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Peptide ligand-based ELISA reagents for antibody detection.

ABSTRACT: Detection of specific antibodies has numerous research, therapeutic and diagnostic applications. Short peptide ligands that bind specifically to antibodies with continuous epitopes can be derived from epitope mapping experiments. Short peptide ligands (mimotopes) specific to antibodies with discontinuous epitopes can be identified by screening complex peptide libraries. In an effort to enhance practical utility of such peptide ligands, we describe here a simple approach to turn such target antibody-specific peptide ligands into specific ELISA detection reagents. We show that a simple addition of biotinylated peptide ligands to commonly available horseradish peroxidase (HRP)-labeled streptavidin (or HRP-anti-biotin antibody), or digoxigenin-labeled peptides to HRP-anti-digoxigenin antibody detection reagents transformed these generic detection reagents into sensitive target antibody-specific reagents. ELISA assays performed using these reagents exhibited excellent analytical properties indicating their practical utility for antibody detection. One generic detection reagent can be readily transformed into many different specific ELISA reagents by a simple mix and match design using an appropriate target-specific peptide ligand. Simplicity of preparation of these ELISA reagents for detecting antibodies should facilitate their practical applications.

SUBMITTER: Heyduk E

PROVIDER: S-EPMC6185743 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Peptide ligand-based ELISA reagents for antibody detection.

Heyduk Ewa E Hickey Rachel R Pozzi Nicola N Heyduk Tomasz T

Analytical biochemistry 20180818

Detection of specific antibodies has numerous research, therapeutic and diagnostic applications. Short peptide ligands that bind specifically to antibodies with continuous epitopes can be derived from epitope mapping experiments. Short peptide ligands (mimotopes) specific to antibodies with discontinuous epitopes can be identified by screening complex peptide libraries. In an effort to enhance practical utility of such peptide ligands, we describe here a simple approach to turn such target antib ...[more]

PMID: 30130491

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Project description:Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA). In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis, and may ultimately enable earlier diagnosis, better prognostication, and improved treatment monitoring.

Project description:BackgroundMost Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.MethodsPhage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.ResultsSeven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.ConclusionsOur high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

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Peptide ligand-based ELISA reagents for antibody detection.

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Peptide ligand-based ELISA reagents for antibody detection.

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