Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

Wang, Chao; Li, Tao; Wang, Zhixin; Feng, Feng; Wang, Hailin

doi:10.1016/j.chroma.2010.02.024

Cited by 7 publications

(3 citation statements)

References 45 publications

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“…This short oligo was modified by a single BPDE−dG adduct in the middle of the chain and fluorescently labeled at the 5′ end by a single tag of FAM. Magnetic particles conjugated with goat anti-mouse IgG were used to immobilize mouse monoclonal anti-BPDE−dG antibody (mAb 8E11), which can specifically recognize single anti -BPDE− N 2 -dG adducts in oligonucleotides and genomic DNA with high affinity and negligible cross-reaction . Due to high-affinity recognition against BPDE−dG adducts, the immobilized mAb 8E11 on magnetic particles can specifically capture the FAM−BPDE 16-mer from the aqueous solution.…”

Section: Resultsmentioning

confidence: 99%

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Wang

Liu

et al. 2010

Anal. Chem.

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Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.Magnetic micro-and nanoparticles have wide applications in bioanalysis, including aptamer selection, proteomics research, and clinical diagnostics, due to their unique separation power endowed by their paramagnetic property. 1,2 When functionalized with specific recognition elements (e.g., affinity substrates, aptamers, DNA, and antibodies), magnetic particles can be used for specific capture of desired analytes from a complex biological matrix, 3,4 including environmental pollutants, 5 various nucleic acids, 6,7 aptamers, 8,9 phosphopeptides, 10-12 cancer marker proteins, [13][14][15] and pathogenic bacteria. [16][17][18] The captured targets can be detected using appropriately designed signal probes, such as quantum dots, 19 carbon nanotubes, 20 Au nanoparticles, 21 and liposomes encapsulated with fluorescent dye. 22 Although some of the designed signal probes may provide an amplified signal, it is still a challenge to quantitate targets simply by particle counting. Despite the complex design and versatile functions, routine flow cytometry based particle counting analysis cannot distinguish specific binding from nonspecific adsorption and is only suitable for particles with a size over micrometers.Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifica-* To whom correspondence should be addressed. Phone/fax: +86-10-62849600. E-mail: hlwang@rcees.ac.cn.(

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Section: Resultsmentioning

confidence: 99%

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Wang

Liu

et al. 2010

Anal. Chem.

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“…The specific binding of antibodies to antigens dictates the applicability of immunoassays. Wang et al used noncompetitive and competitive CE immunoassays to study the binding of mAb 8E11 to anti -benzo[ a ]pyrene diol epoxide (BPDE)-dG adducts . Fluorescent probes containing 90mers with a single anti- BPDE-dG adduct were used for the CE immunoassays.…”

Section: Applicationsmentioning

confidence: 99%

Capillary Electrophoresis

2011

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C apillary electrophoresis (CE) is a field that continues to grow. All areas of CE including theory, separation modes, instrumentation, and applications remain highly active areas of research. This review includes a cross section of references from all areas of the field published in the 2 year period between January 2010 and November 2011. Web of Science reports over 4 000 articles, including 396 reviews, published with CE in the title, abstract, or keywords during this time period. Of these we have chosen 218 papers. We have attempted to choose papers that showcase some of the newest and most exciting developments in the field. It should be noted that papers describing electrophoresis in microfabricated devices were excluded since another review in this issue exclusively covers this topic.

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“…In this case, the more Ag is present in the solution, the more free Ag* will remain and the less immune complex (Ag*‐Ab) will be formed between Ab and Ag*. CE‐LIF detection of the mixture produces two distinct fluorescent peaks corresponding to Ag* and Ag*‐Ab, the intensities of which can be related to the original concentration of Ag []. After CEIA experimental parameters such as running buffer, separation voltage as well as incubation time were optimized, the limit of detection (LOD) of 0.07 μg/L and RSD of less than 5% were obtained in a rapid and simple process.…”

Section: Introductionmentioning

confidence: 99%

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser‐induced fluorescence detector

Liu¹,

Fang²,

Deng³

et al. 2012

Electrophoresis

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A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na₂B₄O₇/NaH₂PO₄ buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.

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Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

Cited by 7 publications

References 45 publications

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Capillary Electrophoresis

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser‐induced fluorescence detector

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