Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining

Moeremans, M.; Daneels, Guy; Dijck, A. Van; Langanger, G.; Mey, J. De

doi:10.1016/0022-1759(84)90303-x

Cited by 262 publications

(69 citation statements)

References 21 publications

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“…Hybridization is followed by extensive washing of the filter to remove unreacted probe. The hybrids are then detected by autoradiography (32P-probes) or various enzyme immunochemical methods (Moeremans et al, 1984;Moeremans, Daniels and De Mey, 1985;Hull and Al-Hakim, 1988). Methods of filter hybridization that are used in detection of plant pathogens include: colony and dot-blot hybridization.…”

Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

New Approaches to the Detection of Microbial Plant Pathogens

Chu

Waterhouse

Martín

et al. 1989

Biotechnology and Genetic Engineering Reviews

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Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

New Approaches to the Detection of Microbial Plant Pathogens

Chu

Waterhouse

Martín

et al. 1989

Biotechnology and Genetic Engineering Reviews

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“…The reaction was terminated with deionized water. Dot blot-A series of concentrations from 5 to 1,000 ng of purified Rubisco was loaded onto nitrocellulose membrane according to Moeremans et al (1984). The immunobinding and detection were carried out as described for the western immunoblotting.…”

Section: Electrophoresis -Cell Lysates or Purifiedmentioning

confidence: 99%

“…Cells were permeabilized in 0.5% DMS0 (dimethylsulfoxide) in PBS for 3-5 min on ice. Tests for potential leakage of Rubisco from the cells during fixation and permeabilization were carried out by dot blot immunoassay (Moeremans et al 1984). Because paraformaldehyde cross-links this soluble enzyme and prevents its leakage from the cell (Ohba et al 1979), no Rubisco was found in the supernatant.…”

Section: Electrophoresis -Cell Lysates or Purifiedmentioning

confidence: 99%

An immunoprobe to measure Rubisco concentrations and maximal photosynthetic rates of individual phytoplankton cells

Orellana

Perry

1992

Limnology & Oceanography

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The cross-reactivity of an immunological probe to the key photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) was characterized as part of a larger effort to determine maximal photosynthetic rates of individual phytoplankton cells. Polyclonal antiserum was produced against purified Rubisco from the marine diatom Chaetoceros gracilis. The results of western immunoblotting demonstrated that the antiserum reacted positively with Rubisco from 38 species of algae and higher plants and failed to react with only three species of dinoflagellates and one prochlorophyte species. However, the binding affinity or the strength of the cross-reaction for the polyclonal antiserum with purified Rubisco varied among species. The antiserum was then affinity purified against spinach Rubisco and its binding affinity for purified Rubisco determined by ELISA. Two taxonomic groupings resulted: one with high-binding affinity (these species included chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes) and the other with low-binding affinity (dinophytes and cyanophytes). Rubisco concentration per cell and light-saturated rates of photosynthesis were highly correlated for cultures of the diatom Tha/ussiosira weissfogii. These results indicate that affinity-purified antiserum can be rigorously characterized for use in quantifying Rubisco concentration and for assessing the maximal photosynthetic potential of individual phytoplankton cells.

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“…Blotting of minislab gels onto nitrocellulose sheets (Schleicher and Schuell, FRG) was performed according to Towbin et al [9] and silver-enhanced immunogold-staining carried out as described by Moeremans et al [10] using a secondary antibody with a 20-nm gold tag (Janssen Pharmaceutica, Belgium). Monoclonal antibodies to vinculin, calponin and caldesmon, used for immunoblotting, were from Sigma (vin 11-5, CP-93 and Clone C 0297, respectively).…”

Section: Immunological Techniquesmentioning

confidence: 99%

Smooth muscle specific expression of calponin

et al. 1990

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Calponin is an actin-, calmodulin-, and tropomyosin-binding protein that has been isolated from smooth muscle tissue. Using a monoclonal antibody specific for avian calponin, we demonstrate a differentiation-linked increase in calponin expression in embryonic chick gizzard. Cultivation of gizzard smooth muscle cells in vitro resulted in a down-regulation of calponin expression after the first 48 h that was paralleled by a loss of synthesis of metavinculin and the high molecular weight isoform of caldesmon. In early cultures of smooth muscle cells calponin was localised in the actin-containing stress fibres but labelling was restricted to the central parts of the actin cytoskeleton. Calponin expression is suggested as a potentially useful index of smooth muscle differentiation.

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Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining

Cited by 262 publications

References 21 publications

New Approaches to the Detection of Microbial Plant Pathogens

New Approaches to the Detection of Microbial Plant Pathogens

An immunoprobe to measure Rubisco concentrations and maximal photosynthetic rates of individual phytoplankton cells

Smooth muscle specific expression of calponin

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