Abstract
The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.
Highlights
Enzyme-linked immunosorbent assay (ELISA) is a widely used method for detection of proteins such as hormones, immunoglobulins and infectious agent antigens because of its sensitivity and easiness to use [1,2,3]
Each enzyme was diluted at 1:10 serial dilutions and mixed with the fluorescent labeled oligonucleotide substrate (FLOS) (10–6 M) to release the fluorophore that was quenched by Iowa Black FQ
We developed a novel nuclease-linked fluorescence oligonucleotide assay (NLFOA) by taking the advantages of the nanoparticles coupled with multiple streptavidin copies, high affinity and specificity of streptavidin-biotin interaction, biotinylated TurboNuclease and the novel fluorescent labeled oligonucleotide substrate (FLOS)
Summary
Enzyme-linked immunosorbent assay (ELISA) is a widely used method for detection of proteins such as hormones, immunoglobulins and infectious agent antigens because of its sensitivity and easiness to use [1,2,3]. The unique properties of nanoparticles (large ratio of surface area-to-volume, stability and high binding capability) were explored to improve ELISA sensitivity [9,10,11,12] Because of their high sensitivity and specificity, many nanoparticles have been generated for diagnosis of diseases [13,14]. In all previous ELISA systems, the enzymes conjugated to the detection antibodies are horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase, glucose oxidase or malate dehydrogenase [15,16,17,18] They share some common features: stable, highly active, and capable of forming cross-links with other proteins (antibody, streptavidin and others). No other enzymes have been used to further improve the ELISA sensitivity
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