Development of a sensitive protein a-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens

Hopley, John; Doane, Frances W.

doi:10.1016/0166-0934(85)90014-x

Cited by 44 publications

(15 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After 3 min, the grid was washed in TBS followed by H 2 O. The sample was negatively stained in 2% phosphotungstic acid (24) and analyzed by electron microscopy. Construction of SCF-Directed Vectors.…”

Section: Methodsmentioning

confidence: 99%

Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens

Smith

Keller

Lohrey

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The inability of adenovirus to infect primitive hematopoietic cells presents an obstacle to the use of adenovirus vectors for gene transfer to these cell types. Therefore, expanding the tropism of adenovirus vectors to unique cell surface antigens would be an important development for gene therapy protocols. In this study, we sought to redirect infection of adenovirus vectors to primitive human hematopoietic cells that universally express the c-Kit receptor on their cell surface. To accomplish this, a vector was constructed by covalently linking biotin molecules to recombinant adenovirus, followed by addition of the biotinylated ligand for the c-Kit receptor, stem cell factor (SCF), through an avidin bridge.

show abstract

Section: Methodsmentioning

confidence: 99%

Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens

Smith

Keller

Lohrey

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Recent developments (Slot & Geuze, 1985) have greatly simplified the procedures for producing homodisperse sols of small particle size which are essential for high resolution ultrastructural studies. Direct coupling of monoclonal antibodies to gold particles was chosen in preference to the indirect use of gold coupled to Protein A (Hopley & Doane, 1985) or anti-IgG (Kjeldsberg, 1985). We found that direct coupling provided specific reagents that bound efficiently and uniformly to single sites on morphologically identifiable structures.…”

Section: Introductionmentioning

confidence: 99%

Herpes Simplex Virus Glycoproteins Associated with Different Morphological Entities Projecting from the Virion Envelope

Stannard

Fuller

Spear

1987

Journal of General Virology

105

View full text Add to dashboard Cite

SUMMARYSpikes of different kinds, distinct in size and appearance were detected on the surfaces of herpes simplex virions by electron microscopy of negatively stained preparations. Use of monoclonal antibodies coupled to colloidal gold permitted identification of viral glycoproteins present in different structures projecting from the virion envelope. Antibodies specific for the glycoprotein designated gB bound to the most prominent spikes, which were about 14 nm long and, in side view, had a flattened T-shaped top. Antibodies specific for gC bound to structures that, in some instances, appeared to extend as much as 24 nm from the surface of the envelope and were too thin to resolve. Antibodies specific for gD bound to structures that extended as much as 8 to 10 nm from the surface of the envelope. The gB spikes were invariably clustered, usually in protrusions of the envelope varying from small bulbous distentions to long tail-like projections. The gC components were randomly distributed and widely spaced and the gD components were irregularly clustered in patterns distinct from those of the gB spikes. These three glycoproteins therefore form structures that are different in size, morphology and distribution in the envelope.

show abstract

“…methods for detecting enteric coronaviruses are more sensitive than conventionnal DEM. techniques; 17,24 however, the sensitivity of these methods is still dependent on the morphologic identification of virus particles or immune complexes on the EM grids. The recently described colloidal gold immunoelectron mi- Presented at the 33rd Annual Meeting of the AAVLD, Denver, CO, October 6-11, 1990.…”

mentioning

confidence: 99%

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Dea

Garzón

1991

J VET Diagn Invest

View full text Add to dashboard Cite

Abstract. Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.Coronaviruses, particularly enteropathogenic strains, from avian and mammalian species are difficult to grow in cell cultures. 2 6 Direct electron microscopy (DEM) of viral particles in clarified fecal specimens and fluorescent antibody techniques for detection of coronavirus antigens in the cells of infected gut are generally used for the diagnosis of enteric coronaviral infections. 11,14,18,19,21 The detection of coronavirus antigens by indirect immunofluorescence (IFF) is dependent on tissue being removed very shortly after death, and most antigen is present early in the course of disease. 19,21 The lack of highly specific antisera can also compromise the specificity of the IFF technique. Coronaviruses are difficult to identify in negatively stained preparations because they are very pleomorphic and the peplomers are lost during processing of clinical specimens. 10,11,14 It is difficult to distinguish coronavirus particles from cell artifacts such as parts of nuclear or cytoplasmic membranes and other fringed bodies 10 Several immunoelectron microscopy (IEM) . methods for detecting enteric coronaviruses are more sensitive than conventionnal DEM. techniques; 17,24 however, the sensitivity of these methods is still dependent on the morphologic identification of virus particles or immune complexes on the EM grids. The recently described colloidal gold immunoelectron mi- Presented at the 33rd Annual Meeting of the AAVLD, Denver, CO, October 6-11, 1990.Received for publication February 13, 1991. croscopy technique improves detection of bovine enteric coronavirus (BCV) in fecal samples. 9 The purpose of the present study was to evaluate the efficiency of combining the concentration of viral suspensions by airfuge and the indirect protein A-colloidal gold immunoelectron microscopy labeling (PAG-IEM) techniques for the detection and differentiation of avian and mammalian enteric and respiratory coronaviruses. Materials and methodsViruses an...

show abstract

Development of a sensitive protein a-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens

Cited by 44 publications

References 25 publications

Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens

Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens

Herpes Simplex Virus Glycoproteins Associated with Different Morphological Entities Projecting from the Virion Envelope

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Contact Info

Product

Resources

About