Antibody-specific aptamer-based PCR analysis for sensitive protein detection

Abstract

Nucleic acid amplification techniques were applied to the enzyme linked immunosorbent assay (ELISA) with an antibody-specific aptamer, R18. This novel detection system is a modification of the original immuno-polymerase chain reaction (immuno-PCR), but oligonucleotide-labeled antibodies are not required in the assay. This method is performed with the usual ELISA protocol, using an RNA aptamer for rabbit IgG instead of the conventional secondary antibody. After the assay plate was washed, quantitative reverse transcription (RT)-PCR was performed. Ribonuclease (RNase) inhibitors are not needed for this method. The detection limit of the quantitative RT-PCR is over 100 times more sensitive than the original ELISA method, even with the same sandwich-antibody combination. Only 1 mg of aptamer is sufficient for more than 10 million assays. This aptamer-based quantitative PCR successfully detected 16 attomoles (16 x 10(-18)) of vascular endothelial growth factor (VEGF). This is a cost-effective and easy method to increase the sensitivity of the rabbit antibody-based ELISA systems. The new method is referred to as immuno-aptamer PCR (iaPCR), to distinguish it from the original immuno-PCR.

Screening ssDNA aptamers against HIV P24 antigen using agarose beads as carriers

“…Aptamers have structural diversity, demonstrating a high specificity and a strong affinity upon binding to a variety of target proteins. In terms of protein detection, aptamers are superior to the conventional antigen-antibody means [11][12], and has great potential for broad applications. This study combined subtractive SELEX and RT-qPCR, using carboxylated agarose beads as the carrier and HIV-P24 antigen as the target molecule, to screen specific oligonucleotide aptamers.…”

“…Preparation of the dsDNA library [12]: A sample of 80 µl of recovered supernatant was used as a template to prepare dsDNA in a 100 µl PCR reaction. Besides the template, the PCR solution included a 10 µl 10×Buffer, fluorescent dye supergreen I 1. deionized water to a total volume of 100 µl.…”

“…Preparation of the ssDNA library [12]: The prepared streptavidin-biotin magnetic beads were rinsed three times in 0.01 mol/L pH7.4 PBS. The biotin-labeled dsDNA from the last step was added to the beads and incubated in a rotating shaker for 30 min at 37 ºC.…”

A fluorescent aptasensor for H5N1 influenza virus detection based-on the core–shell nanoparticles metal-enhanced fluorescence (MEF)

“…Comparing with antibodies, aptamer was capable of improving sensitivities by exponential amplification, with lower production costs, relative ease of modification, tailored binding affinity and reversible denaturation. Resent years, aptamers have been thought to be representing alternative options to antibodies for diagnostics development (Cunningham et al, 2005;Yoshida et al, 2009 and. Recently, various anti-influenza virus protein aptamers have been developed (Cheng et al, 2008;Gopinath et al, 2006;Jeon et al, 2004;.…”

Target replacement strategy for selection of DNA aptamers against the Fc region of mouse IgG

“…Aptamers specifically recognize the highly conservative fragment, crystallizable (Fc) region in IgGs, thus providing a general affinity agent for the IgG family, which is highly desirable . It can be used as a conventional secondary antibody, providing a general, simple, and sensitive tool for an ELISA system, or it can also provide a new affinity agent for mass purification of therapeutic antibodies Yoshida et al, 2009). Using the conventional SELEX method, the rabbit antibody was recognized from a pool of 2 10 -2 14 RNA aptamers (Yoshida et al, 2008).…”