By: Nancy Shine, PhD, Director of R&D, List Labs

A fast, sensitive, specific and accurate detection method to determine active infection by Bacillus anthracis in plasma has been developed at List Biological Laboratories.

Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can kill quickly.  While antibiotic treatment can clear the bacterium from the host, if diagnosis is delayed, the toxin, which is rapidly produced, may already be present in lethal amounts. There is a critical need for a rapid, accurate, sensitive and simple assay to determine whether infection has occurred thereby allowing immediate treatment.

Anthrax Detection Method

Anthrax lethal factor (LF), an endopeptidase, is present in blood in the early stages of the infection.  The use of peptidic substrates in plasma is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate.  A fluorescently labeled peptide substrate, MAPKKide Plus, Prod #532, which is not cleaved by plasma proteases and thus is specific for LF has been designed. The LF is enriched by capture from plasma using an LF antibody-coated microtiter plate, and the captured LF is then exposed to the fluorescent substrate.  The amount of cleaved peptide substrate is determined by HPLC with fluorescence detection. Concentration of the LF using the antibody-coated plates allows for the detection of 5 pg LF/ml of neat plasma after 2 hours of incubation.  Alternately, the MAPKKide Plus may be added directly to diluted plasma and cleavage monitored by an increase in fluorescence as a function of time using a fluorescent microplate reader.  The limit of detection by this simpler method is 1 ng LF/ml of plasma after 5 hours of digestion.  Both methods can be confirmed by analysis of the reaction as a function of time.  These methods are described in the poster Sensitive Detection of Anthrax Lethal Factor in Plasma Using a Specific Biotinylated Fluorogenic Substrate.

What’s Next for Anthrax Detection Method

We are currently working with a biotinylated form of MAPKKide Plus to enhance the sensitivity of the simpler method using the fluorescent plate reader rather than HPLC.

You can see the poster here. To see a complete list of all of List Labs’ posters check out this blog post.

Interested in learning more about this List Labs patented peptide substrate? Contact us!

Over 20 Scientific Posters Available on our website

Scientific posters are a great way to visually display complex scientific issues. List Labs has a large list of scientific posters published on our website under the specific products they pertain to. We have been getting a lot of requests lately to compile a list of all of our published posters into one place. Please see below for a comprehensive list of all List Labs’ scientific posters to date.

Click on the one you are interested in to check it out.

 

Cholera Toxins

• HPLC Method to Assay the ADP-Ribosyltransferase Activity of Cholera Toxin

Botulinum Toxins

• Evaluation of Three Vamptide® Substrates For Botulinum Neurotoxin Type B
• FRET Peptide Substrates for Botulinum Neurotoxin Types A, B, C, E
• Sensitive and Specific Bifunctional Assay for Botulinum Neurotoxin Type A
• Sensitive Detection of Botulinum Neurotoxin Type A Using High Affinity Polyclonal Antibodies
• New FRET substrate for Botulinum Neurotoxin Type A
• Ultra Sensitive HPLC Detection Assay for Botulinum Neurotoxin Type A
• Evaluation of a Small Peptide Inhibitor and A Control Peptide for Botulinum Neurotoxin, Type A
• Sensitive, In Vitro, Bifunctional Potency Assay for Botulinum Neurotoxin Type A
• Ultra Sensitive HPLC Detection Assay for Botulinum Neurotoxin Type A and SYNTAXtide®, FRET Substrate for Botulinum Toxin C.
• Functional Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c
• Capture Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c
• New High Affinity Antibodies Against Botulinum Neurotoxin Type A
• FRET Peptide Substrates for the Botulinum Toxins Type A, B, and E and for Anthrax Lethal Factor 
• SNAP Etide®, a FRET Substrate for Botulinum Toxin Type E
• Sensitive and Specific Assay to Measure the Endoprotease Activity of Botulinum Toxin Type A Holotoxin in Milk
• Comparison of Activity of Botulinum Neurotoxin Type A Holotoxin and Light Chain Using SNAPtide® FRET Substrates
• Rapid, Sensitive, and Specific Assay to Measure the Endoprotease Activity in Botulinum Toxin Type A
• New SV2c Construct for Use in Binding and Detecting Botulinum Neurotoxin Type A

Anthrax Toxins

• Physical Characteristics of rLF and rPA: Effects on Enzymatic Activity and Binding
• Internally Quenched Fluorogenic Substrates for Anthrax Lethal Factor
• Fluorescence Substrate for Specific and Quantitative Detection of Anthrax Lethal Factor in Plasma
• MAPKKide® Plus A Specific Substrate for Sensitive Detection of Anthrax Lethal Factor in Complex Matrices
• Sensitive Detection of Anthrax Lethal Factor in Plasma Using a Specific Biotinylated Fluorogenic Substrate

 

By: Nancy Shine, Ph.D., Director of Research & Development

List Biological Laboratories, Inc. has designed a fluorescently labeled substrate for specific and quantitative detection of anthrax lethal factor in plasma.

Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can kill quickly. While antibiotic treatment can clear the bacterium from the host, if diagnosis is delayed, the toxin, which is rapidly produced, may already be present in lethal amounts. There is a critical need for a rapid, accurate, sensitive and simple assay to determine whether infection has occurred thereby allowing immediate treatment.

MAPKKide® Plus for Anthrax Detection in Plasma

The use of MAPKKide® Plus allows for a fast, sensitive, specific and accurate method to detect active infection by Bacillus anthracis in plasma. Anthrax lethal factor (LF), an endopeptidase, is present in blood early in the infection. The use of peptidic substrates in plasma is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. MAPKKide® Plus is a fluorescently labeled peptide substrate which is not cleaved by plasma proteases and thus is specific for LF.

Methods for Using MAPKKide® Plus

There are two methods to use MAPKKide® Plus for anthrax detection. One method involves the enrichment of LF by capture from plasma using an LF antibody-coated microtiter plate, and the captured LF is then exposed to MAPKKide® Plus. The amount of cleaved peptide substrate is determined by HPLC with fluorescence detection. Concentration of the LF using the antibody-coated plates allows for the detection of 5 pg LF/ml of neat plasma after 2 hours of incubation. Alternately the MAPKKide® Plus may be added directly to diluted plasma and cleavage monitored by an increase in fluorescence as a function of time using a fluorescent microplate reader. The limit of detection by this simpler method is 1 ng LF/ml of plasma after 5 hours of digestion. Both methods can be confirmed by analysis of the reaction as a function of time.

MAPKKide® Plus details are as follows:

• Product: #532
• Price per vial: $250
• Vial size: 100 nmoles
• Not a Select Agent or a controlled export item
• A patent application has been filed for this peptide substrate.

MAPKKide® Plus is in its final stages of release and will be available from stock early next month. We are accepting orders now. Orders may be placed online on the product detail page.

Please do not hesitate to contact us with any other questions about MAPKKide® Plus. More information about all List Labs products and potential future products can be found on the following pages:

• Seach all Research Reagent Products
• Product Pipeline