scholarly journals Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

Sensors ◽  
2009 ◽  
Vol 9 (10) ◽  
pp. 8271-8277 ◽  
Author(s):  
Takehito Ogawa ◽  
Satoka Aoyagi ◽  
Takehiro Miyasaka ◽  
Kiyotaka Sakai
Keyword(s):  
Immunoglobulin G ◽  
Fluorescence Enhancement ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Affinity Binding ◽  
Bovine Plasma

scholarly journals Use of fluorescence flow cytometry to study the binding of various ligands to platelets.

1985 ◽  
Vol 33 (11) ◽  
pp. 1176-1179 ◽  
Author(s):  
R A Dunstan
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Immunoglobulin G ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Staphylococcal Protein A ◽  
Careful Choice ◽  
Fluorescence Studies ◽  
Staphylococcal Protein

Fluorescence flow cytometry can be used to analyze the binding of different ligands to platelets. However careful choice of volume gates is essential in selecting the population of platelets for analysis. The use of fluorescein isothiocyanate conjugated to staphylococcal protein A or F(ab')2 fragments of immunoglobulin G anti-immunoglobulin offers no advantage in sensitivity or specificity in fluorescence studies of platelets and prefixation of washed platelets with paraformaldehyde has no effect on nonspecific fluorescence. The application of this technology to platelets facilitates quantitation of fluorescence intensity and may yield additional useful information.

Use of Protein a Conjugated to Peroxidase to Localize Immunoglobulin G in SSPE Ferret Brain

1982 ◽  
Vol 40 ◽  
pp. 350-351
Author(s):  
Hannah R. Brown ◽  
Anthony F. Nostro ◽  
Halldor Thormar
Keyword(s):  
Immunoglobulin G ◽  
Human Disease ◽  
Measles Virus ◽  
Subacute Sclerosing Panencephalitis ◽  
Protein A ◽  
Inflammatory Lesions ◽  
Sspe Virus ◽  
Specific Immunoglobulin ◽  
Antibody Titers ◽  
The Brain

Subacute sclerosing panencephalitis (SSPE) is a slowly progressing disease of the CNS in children which is caused by measles virus. Ferrets immunized with measles virus prior to inoculation with the cell associated, syncytiogenic D.R. strain of SSPE virus exhibit characteristics very similar to the human disease. Measles virus nucleocapsids are present, high measles antibody titers are found in the sera and inflammatory lesions are prominent in the brains. Measles virus specific immunoglobulin G (IgG) is present in the brain,and IgG/ albumin ratios indicate that the antibodies are synthesized within the CNS.

Differences in the Interactions of Lupus Anticoagulant IgG with Human Prothrombin and Bovine Prothrombin

1995 ◽  
Vol 73 (04) ◽  
pp. 668-674 ◽  
Author(s):  
L Vijaya Mohan Rao ◽  
An D Hoang ◽  
Samuel I Rapaport
Keyword(s):  
Western Blot ◽  
Lupus Anticoagulant ◽  
Protein A ◽  
Phospholipid Complex ◽  
Experimental Conditions ◽  
Bovine Plasma ◽  
Activation System ◽  
Rate Limiting ◽  
Substantial Extent ◽  
Prothrombin Activation

SummaryLupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin than the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell’s viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing >50% of inhibition. In contrast, only one LA IgG markedly inhibited (>50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin. In experiments with 125I-human prothrombin or 125I-bovine prothrombin in a solution containing Ca2+, the addition of PS/PC vesicles enhanced the binding of both human and bovine prothrombin to some LA IgG preparations. The enhanced binding was particularly evident for bovine prothrombin. Although seemingly related for some preparations, the ability of a LA IgG to bind to bovine prothrombin, either in the presence or absence of PS, and the ability of that LA IgG to inhibit the activation of bovine prothrombin was not consistently related for all preparations.

scholarly journals Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.

1976 ◽  
Vol 24 (9) ◽  
pp. 1017-1025 ◽  
Author(s):  
D M Broorsma ◽  
J G Steefkerk ◽  
N Kors
Keyword(s):  
Enzyme Activity ◽  
Immunoglobulin G ◽  
Fluorescein Isothiocyanate ◽  
Antibody Activity ◽  
Positive Staining ◽  
Passive Hemagglutination ◽  
Agarose Bead ◽  
Cryostat Sections ◽  
Peroxidase Conjugate ◽  
Considerable Loss

Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.

scholarly journals Human Immunoglobulin G Cannot Inhibit Fibrinogen Binding by the Genetically Diverse A Domain ofStaphylococcus aureusFibronectin-Binding Protein A

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00590-17
Author(s):  
P. Martijn den Reijer ◽  
Mehri Tavakol ◽  
Nicole Lemmens-den Toom ◽  
Dikra Allouch ◽  
Sheila Thomas ◽  
...  
Keyword(s):  
Antibody Response ◽  
Virulence Factor ◽  
Antibody Production ◽  
Immunoglobulin G ◽  
Binding Protein ◽  
Protein A ◽  
Sequence Diversity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Antibody ◽  
Fibrinogen Binding

ABSTRACTThe fibronectin-binding protein A (FnBPA) is a cell surface-associated protein ofStaphylococcus aureuswhich mediates adherence to the host extracellular matrix and is important for bacterial virulence. Previously, substantial sequence diversity was found among strains in the fibrinogen-binding A domain of this protein, and 7 different isotypes were described. The effect of this sequence diversity on the human antibody response, in terms of both antibody production and antibody function, remains unclear. In this study, we identify five different FnBPA A domain isotypes based on the sequence results of 22 clinicalS. aureusisolates, obtained from the same number of patients suffering from bacteremia. Using a bead-based Luminex technique, we measure the patients’ total immunoglobulin G (IgG) against the 7 FnBPA isotypes at the onset and during the time course of bacteremia (median of 10 serum samples per patient over a median of 35 days). A significant increase in IgG against the FnBPA A domain, including the isotype carried by the infecting strain, is observed in only three out of 22 patients (14%) after the onset of bacteremia. Using a Luminex-based FnBPA–fibrinogen-binding assay, we find that preincubation of recombinant FnBPA isotypes with IgG from diverse patients does not interfere with binding to fibrinogen. This observation is confirmed using an alternative Luminex-based assay and enzyme-linked immunosorbent assay (ELISA).IMPORTANCEDespite the manyin vitroand murinein vivostudies involving FnBPA, the actual presence of this virulence factor during human infection is less well established. Furthermore, it is currently unknown to what extent sequence variation in such a virulence factor affects the human antibody response and the ability of antibodies to interfere with FnBPA function. This study sheds new light on these issues. First, the uniform presence of a patient’s IgG against FnBPA indicates the presence and importance of this virulence factor duringS. aureuspathogenesis. Second, the absence of an increase in antibody production in most patients following bacteremia indicates the complexity ofS. aureus-host interactions, possibly involving immune evasion or lack of expression of FnBPA during invasive infection. Finally, we provide new insights into the inability of human antibodies to interfere with FnBPA-fibrinogen binding. These observations should be taken into account during the development of novel vaccination approaches.

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