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Botulinum Neurotoxin Type A DuoSet ELISA

R&D Systems | Catalog # DY4489-05

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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

46.9-3000 pg/mL

Sample Type

Cell culture supernates, serum, and plasma
Note: Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet

Reactivity

C. botulinum

Botulinum Neurotoxin Type A DuoSet ELISA Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits
View all BoNT-A ELISA Kits »

Product Summary for Botulinum Neurotoxin Type A DuoSet ELISA

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant Botulinum Neurotoxin Type A (BoNT-A). The suggested diluent is suitable for the analysis of most cell culture supernate, serum, and plasma samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Botulinum Neurotoxin Type A DuoSet ELISA

BoNT-A ELISA Standard Curve

BoNT-A ELISA Standard Curve

Kit Contents for Botulinum Neurotoxin Type A DuoSet ELISA

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008C) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Reagent Diluent*

Blocking Buffer*

Substrate Solution: ELISA TMB Substrate (Catalog # DY999B or DY999B-250)

Stop Solution: Methanesulfonic acid (Catalog # DY994B or DY994B-250)

Microplates: (Catalog # DY990), or equivalent

Plate Sealers: (Catalog # DY992), or equivalent

*For the recommended Reagent Diluent and Blocking Buffer for a specific DuoSet ELISA Development Kit, refer to the product datasheet.

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: BoNT-A

There are seven serotypes of Botulinum Neurotoxins (BoNTs) produced by various strains of Clostridium botulinum. BoNTs are synthesized as inactive single chain protein precursors and activated by proteolytic cleavage to generate disulfide-linked two-chain proteins. The 50 kDa light chain contains the catalytic domain, whereas the 100 kDa heavy chain contains an internal translocation domain and a receptor binding domain. BoNTs are the most potent protein toxins for humans. As zinc proteases, they cleave SNARE proteins to elicit flaccid paralysis in botulism by blocking acetylcholine release at the neuromuscular junction.

E. coli-expressed recombinant BoNT light chains are active proteases. However, they are not toxic because they cannot enter into host cells in the absence of the heavy chains.

Long Name

Botulinum Neurotoxin Type A

Alternate Names

BoNTA

Entrez Gene IDs

5185061 (C. botulinum)

Gene Symbol

BONT

Additional BoNT-A Products

Product Documents for Botulinum Neurotoxin Type A DuoSet ELISA

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Botulinum Neurotoxin Type A DuoSet ELISA

For research use only

Related Research Areas

Metalloproteases and Regulators

Citations for Botulinum Neurotoxin Type A DuoSet ELISA

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Protocols

View specific protocols for Botulinum Neurotoxin Type A DuoSet ELISA (DY4489-05):

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

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