Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices

Brown‐Augsburger, Patricia; Yue, Xin; Lockridge, Jennifer A.; McSwiggen, James A.; Kamboj, Dimple; Hillgren, Kathleen M.

doi:10.1016/j.japna.2003.07.002

Cited by 13 publications

(6 citation statements)

References 10 publications

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“…A log/log transformation improved the linearity and is commonly used for ELISA-based assays (20,25). We hypothesized that some plasma or cellular protein (possibly albumin) may interfere with the hybridization of antisense ODNs with capture ODNs at the first hybridization step, therefore causing nonlinearity.…”

Section: Discussionmentioning

confidence: 99%

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

et al. 2006

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Section: Discussionmentioning

confidence: 99%

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

et al. 2006

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“…However, none of these methods were used to characterize exogenous, synthetic miRNAs. Furthermore, hybridizationbased ELISA methods have been successfully developed for the determination of polynucleotides in biological matrices (28)(29)(30)32,(40)(41)(42). However, these ELISA methods have not been applied to quantify endogenous miRNAs and their applicability for measuring these endogenous substances remains uncertain because of the extremely low endogenous miRNA levels.…”

Section: Discussionmentioning

confidence: 99%

A Novel Ultrasensitive Hybridization-Based ELISA Method for 2-Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application

Chan

Liu

Xie

et al. 2010

AAPS J

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Abstract. MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has indicated the involvement of miRNAs in human malignancies. It has been suggested that aberrantly down-regulated or up-regulated miRNAs may be replaced with synthetic miRNAs or antagomiRNAs, respectively, and restore normal cell functions. As therapeutic development requires analytical support, we developed and validated an ultrasensitive and selective assay for quantification of synthetic 2′-methoxyphosphorothiolate-miRNA in mouse plasma and cell lysate for the first time. The method is based on a hybridization-ligation fluorescence enzyme-linked immunosorbent assay and has provided a linear dynamic range of 10-1,000,000 pM for three synthetic miRNAs both singly and in a mixture. The intra-and inter-day coefficients of variation were <20% and the accuracy values nearly 100%. Using this assay, we performed pharmacokinetic studies of three synthetic miRNAs in mice treated with a single i.v. bolus dose of 7.5 mg kg −1. The 2-methoxyphosphorothiolate-miRNAs reached peak concentrations in the μM and nM ranges in plasma and bone marrow, respectively, and remained measurable at 24 h. These concentrations are in a range that shows biological activities. We conclude that this method provides a general and valuable tool for the pharmacologic study and clinical development of synthetic miRNAs.

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“…Given the ability of the ASO in tissue sections to specifically bind the appropriate probe oligonucleotide, it is probable that ASO identified in tissue sections using the FISH method represents largely intact ASO rather than smaller metabolites. In hybridization ELISA studies using oligonucleotide probes similar to those employed in the FISH method, hybridization to truncated ASO metabolites is relatively inefficient (Brown-Augsburger, 2004). This result is also consistent with data from tissue distribution/metabolism studies with second generation ASOs, demonstrating that the majority of tissue associated drug is intact, full length ASO (Yu et al, 2004b).…”

Section: Discussionmentioning

confidence: 99%

“…Since ASOs represent a unique class of macromolecular drug, new analytical methods for analyzing the tissue distribution of these drugs need to be developed and evaluated. Hybridization ELISA methods have been successfully developed for the analysis of ASOs and other nucleic acid-based compounds in biological matrices (Yu et al, 2002; Brown-Augsburger et al, 2004). These assays rely on complementary base pairing of the target drug to labeled oligonucleotide probe sequences immobilized on a 96-well plate format and have been used primarily to quantify drug levels in plasma and serum.…”

Section: Introductionmentioning

confidence: 99%

Development of a Sensitive and Specific in Situ Hybridization Technique for the Cellular Localization of Antisense Oligodeoxynucleotide Drugs in Tissue Sections

et al. 2007

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A sensitive method has been developed for the identification and assessment of phosphorothioate oligonucleotide accumulation in dosed animal tissues using an in situ hybridization approach, which is both sequence specific yet adaptable to every antisense oligonucleotide (ASO), which has been tested to date. Hybridization is accomplished using a digoxigenin-tailed oligonucleotide probe complementary to the ASO target sequence on routinely processed paraffin sections which have been pretreated with a mild target retrieval solution. The DIG-labeled probe is amplified first with an anti-DIG:FITC antibody conjugate followed by an anti:FITC Alexa 488 antibody, then visualized using FITC epifluorescence microscopy. Fluorescent labeling of ASO drug in tissue sections by this method confirms that H&E basophilia previously observed in dosed tissues represents largely intact ASO. However, the fluorescent method enables a wider assessment of tissue distribution in a variety of tissue types due to increased sensitivity and lower signal to noise than can be obtained through an examination of H&E stained tissue sections alone.

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Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices

Cited by 13 publications

References 10 publications

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

A Novel Ultrasensitive Hybridization-Based ELISA Method for 2-Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application

Development of a Sensitive and Specific in Situ Hybridization Technique for the Cellular Localization of Antisense Oligodeoxynucleotide Drugs in Tissue Sections

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