal viruses, especially rotaviruses. We have investigated the use of this solid phase IEM technique (SPIEM) in detecting and identifying enteroviruses (in the form of crude cell culture isolates), and have compared it with a modified “SPIEM-SPA” method in which grids are coated with protein A from Staphylococcus aureus prior to exposure to antiserum.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Comparison of the Potency of 8-L-Arginine, 8-D-Arginine and 8-D-Homoarginine Vasopressin Analogs with Substituted Phenylalanine in Position 2

1994 ◽  
Vol 59 (6) ◽  
pp. 1439-1450 ◽  
Author(s):  
Miroslava Žertová ◽  
Jiřina Slaninová ◽  
Zdenko Procházka
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Basic Amino Acid ◽  
The Other ◽  
Side Chain ◽  
Inhibitory Potency ◽  
Phase Synthesis ◽  
Other Hand ◽  
Order Of Magnitude

An analysis of the uterotonic potencies of all analogs having substituted L- or D-tyrosine or -phenylalanine in position 2 and L-arginine, D-arginine or D-homoarginine in position 8 was made. The series of analogs already published was completed by the solid phase synthesis of ten new analogs having L- or D-Phe, L- or D-Phe(2-Et), L- or D-Phe(2,4,6-triMe) or D-Tyr(Me) in position 2 and either L- or D-arginine in position 8. All newly synthesized analogs were found to be uterotonic inhibitors. Deamination increases both the agonistic and antagonistic potency. In the case of phenylalanine analogs the change of configuration from L to D in position 2 enhances the uterotonic inhibition for more than 1 order of magnitude. The L to D change in position 8 enhances the inhibitory potency negligibly. Prolongation of the side chain of the D-basic amino acid in position 8 seems to decrease slightly the inhibitory potency if there is L-substituted amino acid in position 2. On the other hand there is a tendency to the increase of the inhibitory potency if there is D-substituted amino acid in position 2.

scholarly journals Analysis of the Mechanism by Which Glucose Inhibits Maltose Induction of MAL Gene Expression in Saccharomyces

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 121-132
Author(s):  
Zhen Hu ◽  
Yingzi Yue ◽  
Hua Jiang ◽  
Bin Zhang ◽  
Peter W Sherwood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Repression ◽  
Protein A ◽  
The Other ◽  
Maltose Permease ◽  
Inducer Exclusion ◽  
Glucose Inhibition ◽  
Independent Mechanism ◽  
Mal Genes

Abstract Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

2021 ◽  
Author(s):  
Yasmin Kaveh-Baghbaderani ◽  
Raphaela Allgayer ◽  
Sebastian Patrick Schwaminger ◽  
Paula Fraga-García ◽  
Sonja Berensmeier
Keyword(s):  
Iron Oxide ◽  
Magnetic Separation ◽  
Iron Oxide Nanoparticles ◽  
Binding Capacity ◽  
Protein A ◽  
Oxide Nanoparticles ◽  
High Binding ◽  
A Domains
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