The Use of the Avidin‐Biotin Complex as a Tool in Molecular Biology

Bayer, Edward A.; Wilchek, Meir

doi:10.1002/9780470110461.ch1

Cited by 551 publications

(76 citation statements)

References 81 publications

(16 reference statements)

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“…The avidin-biotin interactions have been used successfully in many biological and analytical systems but have the disadvantage of being highly immunogenic, which limits their applications in vivo [33].…”

Section: Introductionmentioning

confidence: 99%

Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle

Kheirolomoom

Dayton

Lum

et al. 2007

Journal of Controlled Release

207

175

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A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes each containing 5% DSPE-PEG2kBiotin was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. Highspeed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37°C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.

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Section: Introductionmentioning

confidence: 99%

Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle

Kheirolomoom

Dayton

Lum

et al. 2007

Journal of Controlled Release

207

175

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“…Biotinylation of goat anti-mouse C3 Abs was performed as described (31,32). In brief, 20 mg of Abs (4 mg/ml) were dialyzed twice in 1 liter of 0.1 M sodium borate buffer (pH 8.8) for 6 h. After dialysis, 10 mg of Abs (4 mg/ml) were incubated with 2.5 mg of N-hydroxysuccinimide biotin (2.5 mg/ml in dimethyl formamide) (Sigma-Aldrich) at room temperature for 4 h. Next, the Abs were incubated with 200 l of 1 M NH 4 Cl at room temperature for 10 min.…”

Section: Antibodiesmentioning

confidence: 99%

Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack

Kim

Miwa

Song

2006

The Journal of Immunology

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Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry−/−) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 ± 127 DAF molecules/cell (DAFhigh) compared with 249 ± 94 DAF molecules/cell (DAFlow) and 774 ± 135 Crry molecules/cell on control mouse erythrocytes. DAFhigh-Crry−/− erythrocytes were significantly more resistant than either DAFlow-Crry−/−, DAF−/− -Crry+/+ or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry−/− erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAFhigh-Crry−/− erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

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“…The most common substance used to conjugate with biotin is streptavidin. There is a high affinity constant between the glycoprotein avidin and the vitamin biotin, causing the formation of a biotin-streptavidin complex which can be used to indirectly conjugate QDots to biological molecules (Bayer and Wilchek, 1980). In this study, streptavidin coated QDots have been used to develop a simple and quantitative viral binding assay capable of high throughput applications to detect differential binding of HTLV-1 to various pathologically relevant cell types and to conduct screening of small molecule inhibitors of viral attachment.…”

mentioning

confidence: 99%

A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment

Kampani

Quann

Ahuja

et al. 2007

Journal of Virological Methods

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Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated Qdots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4 + and CD8 + T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry. KeywordsHTLV-1; quantum dot; viral binding assay 1.IntroductionViral binding and attachment to a host cell membrane, while seemingly simplistic, is a complex area of research for a wide range of viruses. It is often viewed as the first step in infection, whereby a virion is able to attach to a target cell, fuse to the cell membrane, and deliver the contents of the capsid to the cytoplasm of the newly infected cell. The exact mechanism of binding to a host cell varies between viruses and is usually determined by the composition of attachment proteins located within the viral and cellular membranes. The population of cells † A part of this work was presented during 7th International Symposium on NeuroVirology (ISNV), Philadelphia, PA, USA, May 30 - Street, Philadelphia, PA 19102, USA, Tel.: 215-762-8586; Fax: 215-762-1955, E-mail: pjain@drexelmed.edu, Web site: http://www.drexelmed.edu/ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Dhawan et al., 1991;Inghirami et al., 1988). Occasionally, radioactive labels are also utilized for the quantitative estimation of binding (Hubbard, 2003). However, organic fluorophores conventionally used for labeling nucleotides and proteins have poor pho...

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The Use of the Avidin‐Biotin Complex as a Tool in Molecular Biology

Cited by 551 publications

References 81 publications

Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle

Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle

Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack

A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment

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