Kinetic studies of a quantitative single-tube enzyme-linked immunosorbent assay.

Tsang, Victor C. W.; Wilson, Brett; Maddison, Shirley E.

doi:10.1093/clinchem/26.9.1255

Cited by 59 publications

(23 citation statements)

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“…This is a result of the fact that the relationship between absorbance and the amount of bound enzyme is not linear over time, and thus, absorbance read at end point might be a plateau value, identical for reactions with different slopes. For the present study, we developed a modification of the kinetic ELISA [23,24], in which data are expressed by the linear segment of the change in absorbance curve over time. A detailed description of the modified procedure is presented here.…”

Section: Measuring Peptide-protein Binding By a Kinetic Elisamentioning

confidence: 99%

A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox

Dahan

Smith

Pick

2015

Journal of Leukocyte Biology

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p67(phox) is the paramount cytosolic regulator of the superoxide-generating Nox of phagocytes, by controlling the conformation of the catalytic component, Nox2. The initiating event of this process is a protein-protein interaction between p67(phox) and the part of Nox2 protruding into the cytosol, known as the dehydrogenase region. The aim of this study was to identify and characterize region(s) in Nox2 acting as binding site(s) for p67(phox). For this purpose, we measured the binding of recombinant p67(phox) to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288-570). We found that: 1) p67(phox) binds to a site corresponding to residues 357-383, represented by a cluster of 5 peptides (Nos. 24-28); 2) maximal binding was to peptides 24 (357-371) and 28 (369-383); 3) these shared a (369)Cys-Gly-Cys(371) triad, found to be responsible for binding; 4) the Cys-Gly-Cys triad was present in Nox2 of mammals, birds, and amphibians but was absent in other Nox; 5) substituting a Nox4 or Nox1 sequence for the Nox2 sequence in peptide 24 abolished binding; 6) replacing (369)Cys by Arg in peptide 24 (mimicking a mutation in chronic granulomatous disease) abolished binding; 7) the same replacement in peptide 28 did not affect binding, indicating the existence of an additional binding site. Our results reveal an essential role for the Cys-Gly-Cys triad in Nox2 in binding p67(phox), seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67(phox).

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Section: Measuring Peptide-protein Binding By a Kinetic Elisamentioning

confidence: 99%

A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox

Dahan

Smith

Pick

2015

Journal of Leukocyte Biology

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“…Plates were washed three times with PBST between each incubation period. Changes in optical densities of each well were measured at 650 nm with a V max kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA, USA) and kinetic ELISA protocol (Tsang, Wilson & Maddison 1980). Levels of supernatant cytokines were judged from log 2 dilution standard curves of homologous recombinant cytokines (Bio-Source International, La Jolla, CA, USA) included with each assay.…”

Section: Cytokine Synthesis and Measurementmentioning

confidence: 99%

IL‐10 deficit correlates with chronic, hypersplenomegaly syndrome in male CBA/J mice infected with Schistosoma mansoni

Bosshardt

Freeman

Secor

et al. 1997

Parasite Immunology

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Twenty weeks after moderate level infections with Schistosoma mansoni, approximately 20% of male CBA/J mice develop hypersplenomegaly syndrome (HSS) while the rest present with moderate splenomegaly syndrome (MSS). HSS and MSS mice differ pathophysiologically (degree of splenomegaly, anaemia, ascites, periportal fibrosis, portal hypertension) and immunologically with regard to antibodies (idiotypic expression, isotype levels) to schistosome soluble egg antigens (SEA), and spleen cell phenotypic profiles. This study compared in vitro proliferative responses and IL-2, IFN gamma, IL-4, and IL-10 production by spleen cells from uninfected mice and mice with acute (8 wk), MSS or HSS schistosomiasis mansoni, upon exposure to anti-CD3 epsilon or SEA, Spleen cells from uninfected mice produce Il-2 to anti-CD3 epsilon but exposure of cells from all three groups of infected mice to anti-CD3 epsilon or SEA led to only very low levels of supernatant IL-2. Anti-CD3 epsilon- or SEA-stimulated production of IFN gamma or Il-4, and anti-CD3 epsilon-stimulated production of IL-10, displayed similar patterns: highest cytokine production by cells from mice with acute infections and lower levels of production that did not differ between the two chronic groups. In contrast, while SEA-stimulated IL-10 production was again highest with cells from mice with acute infections, spleen cells from mice with MSS produced significantly more IL-10 than did those from mice with HSS. This association of low levels of antigen-induced IL-10 with severe pathology is consistent with the theory that IL-10 plays a role in the immunoregulation that occurs in chronic schistosomiasis.

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“…These procedures were performed with the peptides being in either the surface‐attached form or in solution, to be followed, in the latter case, by attachment to the wells. Binding of p67 phox to the peptides was assessed by a kinetic ELISA, in which data are expressed by the linear segment of the change in absorbance curve over time, subject to modification, as recently described . All binding experiments were performed with streptavidin‐coated 96‐well plates (BioBind Assembly Strip 1 × 8, streptavidin‐coated, catalogue no.…”

Section: Methodsmentioning

confidence: 99%

Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369Cys-Gly-Cys371 triad in Nox2 and in p67phox

Fradin

Bechor

Berdichevsky

et al. 2018

Journal of Leukocyte Biology

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A central event in the activation of the phagocyte NADPH oxidase involves binding of p67 to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67 to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357-383, as a potential binding site. A key role was attributed to a Cys-Gly-Cys triad, shared by peptides 357-371 (peptide 24) and 369-383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67 ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67 ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67 with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67 to serines abolishes binding and diminishes the ability of p67 to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67 with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the Cys-Gly-Cys triad and cysteine(s) in p67 .

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Kinetic studies of a quantitative single-tube enzyme-linked immunosorbent assay.

Cited by 59 publications

References 0 publications

A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox

A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox

IL‐10 deficit correlates with chronic, hypersplenomegaly syndrome in male CBA/J mice infected with Schistosoma mansoni

Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369Cys-Gly-Cys371 triad in Nox2 and in p67phox

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