Cleaved SLPI as a marker for exacerbation in cystic fibrosis

Matthew Twigg, Simon Brockbank, Philip Lowery, Peter FitzGerald, Sinead Weldon, Cliff Taggart

Research output: Contribution to conferencePoster

Abstract

Chronic infection in CF leads to an increased inflammatory response and disruption of the lung's antiprotease screen. One antiprotease affected is Secretory Leucocyte Protease Inhibitor (SLPI). The role of SLPI is to protect tissues from the detrimental effects of proteases such has neutrophil elastase (NE). In CF patients, chronically infected with P. aeruginosa, SLPI is cleaved due to excess levels of NE. NE cleavage of SLPI results in the generation of a C-terminal polypeptide fragment, cleaved SLPI (C-SLPI). We predict C-SLPI to be a novel biomarker for the detection of exacerbation in CF patients. An amino acid sequence within C-SLPI was used has an immunogen to generate hybridomas expressing monoclonal antibodies to C-SLPI. In silico modelling of the predicted C-SLPI epitope was performed. Monoclonal antibodies purified from the supernatants of these hybridomas were assayed via ELISA for activity against both C-SLPI and full-length native SLPI. Additionally, CF patient sputum samples were assayed for C-SLPI. In silico modelling showed the epitope to be exposed on C-SLPI but not on full-length native SLPI. An ELISA for recombinant C-SLPI demonstrated that these antibodies are able to detect C-SLPI ranging in concentration from 2000 ng/ml to 2.7 ng/ml; cross reactivity with full-length SLPI was less than 0.5% indicating high specificity for C-SLPI. Critically, we showed the monoclonal antibodies are able to detect C-SLPI in sputum samples from CF patients and observed significant differences in C-SLPI between stable and exacerbating CF patients. Successful detection of both recombinant and native C-SLPI by these antibodies we are pursuing further testing with a broader range of CF and COPD patient samples.
LanguageEnglish
Number of pages1
DOIs
Publication statusPublished - 30 Oct 2015

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Secretory Leukocyte Peptidase Inhibitor
Cystic Fibrosis
Leukocyte Elastase
Monoclonal Antibodies
Hybridomas
Protease Inhibitors
Sputum
Computer Simulation
Epitopes
Enzyme-Linked Immunosorbent Assay
Antibodies
Chronic Obstructive Pulmonary Disease
Amino Acid Sequence
Peptide Hydrolases
Biomarkers
Lung

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Twigg, Matthew ; Brockbank, Simon ; Lowery, Philip ; FitzGerald, Peter ; Weldon, Sinead ; Taggart, Cliff. / Cleaved SLPI as a marker for exacerbation in cystic fibrosis. 1 p.
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abstract = "Chronic infection in CF leads to an increased inflammatory response and disruption of the lung's antiprotease screen. One antiprotease affected is Secretory Leucocyte Protease Inhibitor (SLPI). The role of SLPI is to protect tissues from the detrimental effects of proteases such has neutrophil elastase (NE). In CF patients, chronically infected with P. aeruginosa, SLPI is cleaved due to excess levels of NE. NE cleavage of SLPI results in the generation of a C-terminal polypeptide fragment, cleaved SLPI (C-SLPI). We predict C-SLPI to be a novel biomarker for the detection of exacerbation in CF patients. An amino acid sequence within C-SLPI was used has an immunogen to generate hybridomas expressing monoclonal antibodies to C-SLPI. In silico modelling of the predicted C-SLPI epitope was performed. Monoclonal antibodies purified from the supernatants of these hybridomas were assayed via ELISA for activity against both C-SLPI and full-length native SLPI. Additionally, CF patient sputum samples were assayed for C-SLPI. In silico modelling showed the epitope to be exposed on C-SLPI but not on full-length native SLPI. An ELISA for recombinant C-SLPI demonstrated that these antibodies are able to detect C-SLPI ranging in concentration from 2000 ng/ml to 2.7 ng/ml; cross reactivity with full-length SLPI was less than 0.5{\%} indicating high specificity for C-SLPI. Critically, we showed the monoclonal antibodies are able to detect C-SLPI in sputum samples from CF patients and observed significant differences in C-SLPI between stable and exacerbating CF patients. Successful detection of both recombinant and native C-SLPI by these antibodies we are pursuing further testing with a broader range of CF and COPD patient samples.",
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Cleaved SLPI as a marker for exacerbation in cystic fibrosis. / Twigg, Matthew; Brockbank, Simon; Lowery, Philip ; FitzGerald, Peter; Weldon, Sinead; Taggart, Cliff.

2015.

Research output: Contribution to conferencePoster

TY - CONF

T1 - Cleaved SLPI as a marker for exacerbation in cystic fibrosis

AU - Twigg, Matthew

AU - Brockbank, Simon

AU - Lowery, Philip

AU - FitzGerald, Peter

AU - Weldon, Sinead

AU - Taggart, Cliff

PY - 2015/10/30

Y1 - 2015/10/30

N2 - Chronic infection in CF leads to an increased inflammatory response and disruption of the lung's antiprotease screen. One antiprotease affected is Secretory Leucocyte Protease Inhibitor (SLPI). The role of SLPI is to protect tissues from the detrimental effects of proteases such has neutrophil elastase (NE). In CF patients, chronically infected with P. aeruginosa, SLPI is cleaved due to excess levels of NE. NE cleavage of SLPI results in the generation of a C-terminal polypeptide fragment, cleaved SLPI (C-SLPI). We predict C-SLPI to be a novel biomarker for the detection of exacerbation in CF patients. An amino acid sequence within C-SLPI was used has an immunogen to generate hybridomas expressing monoclonal antibodies to C-SLPI. In silico modelling of the predicted C-SLPI epitope was performed. Monoclonal antibodies purified from the supernatants of these hybridomas were assayed via ELISA for activity against both C-SLPI and full-length native SLPI. Additionally, CF patient sputum samples were assayed for C-SLPI. In silico modelling showed the epitope to be exposed on C-SLPI but not on full-length native SLPI. An ELISA for recombinant C-SLPI demonstrated that these antibodies are able to detect C-SLPI ranging in concentration from 2000 ng/ml to 2.7 ng/ml; cross reactivity with full-length SLPI was less than 0.5% indicating high specificity for C-SLPI. Critically, we showed the monoclonal antibodies are able to detect C-SLPI in sputum samples from CF patients and observed significant differences in C-SLPI between stable and exacerbating CF patients. Successful detection of both recombinant and native C-SLPI by these antibodies we are pursuing further testing with a broader range of CF and COPD patient samples.

AB - Chronic infection in CF leads to an increased inflammatory response and disruption of the lung's antiprotease screen. One antiprotease affected is Secretory Leucocyte Protease Inhibitor (SLPI). The role of SLPI is to protect tissues from the detrimental effects of proteases such has neutrophil elastase (NE). In CF patients, chronically infected with P. aeruginosa, SLPI is cleaved due to excess levels of NE. NE cleavage of SLPI results in the generation of a C-terminal polypeptide fragment, cleaved SLPI (C-SLPI). We predict C-SLPI to be a novel biomarker for the detection of exacerbation in CF patients. An amino acid sequence within C-SLPI was used has an immunogen to generate hybridomas expressing monoclonal antibodies to C-SLPI. In silico modelling of the predicted C-SLPI epitope was performed. Monoclonal antibodies purified from the supernatants of these hybridomas were assayed via ELISA for activity against both C-SLPI and full-length native SLPI. Additionally, CF patient sputum samples were assayed for C-SLPI. In silico modelling showed the epitope to be exposed on C-SLPI but not on full-length native SLPI. An ELISA for recombinant C-SLPI demonstrated that these antibodies are able to detect C-SLPI ranging in concentration from 2000 ng/ml to 2.7 ng/ml; cross reactivity with full-length SLPI was less than 0.5% indicating high specificity for C-SLPI. Critically, we showed the monoclonal antibodies are able to detect C-SLPI in sputum samples from CF patients and observed significant differences in C-SLPI between stable and exacerbating CF patients. Successful detection of both recombinant and native C-SLPI by these antibodies we are pursuing further testing with a broader range of CF and COPD patient samples.

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DO - 10.1183/13993003.congress-2015.PA3876

M3 - Poster

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