P Leonard

Publications (3)16.26 Total impact

• Article: Overcoming antibody expression and screening limitations by smart design: applications to PSA immunoassay development.

  C J Hayes, P Leonard, R O'Kennedy
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  ABSTRACT: Improving the functional and structural properties of target proteins can often be a challenge for researchers. This paper highlights the importance of antibody construct on screening performance, and ultimately, the clone that is selected. We report the reformatting of phage-selected single chain antibody variable region fragments (scFvs) into single chain antibody fragments (scAbs) for improved screening and binding studies. The generation of a scAb, which had a fused human kappa light chain constant domain (C(k)), was shown to significantly improve expression levels in Escherichia coli. Antibody expression levels were compared between the two antibody constructs (scFv and scAb) by ELISA and a 100-fold improvement was observed. The C(k) domain in the expressed scAb also facilitated high throughput analysis by a Biacore capture assay approach. Individual functional scAbs were ranked on the basis of their remaining binding percentage after 5 min dissociation. Selected antibodies were further characterised by kinetic analysis and a sandwich-based immunoassay developed. The scAb construct enhanced expression levels significantly, facilitating antibody screening and immunoassay development for prostate-specific antigen (PSA), a marker for prostate cancer.
  Protein Expression and Purification 03/2012; 83(1):84-91. · 1.59 Impact Factor
• Article: Rapid temperature-dependent antibody ranking using Biacore A100.

  P Leonard, C J Hayes, R O'Kennedy
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  ABSTRACT: The ability to select antibodies with stable thermodynamic profiles can greatly enhance the performance of point-of-care (POC) devices allowing reproducible "on-the-spot" analysis for markers of clinical and environmental relevance. We show that by ranking antibodies based on their kinetic profiles at two different temperatures, greater information content is acquired, facilitating the selection of antibodies with desired binding characteristics. Observed binding patterns highlight the importance of temperature to assay design, which should be fully taken into consideration to ensure that the appropriate antibody is selected for the desired test.
  Analytical Biochemistry 11/2010; 409(2):290-2. · 3.00 Impact Factor
• Article: Helicobacter pylori lipopolysaccharide interacts with TFF1 in a pH-dependent manner.

  Emer P Reeves, Tehmeena Ali, Paul Leonard, Stephen Hearty, Richard O'Kennedy, Felicity E B May, Bruce R Westley, Christine Josenhans, Melanie Rust, Sebastian Suerbaum, Angeline Smith, Brendan Drumm, Marguerite Clyne
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  ABSTRACT: Little is known about how bacteria establish chronic infections of mucosal surfaces. Helicobacter pylori (H. pylori), a chronic pathogen that lives in the gastric mucosa of humans, interacts with the trefoil factor family (TFF) protein TFF1, which is found in gastric mucus. We aimed to characterize the interaction of H. pylori with TFF1 and to assess the role of this interaction in mediating colonization. Subcellular fractions of H. pylori were immobilized and then probed with TFF1, TFF2, or TFF3. The effect of glycosidases and preincubation with monosaccharides on the interaction and binding of TFF1 to a H. pylori adhesin was assessed. The interaction between H. pylori adhesin and TFF1 was characterized using surface plasmon resonance, flow cytometry, nondenaturing polyacrylamide gel electrophoresis, coimmunofluoresence, and incubation with tissue sections. The H. pylori core oligosaccharide portion (rough form) of lipopolysaccharide (RF-LPS) bound to TFF1 and to a lesser extent TFF3; this interaction was inhibited by incubation of RF-LPS with mannosidase, glucosidase, or mixed monosaccharides. TFF1 also bound to human serum albumin-conjugated mannose and glucose. The optimum pH for binding was 5.0-6.0 for TFF1 and 7.0 for TFF3. H. pylori bound TFF1 in gastric mucus ex vivo; binding of LPS-coated latex beads to human antral gastric tissue was inhibited by TFF1. TFF1 interacts specifically with H. pylori RF-LPS. The pH dependence of this interaction indicates that binding of H. pylori to TFF1 in the stomach could promote colonization of the mucus layer adjacent to the gastric epithelial surface.
  Gastroenterology 09/2008; 135(6):2043-54, 2054.e1-2. · 11.68 Impact Factor