Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Aoyagi, Satoka; Imai, Rika; Sakai, Kiyotaka; Kudo, Masahiro

doi:10.1016/s0956-5663(03)00046-0

Cited by 24 publications

(21 citation statements)

References 8 publications

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“…The amount of plasminogen binding was quantified as total fluorescence intensity per slice, because fluorescence intensity is greater for FITC-labeled proteins bound to their target than for FITC-labeled proteins in solution. 27 Total fluorescence was significantly lower for A␤-containing clots ( Figure 6C). These results demonstrate that A␤-modified fibrin binds less plasminogen, but they do not prove that A␤ is blocking plasminogen's access to fibrin, since changes in fibrin fiber thickness may be an alternative explanation.…”

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confidence: 94%

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Zamolodchikov

Strickland

2012

Blood

157

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Alzheimer disease is characterized by the presence of increased levels of the ␤-amyloid peptide (A␤) in the brain parenchyma and cerebral blood vessels. This accumulated A␤ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that A␤ 42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, A␤ 42 -induced structural changes cannot be the sole mechanism of delayed lysis because A␤ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that A␤ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing A␤ 42 , and clot lysis by plasmin, but not trypsin, is IntroductionCerebrovascular dysfunction has been implicated as an early event in Alzheimer disease (AD) progression, 1-4 but the origins and mechanisms of vascular dysfunction in AD are not clear. The ␤-amyloid peptide (A␤) accumulates in the brain parenchyma and blood vessel walls of AD patients and has been genetically and clinically linked to AD. We have previously shown that fibrinogen, the main protein component of blood clots, can bind A␤ 42 (henceforth designated A␤) specifically with a K d of 26.3 Ϯ 6.7nM. 5 We also found that fibrin clots formed in the presence of A␤ are structurally altered and more resistant to fibrinolysis than normal clots. 6 However, the mechanism by which A␤-fibrin(ogen) binding delays fibrin clot lysis has not been defined.Fibrin clot lysis is mediated by plasmin, a serine protease that cleaves the fibrin network at specific sites. Plasmin is derived from plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin, which itself enhances the rate of the reaction. One fibrin site initially involved in plasminogen activation by tPA includes residues 148-160 on the A␣-chain (reviewed by Medved and Nieuwewnhuizen 7 ). This site becomes exposed and available for plasminogen binding after the conversion of fibrinogen to fibrin, 8 but could remain hidden if clots are formed in the presence of A␤, leading to delayed clot lysis. This hypothesis is derived from our finding that A␤ binds the fibrinogen ␤-chain near the ␤-hole, 5 which is in close spatial proximity to residues 148-160 of the A␣-chain. 9 Another potential explanation for delayed clot lysis is based on the relationship between fibrin structure and its susceptibility to fibrinolysis. Tighter fibrin networks composed of thin fibers are degraded less efficiently by plasmin than those composed of thick fibers 10-13 because: (1) there are more fibers to be cleaved, 11,14 requiring plasmin to detach from and move between fibers more frequently 15 ; and (2) decreased network porosity of tighter fibrin networks results in impeded diffusion of fibrinolytic enzymes throughout the clot (...

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confidence: 94%

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Zamolodchikov

Strickland

2012

Blood

157

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“…On the other hand, the remarkable increase of the largest intensity was found by the addition of gPGA, indicating that the surrounding environment of the FITC moiety becomes hydrophobic in the presence of gPGA. 43,44 These data suggest that gPGA wraps FITC-CA by their physical interaction to locate FITC-CA in the hydrophobic region, which may lead to the improved activity of CA by gPGA. Furthermore, 1-anilinonaphthalene-8-sulfonate was used as hydrophobic probe for the investigation with gPGA.…”

Section: Resultsmentioning

confidence: 84%

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

Lee

Tsujimoto

Uyama

et al. 2010

Polym J

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The effects of poly(c-glutamic acid) (cPGA), a water-soluble poly(amino acid), on the enzyme activity and stability have been investigated. The activity of carbonic anhydrase (CA) was distinctly improved in the presence of 0.30-2.0% cPGA at pH 7. cPGA was also effective to enhance the activity of lipase and a-amylase. cPGA efficiently suppressed denaturation of the enzyme by the thermal treatment and repeated freeze-thaw process. The interaction of cPGA and CA was investigated by using fluorescence probe-labeled protein. The Michaelis-Menten kinetics on the CA-catalyzed hydrolysis of p-nitrophenyl acetate in the absence or presence of cPGA were performed, which demonstrated that the enhancement of the CA activity by cPGA is ascribed to the increase of the catalytic constant k cat .

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“…Immobilized SpA samples were prepared on a regenerable optic fluorescent immunosensor using SpA in the same manner as described in the previous study [14]. An indium tin oxide (ITO) coated glass slide (SigmaAldrich Co., St Louis, MO, USA) of 8 mm 2 was aminosilanized with aminopropyltrimethoxysilan (Tokyo Kasei, Tokyo, Japan).…”

Section: A Sample Preparationmentioning

confidence: 99%

“…Staphylococcal protein A (SpA), a cell wall component of Staphylococcus aureus, binds to the Fc region of immunoglobulin (Ig), especially immunoglobulin G (IgG) [9][10][11][12], and therefore it has been widely employed to immobilize or detect IgG [13,14]. SpA contains five homologous Ig-binding domains, E, D, A, B and C, in tandem, and each domain is a three-helix bundle consisting of 57-60 residues [15].…”

Section: Introductionmentioning

confidence: 99%

“…The reaction between SpA and IgG is reversible, and they are separated in a low pH of approximately pH 4-2 [16]. In the previous study, the regenerablity of IgG-binding function of SpA after separation of IgG in an acid solution was evaluated in application to an optic-biosensor [14]. In the present study, SpA in a solution at pH 7, SpA in an acidic solution and SpA in a pH 7 solution after having been soaked in an acidic solution, were compared in order to investigate the structural change in the B and E domains of SpA which depend on pH.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of pH-Dependent Staphylococcal Protein A Structural Change Using TOF-SIMS

Aoyagi

Higuchi

Kato

et al. 2009

e-J. Surf. Sci. Nanotechnol.

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The evaluation of protein structural change is crucial for the study of protein-protein interactions and protein conformational changes. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to detect pH dependent structural change in staphylococcal protein A (SpA), which is well known to the bind Fc region of immunoglobulins. TOF-SIMS spectra of SpA-immobilized samples in a pH 7 solution, in an acidic solution, and in a pH 7 solution after having been soaked in an acidic solution, respectively, were compared to select fragment ions specific to normal SpA at pH 7 using spectral analysis techniques such as principal component analysis (PCA) and the mutual information method. A fragment ion depending on change in the B and E domains of SpA were detected with this method. These findings demonstrate that structural change of SpA depending on pH change can be evaluated with TOF-SIMS together with spectrum analysis methods for the multivariate data.

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Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Cited by 24 publications

References 8 publications

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

Evaluation of pH-Dependent Staphylococcal Protein A Structural Change Using TOF-SIMS

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