ABTS One Component HRP Microwell Substrate

Cat. No.: A572873

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Overview

The ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) substrate is supplied as a one component, ready-to-use solution for ELISA applications. A soluble blue-green reaction product is obtained when this substrate system is reacted with horseradish peroxidase. 

Usage suggestions

The product should be allowed to reach room temperature (25°C) prior to use.    
1) For immunoassay microwell applications, 100 µl of substrate solution is added to each well producing a soluble, blue‐green product.  
 Color development time can range between 10‐40 minutes depending on the assay.   
 It is recommended that development time be optimized for your assay conditions.
2) For best results, sample absorbance values should be monitored and read before absorbance values exceed 2.0 OD units.  
 Sample OD values can be read with or without the addition of stop solution in the 405‐410 nm
range.
The substrate reaction can be stopped using 650 nm Liquid Stop Solution for TMB Microwell Substrates (LBSP).
3) Dilution of the substrate is not recommended. It is recommended that the antibodies or conjugates be diluted to reduce the intensity of the reaction.   
The product is not recommended for membrane or IHC applications that require a precipitating reaction product.

Specifications

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Chemical and Physical Properties

• Sensitivity: Light sensitive

Application Protocols

Tested application details, recommended dilutions, and validated protocols are Not specified for this item; refer to CoA/Spec Sheet.

General note: In ELISA workflows, the substrate is typically added after final wash, incubated for a defined time at room temperature protected from light, and read at ~405–410 nm. However, specific incubation times, stopping procedures, and read settings must be established empirically for your assay and should follow any manufacturer-provided instructions when available.

Biological Roles

This is an assay reagent and does not have intrinsic biological roles in living systems as supplied. Its relevance is through the enzymatic reporting system used in immunoassays.

• HRP system (literature): Horseradish peroxidase is a plant heme enzyme that catalyzes peroxide-dependent oxidation of diverse substrates. In ELISA, HRP is conjugated to antibodies or streptavidin to transduce binding events into measurable color changes with chromogens such as ABTS.
• ABTS chromogen (literature): ABTS functions as an electron donor/acceptor pair that, upon HRP-catalyzed oxidation by H2O2, forms a stable radical cation with intense visible absorption. This provides a quantitative readout of enzyme activity.
• Interferences (general): Biological buffers containing azide (peroxidase inhibitor), high concentrations of thiols (e.g., DTT), or radical scavengers can suppress color development. Hemoglobin, bilirubin, and other colored species may add background absorbance if present in wells.

Note: This product is intended for research use only and is not designed for in vivo or diagnostic clinical use.

Buffer Applications

Not a buffering reagent. This product is a ready-to-use HRP substrate solution and is typically added after blocking/washing steps in ELISA workflows.

• Buffer compatibility (general): Perform antigen/antibody incubations in validated buffers free of peroxidase inhibitors (e.g., avoid sodium azide). Rinse thoroughly before substrate addition to prevent carryover.
• pH considerations (general literature): ABTS/HRP reactions proceed in mildly acidic to neutral buffers; exact pH of this formulated substrate is not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

This product is an aqueous, ready-to-use HRP substrate; as such, it aligns well with green chemistry principles regarding solvent choice and operator safety. Selection among HRP substrates can still reflect green and safety trade-offs.

Comparison of common HRP substrates (literature, general):

• ABTS (this product type)
  • Pros: Aqueous, low odor, non-volatile; green soluble product; suitable for kinetic reads without strong acids; generally lower acute hazards than some aromatic diamines.
  • Cons: Often lower sensitivity than TMB in endpoint acid-stopped ELISAs; color may continue to develop, requiring tight timing for endpoints.
• TMB
  • Pros: High sensitivity; strong signal at 450 nm after acid stop; widely standardized.
  • Cons: Requires acid stop (corrosive waste stream); some formulations classified as irritants or sensitizers; can be light/metal sensitive.
• OPD/o-phenylenediamine
  • Pros: Legacy workflows at 492 nm.
  • Cons: Aromatic diamine safety concerns; typically less favored in modern green protocols.

Greener practice tips (general):

• Use the smallest feasible reaction volumes and microplate formats to reduce waste.
• Avoid strong acid stops when kinetic monitoring suffices; if stopping is necessary, consider milder quenchers validated for ABTS systems.
• Consolidate rinses and neutralize aqueous waste per institutional guidelines to minimize environmental impact.
• Store at 2–8°C, protected from light, to maximize shelf life and reduce disposal due to premature degradation.

Pharmaceutical Uses

This product is an analytical laboratory reagent for colorimetric detection of HRP activity. It is not intended for pharmaceutical formulation or therapeutic use.

• Pharmacopeial status: Not specified for this item; refer to CoA/Spec Sheet.
• Typical role in regulated environments (general): May be used in research-stage ELISAs for process development, analytical method development, or non-clinical characterization where HRP-conjugates are employed. Any GMP or QC use would require internal validation and supplier documentation.
• Excipients/clinical formulation: Not applicable.

Physical Properties

As supplied, this is a formulated aqueous solution, not a neat compound; item-specific physicochemical specifications are not provided.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Density, pH, refractive index, viscosity: Not specified for this item; refer to CoA/Spec Sheet.
• Miscibility: Aqueous solution; miscible with water (general).
• Storage conditions: Store at 2–8°C, protected from light (per product data).
• Spectral behavior (literature, ABTS/HRP system):
  • The oxidized ABTS radical cation exhibits strong absorbance in the visible range; common monitoring wavelengths are 405–410 nm for ELISA plates and additional bands near ~650–734 nm in cuvette assays, depending on buffer and pathlength.
  • The green product is water-soluble and generally does not require a stop solution for short kinetic readings; absorbance may continue to increase over time in the presence of active HRP.
• Solubility (literature): ABTS salts are highly water-soluble due to sulfonate groups.

Important: Do not treat the literature spectral features above as product specifications. For lot-specific properties (e.g., pH, extinction characteristics in the supplied buffer), consult the CoA/Spec Sheet.

Quality & Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• Product type: One-component HRP microwell substrate (ready-to-use). These products are typically qualified for ELISA applications emphasizing low background, high sensitivity, and lot-to-lot consistency rather than classical chemical purity metrics.
• Typical QC attributes for HRP substrates (general, not item-specific):
  • Absorbance baseline (blank) in microwells.
  • Signal development kinetics with a defined HRP calibrator at 405–410 nm.
  • Linearity and dynamic range across HRP dilutions.
  • Stability under recommended storage (2–8°C, protected from light).
• Stabilizers and buffer system: Not specified for this item; refer to CoA/Spec Sheet. One-component systems commonly include peroxide and chromogen in a proprietary buffered matrix to balance background and sensitivity.
• Use implications:
  • Ready-to-use format minimizes pipetting errors and inter-well variability compared with two-component mixes.
  • Formulation is optimized for microwell pathlengths; do not assume equivalent behavior in cuvettes or membrane assays without verification.
• Documentation: For assay-specific performance criteria (limit of detection, S/N, lot acceptance ranges), consult the lot-specific Certificate of Analysis or internal validation data.

Reaction & Applications

Primary application: Colorimetric detection of horseradish peroxidase (HRP) activity in ELISA microwell assays.

• Mechanism (literature): In the presence of trace H2O2, HRP catalyzes one-electron oxidation of ABTS to a stable green radical cation. The reaction is monitored spectrophotometrically (commonly at 405–410 nm for microplates). Signal is proportional to HRP (and thus analyte) amount within the linear range.
• Use cases:
  • Sandwich or indirect ELISAs employing HRP-labeled detection antibodies or streptavidin-HRP.
  • Kinetic HRP activity assays and QC of conjugates, where continuous readout without strong acid stop is advantageous.
• Practical tips (general):
  • Equilibrate substrate to room temperature in the dark before use to minimize condensation and edge effects; do not prewarm excessively.
  • Thorough washing is essential; residual HRP-conjugate or contaminants can elevate background.
  • Avoid sodium azide in buffers that contact HRP; azide inhibits peroxidases.
  • Use clean plasticware; trace metals or peroxidase-like contaminants can increase non-enzymatic oxidation.
  • Read plates promptly and consistently; ABTS can continue to develop signal over time in the presence of HRP. For endpoint work, standardize incubation times and temperature.
• Comparison (general): ABTS provides a soluble green product suitable for kinetic monitoring. TMB typically achieves lower limits of detection with acid stop and 450 nm readouts, whereas ABTS is convenient for non-destructive kinetic measurements.

Reaction Conditions

General guidance for HRP–ABTS color development in microwell assays (literature; not product-specific):

• Temperature: Room temperature (20–25°C) is commonly used; maintain consistent temperature across plates for reproducibility.
• Wavelength: Monitor at 405–410 nm in microplates. Verify using your instrument’s recommended filter set.
• Incubation time: Typically 5–30 minutes, depending on HRP activity and desired signal window. ABTS can continue to develop; standardize incubation time across experiments.
• Stopping the reaction: Many ABTS assays are read kinetically without a strong acid stop. If an endpoint is needed, validated quenchers (e.g., surfactant such as SDS) may be used; confirm compatibility with your assay. Acidic stops used for TMB are not always applied to ABTS.
• Buffers and additives: Avoid sodium azide and strong reducing agents. Ensure thorough washing before adding substrate.
• Expected performance: Sensitivity and linear range depend on conjugate activity, plate type, and blocking efficacy. Establish standard curves with your specific system.

Important: The exact working conditions for this specific product (e.g., recommended incubation time, optimal wavelength in its proprietary buffer) are Not specified for this item; refer to CoA/Spec Sheet and perform in-house optimization.

Safety & Handling

• GHS classification, signal word, hazard and precautionary statements: Not specified for this item; refer to SDS for authoritative information.
• Likely hazards (general guidance for HRP peroxidase substrates): May cause eye/skin irritation. Formulations often contain low levels of hydrogen peroxide and stabilizers; handle with care, avoid contact with incompatible reducers or transition metals that may catalyze decomposition.
• Light sensitivity: Protect from light as specified; chromogens and peroxides are light-sensitive and can degrade or auto-oxidize.
• Incompatibilities (general): Strong reducing agents (consume peroxide or reduce oxidized chromophore), strong oxidizers (over-oxidation), metal contaminants, and elevated temperatures. Avoid contamination from HRP-containing solutions during storage.
• PPE: Laboratory coat, safety glasses, and chemical-resistant gloves (e.g., nitrile). Use in a clean area to prevent cross-contamination of assay reagents.
• First aid overview (general):
  • Skin contact: Rinse with water, remove contaminated clothing.
  • Eye contact: Rinse cautiously with water for several minutes; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; seek medical attention if symptoms occur (aerosols/sprays should be avoided).
  • Ingestion: Rinse mouth; seek medical advice.
• Spill/cleanup: Wipe with absorbent material; rinse area with water. Avoid releasing bulk solutions to drains without proper authorization.
• Disposal: Follow institutional and local regulations for aqueous chemical waste containing peroxides and dyes.

Always consult the product SDS for full and current hazard classifications and response measures.

Solvent Selection

This is an aqueous, ready-to-use substrate solution intended for direct addition to microwells.

• Solvent/polarity: Water-based buffer (highly polar, protic). Dielectric constant and buffer composition are formulation-dependent and not disclosed; do not dilute with organic solvents.
• Compatibility:
  • Compatible with polystyrene ELISA plates and common blocking agents when thoroughly rinsed prior to substrate addition.
  • Avoid residual azide, high concentrations of reducing agents (e.g., DTT, β-mercaptoethanol), or metal chelates that can inhibit HRP activity or alter color development.
• When to use this substrate vs alternatives (general):
  • Choose ABTS for soluble, green end-point or kinetic reads at ~405–410 nm, particularly where non-hazardous stop solutions or continuous monitoring are favored.
  • Consider TMB for higher sensitivity at 450 nm with acid stop; OPD for legacy 492 nm workflows; o-phenylenediamine and other substrates may have different safety/tuning profiles.
• Dilution: Use as supplied. If a lower reaction rate is desired, evaluate dilution only with the recommended buffer (if provided by the manufacturer). Water or non-buffered diluents can shift pH and performance.

Storage & Reconstitution

• Storage: Store at 2–8°C, protected from light (per product data). Minimize time at room temperature and keep the container tightly closed when not in use.
• Shipping: Shipped on wet ice (per product data).
• Reconstitution: Not applicable. This is a one-component, ready-to-use solution; do not dilute unless the CoA/Spec Sheet specifically recommends a compatible diluent.
• Handling tips:
  • Equilibrate to room temperature in the dark before dispensing to reduce condensation.
  • Do not freeze unless explicitly permitted in the CoA/Spec Sheet; freeze–thaw cycles may degrade performance.
  • Avoid microbial contamination; dispense using clean tips and do not return used substrate to the original container.
  • Protect from light during storage and use to preserve substrate integrity.
• Shelf life: Not specified for this item; refer to CoA/Spec Sheet.

Research Use Only: For research use only. Not for human or veterinary diagnostic or therapeutic use.

Structure & Identity

This product is a formulated, ready-to-use aqueous substrate solution for horseradish peroxidase (HRP) microwell assays (ELISA). As a mixture/solution, a single CAS/SMILES/InChIKey and a unique molecular formula do not apply to the product as sold.

• Item-specific identifiers (CAS, CID, InChIKey, SMILES, molecular formula, molecular weight): Not specified for this item; refer to CoA/Spec Sheet.
• Core chromogen (literature): ABTS stands for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), typically used as its diammonium salt in HRP colorimetric assays.
• Structural features (literature): ABTS consists of two benzothiazoline rings linked via an azo/azine-type bridge, each ring bearing an ethyl substituent and a sulfonate group for high aqueous solubility. Oxidation by HRP/H2O2 yields a stable green radical cation with strong visible absorption.
• Format: One-component substrate (ready-to-use), no mixing of separate peroxide/chromogen solutions required.

2D structure description (literature, ABTS chromogen): Two fused aromatic/heteroaromatic systems (benzothiazolines) connected symmetrically; each ring carries an electron-withdrawing sulfonate and an ethyl group at the 3-position. The conjugated system supports formation of an intensely colored radical cation upon enzymatic oxidation.

Notes:

• Exact buffer composition, stabilizers, and concentrations are formulation-specific and are not disclosed here. Refer to the product CoA/SDS for definitive identity and composition details.

Synthetic Utility

Not a synthetic building block or reagent for chemical transformations in organic synthesis. This is a formulated analytical substrate for HRP-based colorimetric assays.

• If a chromogenic peroxidase substrate is needed for synthetic or biocatalytic process monitoring, ABTS can serve as a convenient reporter (literature), but the present product is optimized for microwell detection rather than preparative chemistry.
• For chemical synthesis applications involving peroxidase-mediated oxidations, consider sourcing neat ABTS salts or alternative redox mediators specifically intended for process chemistry.

Target Specificity

Not applicable. This is a generic HRP enzyme substrate and does not possess antigen/epitope specificity.

• Species reactivity, clone, isotype: Not applicable.
• Any target-related details would derive from the HRP-conjugated antibodies or proteins used in the assay, not from the substrate.