Enzyme-Linked Immunoelectrotransfer Blot (EITB)

Tsang, Victor C. W.; Bers, George; Hancock, Kathy

doi:10.1007/978-1-4684-5012-5_22

Cited by 30 publications

(18 citation statements)

References 64 publications

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“…Antigens separated by SDS-PAGE were transferred to nitrocellulose paper (0.2 m pore size) in a blotting medium consisting of 212 mM Tris and 20% (vol/vol) methanol (pH 9.2) for 2 h at 1.4 mA (27). The blots were washed four times in 0.3% (vol/vol) Tween 20 (Sigma) in 0.1 M PBS (pH 7.2) and twice with PBS alone.…”

Section: Methodsmentioning

confidence: 99%

Sonicated Diagnostic Immunoblot for Bartonellosis

Mallqui¹,

Speelmon

Verástegui

et al. 2000

Clin Diagn Lab Immunol

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Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction. Both methods were then tested for sensitivity and specificity. Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In a healthy group, it is 100% specific. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity.

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Section: Methodsmentioning

confidence: 99%

Sonicated Diagnostic Immunoblot for Bartonellosis

Mallqui¹,

Speelmon

Verástegui

et al. 2000

Clin Diagn Lab Immunol

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“…Hydatid cyst fluid (HCF) was obtained from lung and liver fertile cysts of sheep from abattoirs in Lima. Sera were tested by Arc-5 double diffusion assay (DD5), immunoelectrophoresis (IEF), as previously described (Coltorti & Varela-Diaz 1978, Centro Panamericano de Zoonosis 1979 and immunoelectrotransfer blot technique (EITB), as reported (Tsang et al 1983, Tsang 1987 de Zoonosis 1979). Demographic and clinical data were obtained from each individual.…”

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confidence: 99%

Notes on human cases of cystic echinococcosis in Peru

Romani

Rodrigues-Silva²,

Maldonado

et al. 2006

Mem. Inst. Oswaldo Cruz

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“…Proteins electrophoresed in a 10% SDSpolyacrylamide gel were electroeluted onto nitrocellulose (2) and developed (35) by published methods. The nitrocellulose filters were exposed to 1:1,000 dilutions of a rabbit antisera against DnaK and then a 1:1,000 dilution of anti-rabbit donkey immunoglobulin G antibodies coupled to horseradish peroxidase (Amersham Corp.) and developed with diaminobenzidine and hydrogen peroxide.…”

mentioning

confidence: 99%

Escherichia coli dnaK null mutants are inviable at high temperature

Paek¹,

Walker²

1987

J Bacteriol

227

169

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DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp7Os) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37°C) and could not form colonies at a high temperature (42°C); furthermore, they also formed long filaments at 42°C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42°C for 2 h were no longer capable of forming colonies at 30°C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42°C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The wellcharacterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or suiB mutations, which suppress SOS-induced filamentation. Our results indicate that DnaK function is normally essential only at elevated temperatures. Nevertheless, cells did not have an absolute requirement for DnaK function for growth at 42°C since we were able to isolate spontaneously arising suppressors of dnaK deletion mutations that permitted growth at that temperature.The heat shock response is an example of a biological response to an environmental change that has been extremely strongly conserved during evolution (5,25,31). A characteristic feature of the heat shock responses of organisms as diverse as Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, and humans is the induction of a ca. 70-kilodalton (kDa) protein. Furthermore, many eucaryotic organisms encode families of related ca. 70-kDa proteins (Hsp7Os), some members of which are not induced by heat. In contrast, E. coli encodes only one Hsp7O protein, the product of the dnaK gene, and its synthesis is induced by heat and other forms of stress (14,19,34). DNA sequencing studies have shown that the amino acid sequence of DnaK is about 50% homologous with the heat-induced Hsp7Os of D. melanogaster and humans (1,17).The strong conservation of Hsp7O structure throughout evolution suggests that the Hsp7O proteins induced by heat may carry out common or related roles in various organisms and, moreover, that there may be common elements of function between various members of the Hsp7O family within a given organism. To date, the E. coli DnaK protein has been the heat-induced Hsp7O protein most extensively characterized a...

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Enzyme-Linked Immunoelectrotransfer Blot (EITB)

Cited by 30 publications

References 64 publications

Sonicated Diagnostic Immunoblot for Bartonellosis

Sonicated Diagnostic Immunoblot for Bartonellosis

Notes on human cases of cystic echinococcosis in Peru

Escherichia coli dnaK null mutants are inviable at high temperature

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