High affinity monoclonal antibodies for detecting amanitins
Inventors
Bever, Candace R. • Stanker, Larry H. • Hnasko, Robert M. • Cheng, Luisa W.
Assignees
US Department of Agriculture USDA
Publication Number
US-11802147-B2
Publication Date
2023-10-31
Expiration Date
2040-06-19
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Abstract
Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species, often suspected in mushroom poisonings. Provided herein are novel, sensitive monoclonal antibodies (mAbs) detection and purification techniques utilizing the mAbs that show selectivity for α-amanitin (α-AMA), β-amanitin (β-AMA) and γ-amanitin (γ-AMA).
Core Innovation
The invention provides novel, sensitive monoclonal antibodies (mAbs) that show selectivity for alpha-amanitin (α-AMA), beta-amanitin (β-AMA), and gamma-amanitin (γ-AMA), which are lethal amatoxin toxins found in various mushroom species. These mAbs are produced by hybridoma cell lines with specific ATCC deposit accession numbers and can be used for detection and purification techniques to identify and isolate these toxins from samples. The invention also includes methods, kits, and devices utilizing these mAbs for the selective detection and measurement of amanitins.
The problem addressed is the need for improved detection methods for amatoxins in mushroom species, which are often involved in mushroom poisonings worldwide. Existing detection methods, such as liquid chromatography with UV detection or mass spectrometry, are sensitive but time-consuming, require expensive instrumentation, and trained personnel. Available antibody-based assays rely on polyclonal antibodies with drawbacks such as batch-to-batch variability. There is a need for monoclonal antibodies that provide high affinity, specificity, and reproducibility, enabling rapid, sensitive, and field-portable detection assays as well as purification techniques applicable in clinical, veterinary, and environmental settings.
Claims Coverage
The patent claims cover several inventive features related to monoclonal antibodies, their production, detection methods, kits, devices, and purification methods involving amanitins. There are multiple independent claims addressing these features.
Monoclonal antibodies produced by specified hybridoma cell lines
Monoclonal antibodies produced by hybridoma cell lines with ATCC deposit accession numbers PTA-125922 or PTA-125923.
Methods for detecting amatoxins in samples using monoclonal antibodies
A method comprising incubating a sample with the monoclonal antibody and detecting an immunological complex to indicate presence or absence of alpha-amanitin, beta-amanitin, or gamma-amanitin in the sample.
Kits for detecting amatoxins using monoclonal antibodies
Kits comprising containers with monoclonal antibodies produced by hybridoma cell lines PTA-125922 and/or PTA-125923, together with instructions to detect formation of immunological complexes indicating presence or absence of amanitins in samples.
Lateral flow devices incorporating monoclonal antibodies for amanitin detection
Lateral flow assay devices comprising monoclonal antibodies produced by hybridoma cell lines PTA-125922, PTA-125923, or their mixtures for detecting amanitins.
Methods for purifying amanitins from samples using monoclonal antibodies
A purification method involving incubation of a sample with monoclonal antibodies to form an immunological complex, isolation of the complex, decoupling to release amanitin, and separation from the antibody, wherein the antibodies correspond to PTA-125922 or PTA-125923 hybridoma cell lines.
The claims collectively cover the novel monoclonal antibodies from two hybridoma cell lines, methods employing these antibodies for sensitive amanitin detection, kits and lateral flow devices embodying these antibodies for practical use, and methods for purification of amanitins using antibody-based immunological complex formation and separation.
Stated Advantages
Monoclonal antibodies provide long-term consistency and scalability over polyclonal antibodies, overcoming batch-to-batch variability issues.
The mAbs developed have high affinity and specificity for α-AMA, β-AMA, and γ-AMA, enabling sensitive detection with limits of detection around 1 ng/mL for α- and γ-amanitin.
Immunoassays using these mAbs are faster, field portable, require less sophisticated instrumentation, and allow rapid sample extraction using simple aqueous methods.
The mAbs enable development of lateral flow devices providing rapid, point-of-care detection with clear visual signals within ten minutes, applicable to diverse samples including human and animal urine as well as mushroom extracts.
The mAbs can be used for immunoaffinity purification of amanitins from complex samples, concentrating and isolating toxins for therapeutic and analytical purposes.
Documented Applications
Detection of amatoxins in mushroom extracts for identifying poisonous mushroom species.
Clinical diagnostics for amanitin poisoning in human and veterinary samples, especially using urine samples.
Rapid, field-deployable lateral flow immunoassays for point-of-care detection of amatoxins in various sample types.
Purification of α-amanitin and β-amanitin from samples for use as chemical standards or in therapeutic antibody-drug conjugates.
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