Cleavage sensitive antibodies and methods of use thereof
Inventors
Bavari, Sina • Nuss, Jonathan E.
Assignees
United States Department of the Army
Publication Number
US-8936915-B2
Publication Date
2015-01-20
Expiration Date
2030-08-17
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Abstract
We disclose cleavage-sensitive antibodies with epitopes spanning the scissile bond of the toxins molecular target protein, enabling toxin-associated proteolysis to be measured in a variety of assay formats.
Core Innovation
This invention discloses cleavage-sensitive antibodies designed with epitopes spanning the scissile bond of the toxin's molecular target protein, specifically SNAP-25 for botulinum neurotoxin serotype A (BoNT/A). These antibodies enable direct measurement of toxin-associated proteolysis in various assay formats, including ELISA and immunofluorescence methods. They exhibit high specificity for full-length SNAP-25, allowing distinctions between intact and cleaved protein to be made in biological samples, enabling assays amenable to high-throughput analysis.
The problem addressed arises from the limitations of existing in vitro BoNT protease assays that use synthetic substrates and do not function in living systems. These conventional assays only measure the proteolytic activity of BoNT light chain isolated enzymatic activity and cannot detect toxin entry into cells or assess the toxin's combined biological activity. There is a need for assays that can measure endogenous substrate cleavage in cellular models and living systems to better evaluate toxin activity and therapeutic candidates.
Claims Coverage
The patent includes multiple independent claims focused on a proteolytic toxin assay methodology using cleavage-sensitive antibodies with various detection techniques and substrate specificities.
Proteolytic toxin assay in cells with cleavage-sensitive antibodies
A method comprising combining a test compound with a substrate having a cleavage site for a toxin and an antibody that binds to substrate but not cleavage product, where the substrate is within a cell, and detecting antibody binding to the substrate.
Use of specific substrates from the SNAP-25 family
The assay employs substrates selected from intact peptides or fragments of SNAP-25, including its analogs and isoforms, to measure toxin cleavage.
Diverse antibody detection formats
The antibody detection is accomplished through multiple means, including solid-phase matrices (e.g., nitrocellulose, polystyrene), immunohistochemical labels (e.g., reporter enzymes, radioisotopes, fluorescent, chemiluminescent, bioluminescent compounds), secondary antibodies, and immunofluorescence imaging techniques such as high- and low-resolution imaging.
Screening strategy employing sequential immunofluorescence assays
Use of a primary low-resolution BACS immunofluorescence assay for screening, followed by a secondary high-resolution BACS immunofluorescence assay to enhance detection and evaluate inhibitors more precisely.
Antibody specificity to peptides spanning BoNT cleavage sites
The antibodies selectively bind peptides having sequences corresponding to the cleavage site of the toxin's molecular target, allowing differentiation between full-length substrate and cleavage products.
Normalized response quantification
Detection of decreased antibody signals corresponding to cleaved substrate is enhanced by calculating normalized responses as the ratio between levels of full-length substrate and total substrate to provide quantitative measurements.
Together, the independent claims cover a method for sensitive detection of proteolytic toxin activity in cellular contexts using cleavage-sensitive antibodies specific to intact substrates, employing various immunodetection techniques and assay formats, including high-throughput screening applications.
Stated Advantages
Enables direct measurement of BoNT/A catalyzed proteolysis of endogenous substrate SNAP-25 in living cells, overcoming limitations of prior in vitro synthetic substrate assays.
Assays exhibit excellent sensitivity and reproducibility and are adaptable to multi-well high-throughput formats for rapid screening.
Can measure multiple steps in toxin intoxication including toxin entry into cells, allowing evaluation of compound bioavailability, toxicity, and intracellular efficacy in primary screens.
Applicable in various assay formats including ELISA and immunofluorescence, providing versatility and robustness.
Improves evaluation and quality control of BoNT biopharmaceutical manufacturing by assessing both heavy chain-mediated cell entry and light chain proteolytic activity.
Documented Applications
High-throughput screening of small molecule inhibitors and activators of botulinum neurotoxins in cellular models of intoxication.
BoNT biopharmaceutical manufacturing assays including quality control and product formulation evaluation.
In vivo measurement of toxin activity and pharmacokinetics via detection of substrate cleavage in laboratory animals or human patient biopsies.
Evaluation of therapeutic candidates and determination of inhibition potency (IC50) in cell-based systems.
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