Immunoassay for venom detection including noninvasive sample collection
Inventors
Stoecker, William V. • Gomez, Hernan F. • Green, Jonathan A. • McGlasson, David L.
Assignees
Spidertech A Division Of Stoecker & Associates A Subsidiary Of Dermatology Center LLC • Stoecker and Associates LLC • University of Michigan Ann Arbor • University of Missouri St Louis • United States Department of the Air Force
Publication Number
US-7927828-B2
Publication Date
2011-04-19
Expiration Date
2026-10-17
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Abstract
Methods and immunoassays for diagnosing a bite or sting of a venomous organism in a patient having symptoms consistent with such a bite or sting are provided. A sample of venom is collected from the area of the suspected bite or sting using a swab and then contacted with an antibody that specifically binds to an antigenic site on venom present in the sample. Binding is then detected. The invention is illustrated by examples showing diagnosis of brown recluse spider bite, distinguishing it from other diagnoses with which it is often confused. This extremely sensitive test can detect venom antigens down to about 20 picograms even after the sample has been shipped and stored for periods of up to three weeks during the summer.
Core Innovation
This invention provides methods and immunoassays for diagnosing a bite or sting of a venomous organism in a patient having symptoms consistent with such a bite or sting. The method involves collecting a sample comprising venom from the area of the suspected bite or sting using a swab and then contacting the sample with an antibody that specifically binds to an antigenic site on venom present in the sample. Binding between the antibody and venom antigen is then detected. The invention is particularly illustrated by examples showing diagnosis of brown recluse spider bites, distinguishing them from other conditions with similar presentations.
The invention addresses the significant challenge of developing an accurate diagnostic test for Loxosceles venom. Unlike typical ELISAs that detect single proteins, this ELISA detects sphingomyelinase D (SMD), a unique physiologically active protein abundantly present in Loxosceles venom, which contains many proteins with varying physiological properties, some of which resemble proteins of other arthropods causing potential cross-reactivity. SMD is the major venom component responsible for dermal necrosis and is unique to Loxosceles spiders. The assay achieves high sensitivity, able to detect venom antigens down to about 20 picograms, even after samples have been shipped and stored for up to three weeks in summer temperatures.
The method uses a non-invasive sample collection technique with swabs comprising absorbent or adsorbent material such as cotton, gauze, polymeric foam, or brushes, which can be dry or premoistened. The collected sample can be analyzed by various immunoassay techniques including ELISA, sandwich immunoassays, or electrochemical immunoassays, and the assay can be conducted in the field using portable devices such as cartridge or dipstick test devices with antibodies immobilized on a support or the swab itself. The assay can include steps for cell lysis if the sample contains cells, and employs controls including testing a control sample from an unexposed body site.
Claims Coverage
The patent claims cover an immunoassay kit with multiple inventive features related to venom detection from Loxosceles spider bites.
Immunoassay kit components
An immunoassay kit comprising at least one antibody capable of binding to an antigen present in Loxosceles venom, a swab for collecting venom-containing samples from the bite area, and a tracer for detecting binding between the antibody and antigen.
Antibody immobilization on solid substrate
The antibody in the kit is immobilized on a solid substrate, which can be the swab itself.
Swab composition and configuration
The swab comprises an absorbent or adsorbent material selected from cloth, gauze, sponge, polymeric foam, cotton, rayon, Dacron, nylon fibers, polyurethane foam, paper, or absorbent brushes, and may be attached to a handle or comprise natural or synthetic fibers or bristles.
Use of polyclonal and monoclonal antibodies
At least one antibody capable of binding to Loxosceles venom antigen is a polyclonal antibody, optionally raised in rabbits, and the tracer can comprise a monoclonal antibody capable of binding to the venom antigen.
Portable colorimetric assay
The kit can include a portable colorimetric assay for detection.
Labels and blocking agents
The kit includes labels attached to tracers such as enzymes, radioactive isotopes, or color particles, specifically alkaline phosphatase or horseradish peroxidase, and includes blocking agents such as nonfat milk solids.
Means for lysing cells in the sample
The kit includes means for lysing cells present in the sample to release venom antigen.
Antibodies against specific venom proteins
The antibodies are against protein components of Loxosceles reclusa venom, including antibodies targeting sphingomyelinase.
Instructions for use
The kit further comprises instructions for its use.
The claims cover an immunoassay kit for detecting Loxosceles venom from a bite site using a swab-collected sample, with specific antibody and tracer components, swab materials, detection methods including colorimetric assays with enzymatic labels, blocking agents to improve specificity, and components enabling cell lysis and targeting of key venom proteins.
Stated Advantages
Allows non-invasive collection of venom samples, avoiding cutting or needles.
Enables accurate diagnosis of Loxosceles envenomation, reducing morbidity and costly misdiagnoses.
Highly sensitive ELISA that detects venom antigens down to about 20 picograms, even after sample storage for up to three weeks at elevated temperatures.
Provides ability to distinguish Loxosceles venom from other species' venoms due to specificity for unique proteins like sphingomyelinase D.
Adaptable to field use with portable and rapid immunoassay devices.
Facilitates clinical decision-making and appropriate treatment by providing definitive venom presence confirmation.
Techniques can be broadly applied to detect other clinically relevant protein markers and venom types.
Documented Applications
Diagnosis of brown recluse (Loxosceles reclusa) spider bites in humans by detecting venom proteins from swab samples collected non-invasively from suspected bite sites.
Differentiating brown recluse spider envenomation from other skin lesions and conditions with similar presentations, including bacterial cellulitis, pyoderma gangrenosum, other spider bites, and necrotizing fasciitis.
Detection of venom antigens from other venomous organisms such as other Loxosceles species, scorpions, snakes, wasps, bees, and jellyfish by adapting the immunoassay method.
Use in field and clinical settings with portable devices for timely diagnosis.
Testing swab samples shipped and stored for extended periods without loss of assay sensitivity, as demonstrated by samples shipped internationally and tested after days in ambient temperatures.
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