Anthrax lethal factor substrates and methods of use thereof
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Publication Number
US-9932570-B2
Publication Date
2018-04-03
Expiration Date
Abstract
Disclosed herein is a peptide substrate that is cleaved specifically by anthrax lethal factor (LF), and assays for detecting the presence of anthrax lethal factor in a sample using the peptide substrate. In some cases, the sample may be obtained from an individual at an early stage of an anthrax infection. Kits that include the peptide substrate and that find use in carrying out the assays are also disclosed.
Core Innovation
An anthrax lethal factor-specific peptide substrate is provided comprising an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO:8). X1 is either present or absent, and when present X1 is Arg, X2 is Arg, X3 is Lys, X4 is Lys, X5 is Val, X6 is Tyr, and X7 is Pro. X7* has the formula X7–Z, where Z is a detectable label covalently linked by an amide linkage or an ester linkage to an alpha carboxyl group of X7.
The peptide substrate includes stereochemical constraints, wherein at least one of X1, X2, X3 and X4 is a D isomer of the amino acid. The substrate is therefore defined by the X1–X7 template (SEQ ID NO:8), a Pro-X7–Z cleavage site relationship, and a detectable label Z linked at X7, with at least one of the N-terminal residues in the D configuration.
A detection approach is described for measuring anthrax lethal factor in biological samples by detecting labeled cleavage products generated when the lethal factor acts on the lethal factor-specific peptide substrate. Biological samples include blood, plasma, and serum, and detection includes measuring a labeled cleavage product by fluorescence, HPLC with fluorescence detection, or mass spectrometry.
Claims Coverage
The independent claim identified is clm-00001, which covers the anthrax lethal factor-specific peptide substrate defined by the SEQ ID NO:8 X1–X7 template, the X7–Z detectable label linkage, and stereochemical constraints involving D isomers. Dependent claims further refine the label choice and/or residue stereochemistry and also include kit-level coverage for detection and optional enrichment.
Anthrax lethal factor-specific peptide substrate template with detectable label linkage
An anthrax lethal factor-specific peptide substrate comprising an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO:8) in which X7* has the formula X7–Z, where X7 is Pro and Z is a detectable label covalently linked by an amide linkage or an ester linkage to an alpha carboxyl group of X7.
Stereochemical constraint on at least one of X1–X4 as D isomer
At least one of X1, X2, X3 and X4 is a D isomer of the amino acid.
Detection kit with standardization element
A detection kit that identifies anthrax lethal factor in a biological sample using an anthrax lethal factor-specific peptide substrate and a standardization element.
Kit including an anthrax lethal factor enrichment element
The kit further includes an anthrax lethal factor enrichment element configured to enrich lethal factor in a biological sample when the sample is contacted with the element.
Overall, the claim coverage centers on an anthrax lethal factor-specific peptide substrate defined by the SEQ ID NO:8 X1–X7 template with a Pro-X7–Z detectable label linkage, together with stereochemical constraints requiring at least one of X1–X4 to be a D isomer. The dependent coverage includes detection kit embodiments with standardization elements and optional lethal factor enrichment elements.
Stated Advantages
Improved detection performance with D-amino acid substitution to reduce non-specific cleavage.
Demonstrated anthrax lethal factor specificity versus plasma background.
Provided kinetic readouts correlating LF concentration with cleavage rates.
Lower detection limits are reported, including very low levels in neat plasma using enrichment and low- to ng/mL level detection using microplate approaches.
Documented Applications
Detecting anthrax lethal factor (LF) in biological samples including blood, plasma, and serum by measuring labeled cleavage products.
Using lethal factor-specific binding partners such as LF antibodies or PA63 on solid supports for optional LF enrichment prior to detection.
Providing a kit for identifying anthrax lethal factor in a biological sample, including standardization elements such as positive/negative reference samples and conversion chart/table elements.
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