Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Kenna, J. Gerry; Major, Glenn N.; Williams, Roger

doi:10.1016/0022-1759(85)90150-4

Cited by 125 publications

(53 citation statements)

References 17 publications

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“…Crude supernatants were diluted in 50 mM carbonate buffer (1 : 50 v\v) and loaded into ELISA plates. Plates were incubated 18 h at 4mC, washed three times with Tris-casein buffer pH 7.6 (Kenna et al, 1985) for 5 min each, and then incubated for 1.5 h with Tris-casein buffer. A. euteiches antiserum was diluted 1 : 5000 in Tris-casein and incubated for 2 h at 25mC.…”

Section: Quantification Of Arbuscular Mycorrhizal Colonization and Pamentioning

confidence: 99%

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

et al. 1999

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Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches. First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches, and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae. By contrast, in roots inoculated with A. euteiches, a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as ' trypsin-like ' serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.

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Section: Quantification Of Arbuscular Mycorrhizal Colonization and Pamentioning

confidence: 99%

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

et al. 1999

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“…For immunolabelling, all dilutions of primary and secondary antibodies were made in CTM buffer (Kenna, Major & Williams, 1985) modified to contain 0-015",, Tween-20* and 0-2% BSA-C (BioDiagnostics Ltd, Worcester, UK). Thin sections (silver/gold; c. O'l fim thickness) on gold grids were floated on 10-20 mm^ of solution on sealing film on moist filter paper in a Petri dish to prevent drying.…”

Section: Immunogold Labellingmentioning

confidence: 99%

The location of sucrose synthase in root nodules of white clover

1995

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SUMMARYImmunogold labelling was used to identify the location of the sucrose hydrolytic enzyme sucrose synthase in the Nj-fixing root nodules of white clover. This is the major enzyme of sucrose cleavage in clover nodules and might be involved in the modulation of N^ fixation by controlling the rate of utilization of photosynthetic products.Knowledge of the precise cellular location of this enzyme in relation to the point of delivery (the vascular bundles) and the site of utilization of the catabolic product (malate in the bacteroids) might aid our understanding of the metabolic communication between different cell types of the nodule. Gold particle density was greatest in the cytosol of uninfected cells of the central region and in the layer of cortical cells in direct contact with infected cells. By contrast, relative gold particle density was 2-3 fold lower in the cytosol of infected cells. The distribution of sucrose synthase is discussed in relation to sucrose transport, starch metabolism and the provision of carbon and energy for N^ fixation.

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“…The primer fd-tet. 27.p (5Ј-GTAGCATTCCACAGACAGCCCTCATAG-3Ј) was used to sequence the PCR products. All PCR products were sequenced in both directions with the Prism Dye terminator kit (Applied Biosystems Incorporated) by using an ABI-Prism model 377 autosequencer (Applied Biosystems Incorporated).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoslot blot analyses were performed as previously described (36). Briefly, 5 l of antigen was spotted onto nitrocellulose (Invitrogen, Carlsbad, Calif.), air dried, and blocked with casein-thimerosal buffer (27) for 0.5 h. The blots were incubated with 8A6 MAb (1:1,000) diluted in phosphate-buffered saline with 0.005% Tween. The blots were incubated for 2 h at 37°C with shaking and then washed 3 times in phosphate-buffered saline with 0.005% Tween for 5 min (each wash).…”

Section: Ch/tools/)mentioning

confidence: 99%

Newly Characterized Species-Specific ImmunogenicChlamydophila pneumoniaePeptide Reactive with Murine Monoclonal and Human Serum Antibodies

Marston¹,

James²,

Parker

et al. 2002

Clin Vaccine Immunol

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A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.Chlamydophila pneumoniae, formerly known as Chlamydia pneumoniae (12), was first isolated in 1965 (15) and is established as an etiologic agent of respiratory tract diseases and related sequelae (5,14,16,18,19). Chlamydophila and Chlamydia species are gram-negative obligate intracellular bacteria having a biphasic life cycle, consisting of a metabolically inert infectious elementary body (EB) and a metabolically active reticulate body (RB). C. pneumoniae is a respiratory pathogen believed to cause 5% to 20% of community-acquired pneumonias and 5% of bronchitis and sinusitis in adults and children (22,33,40,41,52).Seroepidemiology has shown that most C. pneumoniae infections are asymptomatic. Regional and international serology-based epidemiologic studies of C. pneumoniae have shown high prevalences and ubiquitous infection. These studies have indicated that most persons have had their first C.

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Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Cited by 125 publications

References 17 publications

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

The location of sucrose synthase in root nodules of white clover

Newly Characterized Species-Specific ImmunogenicChlamydophila pneumoniaePeptide Reactive with Murine Monoclonal and Human Serum Antibodies

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