Human Thrombin ELISA Kit

Cat. No.: H1510083

GRADE & PURITY BioReagent

Descripción general

This kit employs a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of Human Thrombin concentration in samples. The Human Thrombin capture antibody is pre-coated onto the microtiter plate. Upon addition of samples or standards, Human Thrombin present therein binds to the immobilized capture antibody, while other unbound components are removed through washing. Subsequently, a biotin-conjugated detection antibody specific for Human Thrombin is added. This antibody binds to the captured Human Thrombin, forming a "sandwich" immunocomplex. Any excess, unbound material is again removed by washing. Next, an enzyme conjugate (typically Streptavidin-Horseradish Peroxidase, SA-HRP) is added. The biotin on the detection antibody and streptavidin on the enzyme conjugate exhibit high-affinity binding, thereby linking the HRP enzyme to the immunocomplex. Following another wash step to remove unbound conjugate, a colorimetric substrate (e.g., TMB) is added. If Human Thrombin is present in the sample, the HRP linked to the complex catalyzes the oxidation of the colorless substrate, yielding a blue product. The reaction is then stopped by adding a stop solution (usually an acid), which changes the color from blue to yellow. The optical density (OD) of each well is measured at 450 nm using a microplate reader. The concentration ofHuman Thrombin is directly proportional to the OD450 value. A standard curve is generated by assaying known concentrations ofHuman Thrombin, and the concentration of Human Thrombin in unknown samples is interpolated from this curve based on their OD values.

Specifications

Especificaciones y pureza

BioReagent

Grado

BioReagent

Aplicación

ELISA

Tipo de muestra

Plasma, Suero

Rango de detección

1.56 ng/mL~100 ng/mL

Método de detección

Sandwich ELISA

Especie detectada Objeto

Human

Especificidad

Detecting Human Thrombin in samples, with no significant cross-reactivity to other homologs.

Detection instrument

Microplate reader

Detection wavelength

450nm

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ for 6 months.

Application Protocols

Vendor-tested applications, sample types, and recommended dilutions for SKU H1510083 are not specified for this item; refer to the official kit insert.

General ELISA workflow (literature/general) for planning purposes:

• Preparation:
  • Bring all reagents to room temperature. Prepare a standard curve via serial dilution from the provided standard.
  • Determine sample dilutions to place unknowns within the dynamic range; avoid matrix extremes.
• Assay steps (pre-coated sandwich ELISA example):
  1. Add standards, controls, and samples to wells; incubate per protocol.
  2. Wash 3–5× thoroughly.
  3. Add HRP-conjugated detection reagent; incubate.
  4. Wash 3–5×.
  5. Add TMB substrate; develop color protected from light.
  6. Add stop solution; read A450 (with reference, if applicable) within 5–10 min.
• Controls and QC:
  • Include blank, zero standard, and at least two internal controls.
  • Fit data with a 4-parameter logistic (4PL) curve; review residuals and back-calculated concentrations.
• Troubleshooting tips:
  • High background: improve washing, check contamination, lower incubation time/temperature.
  • Weak signal: verify reagent integrity, increase incubation time, confirm instrument wavelength.

These steps are generic and must be adapted to the instructions specific to this kit.

Biological Roles

Analyte background (general/literature):

• Thrombin (human) is a central serine protease in the coagulation cascade, produced by proteolytic activation of prothrombin (Factor II) via the prothrombinase complex.
• Functional activities:
  • Cleaves fibrinogen to fibrin monomers; activates Factor XIII, V, VIII, XI; modulates platelet activation via PAR receptors.
  • Exhibits exosite-mediated interactions that regulate specificity and cofactor binding.
• Regulation:
  • Antithrombin and heparin cofactor pathways inhibit thrombin; thrombomodulin-bound thrombin activates protein C.
• Cellular and matrix context:
  • Acts on protease-activated receptors (PAR1, PAR3/4) affecting endothelial and platelet signaling.
• Analytical implications for ELISA (general):
  • Thrombin may exist free or in complexes (e.g., with antithrombin); epitope availability can differ among complexes and zymogen/active forms.
  • Pre-analytical variables (anticoagulant type, time-to-spin, temperature) can influence measured levels.

Item-specific notes:

• Target and epitope specificity, calibration standard (recombinant/native), and cross-reactivity profile for SKU H1510083 are not specified for this item; consult the kit insert and CoA.

Use this kit strictly for research purposes as indicated by the listing: For research use only.

Buffer Applications

This section pertains to buffers as they relate to ELISA performance rather than classical buffering of reactions.

General ELISA buffer systems (literature/general):

• Coating buffer: carbonate–bicarbonate, pH ~9.4 for plate coating (if plates are not pre-coated). For pre-coated plates, this step is not required.
• Blocking buffer: protein-based blockers (e.g., BSA, casein) in PBS or TBS to reduce nonspecific binding.
• Wash buffer: PBS or TBS with 0.05–0.1% Tween-20 to minimize background while preserving binding.
• Sample/standard diluent: buffered protein/carbohydrate matrix to match the standard matrix and mitigate matrix effects.
• Substrate buffer: proprietary formulations optimized for HRP/TMB stability and kinetics.

For this SKU:

• Specific buffer compositions, pH, and surfactant concentrations are not specified for this item; refer to the kit insert.

Practical tips:

• Maintain consistent ionic strength and detergent levels between standards and samples.
• Filter or degas wash solutions to improve dispensing consistency and reduce bubble formation.
• Validate alternative blockers if changing sample types; different blockers can mask epitopes.

Note: If handling plasma/serum, select anticoagulants that do not interfere with the assay chemistry (e.g., avoid EDTA if calcium-dependent interactions are critical), unless the kit explicitly validates them.

Green Alternatives

While ELISA kits are largely aqueous and inherently greener than many organic syntheses, improvements can reduce waste and hazards.

Greener practice options (general):

• Minimize hazardous reagents:
  • Prefer non-acid stop solutions (e.g., alternative substrates with non-corrosive stops) where validated; if acid stops are required, neutralize before disposal per local regulations.
• Waste reduction:
  • Optimize wash volumes and number of washes to specification; avoid overfilling reservoirs.
  • Batch assays to fully utilize 96-well plates and reduce partial-plate waste.
• Material choices:
  • Use recyclable polypropylene reservoirs and low-waste tip systems where acceptable.
  • Implement plate sealers that are compatible with recycling streams when possible.
• Energy and cold-chain:
  • Store at 2–8°C (item-specific). Consolidate cold storage and reduce door-open time. Shipments already use wet ice (item-specific), which has a lower environmental burden than dry ice for 2–8°C goods.
• Safer preservatives:
  • If azide-free or isothiazolinone-free options are needed, confirm availability; for this SKU, specific preservatives are not specified.

Comparison (general overview):

• Conventional ELISA: HRP/TMB with acid stop; aqueous, small volumes, but corrosive stop solution.
• Alternative readouts: fluorescent or chemiluminescent substrates can increase sensitivity and reduce acid waste, though they may require more energy-intensive instrumentation.

Always follow institutional EHS guidance for aqueous biological and acidic waste streams.

Pharmaceutical Uses

This product is not a therapeutic and is labeled For research use only.

Relevant non-clinical uses (general/literature):

• Process and product analytics in biopharmaceutical R&D to characterize coagulation-related pathways in cell-based systems.
• In vitro potency or mechanism-of-action studies where thrombin levels serve as a research biomarker in preclinical models.
• Raw-material or in-process control studies in research manufacturing environments (e.g., testing conditioned media or purified proteins), where permitted by institutional SOPs.

Item-specific regulatory status:

• No pharmacopeial monograph or diagnostic claims are provided for this SKU. It is not cleared for clinical diagnostic use.

Documentation expectations:

• For regulated research settings, maintain kit CoA, lot verification data, and establish internal method suitability (accuracy, precision, linearity, spike-recovery) before routine use. Specific performance claims for this kit (LOD/LOQ, accuracy, precision) are not specified here; refer to CoA/Spec Sheet.

Note: Any deployment in GMP/QC environments requires user-led method validation and risk assessment. Follow site-specific quality procedures.

Physical Properties

Item-specific physical specifications (BP, MP, density, refractive index, pKa, logP) are not applicable to a multi-component ELISA kit and are not specified for this item; refer to CoA/Spec Sheet for any component-level details.

General characteristics (literature/general):

• Typical physical forms: pre-coated 96-well microplate, aqueous buffers (phosphate/Tris-based), lyophilized or liquid standards, HRP-conjugated detection reagent, chromogenic substrate (e.g., TMB).
• Solution properties: aqueous, near-neutral pH; may contain preservatives (e.g., ProClin or Kathon) and stabilizers (BSA/sugars). Exact compositions: Not specified for this item.
• Optical readout: absorbance at 450 nm (TMB/H2SO4 system) or 450/570–620 nm with reference (literature). The exact wavelength requirement for this SKU is not specified; consult the kit insert.
• Sample compatibility (general): plasma or serum often require defined anticoagulants and dilutions; conditioned media and cell lysates may be compatible with proper diluents. Specific validated matrices for this SKU: Not specified.

Storage temperature for this item: Store at 2–8°C (item-specific). Shipping: Wet ice (item-specific). Avoid freezing components unless explicitly allowed by the insert (general guidance).

Quality and Grades

• Item-specific designation: BioReagent (as provided). This denotes suitability for biological laboratory procedures, prepared under controlled conditions to minimize contaminants that could interfere with immunoassays.
• What this means in practice (general):
  • Components are typically QC-tested for performance in ELISA format, including sensitivity, signal-to-noise, and background metrics. Exact QC parameters for this SKU: Not specified for this item; refer to CoA/Spec Sheet.
  • Lot-to-lot consistency is crucial for standard curve linearity and inter-assay precision; BioReagent grade supports reproducibility.
• UV cutoff, metal content, microbial bioburden, and preservative concentrations: Not specified for this item; refer to CoA/Spec Sheet.
• Stabilizers and blockers:
  • ELISA kits often incorporate protein stabilizers (e.g., BSA) and blockers to reduce nonspecific binding (literature/general). Specific stabilizers for this SKU are not specified.
• Documentation:
  • Certificate of Analysis (CoA) and kit insert typically provide acceptance criteria (e.g., dynamic range, LOD/LOQ, linearity recovery). For this item, consult the CoA/Spec Sheet for details.

Summary: BioReagent grade underscores that the kit is configured for reliable immunoassay performance in research workflows; however, end-users should verify compatibility with their sample matrix and adhere to the provided instructions.

Reaction and Applications

As an immunoassay, the kit employs antigen–antibody binding (capture and detection) followed by an enzyme-catalyzed chromogenic reaction.

• Application scope (research use only):
  • Quantitative measurement of human thrombin protein in biological samples such as plasma, serum, cell culture supernatants, or lysates (general/literature). Validated matrices and dilution factors for this SKU: Not specified; refer to the insert.
• Assay principle (general):
  • A capture antibody immobilizes thrombin.
  • A detection antibody (often HRP-conjugated directly or via a streptavidin–biotin system) binds a distinct epitope.
  • Addition of substrate (e.g., TMB) yields a colorimetric signal proportional to thrombin concentration.
• Practical tips (literature/general):
  • Establish a fresh standard curve each run; include blanks and quality controls.
  • Equilibrate all reagents to room temperature before starting; avoid bubbles.
  • Use calibrated pipettes; run standards and samples in duplicate or triplicate.
  • Control for heterophilic antibody interference (e.g., include blockers) when working with complex matrices.
  • Avoid azide with HRP; protect TMB from light.
• Performance expectations:
  • Typical dynamic ranges for ELISAs span low pg/mL to ng/mL levels (literature). Exact LOD/LOQ for this item: Not specified; see CoA/Spec Sheet.

This section provides general guidance. For definitive operating conditions and validated applications, follow the kit’s official instructions.

Reaction Conditions

Item-specific incubation times, temperatures, and volumes are not specified for this kit; refer to the official insert. The following are general ELISA conditions from the literature for planning and benchmarking:

• Temperature: Many ELISAs run at room temperature (20–25°C) for binding steps; some benefit from 37°C to accelerate kinetics, while sensitive interactions prefer 2–8°C overnight incubations to improve affinity.
• Incubation times (typical):
  • Sample binding: 1–2 h at RT or overnight at 4°C (pre-coated plates).
  • Detection reagent: 30–60 min at RT.
  • Substrate development: 5–20 min protected from light; stop when the highest standard approaches the upper linear range.
• Wash cycles: 3–5 washes between steps using PBS/TBS + 0.05–0.1% Tween-20; ensure complete removal to reduce background.
• Readout: Measure absorbance at 450 nm (reference 570–620 nm if instrument supports). Exact wavelengths for this SKU are not specified.
• Performance checkpoints:
  • Standard curve R2 ≥ 0.99 with 4PL fit (literature); back-calculated standards within ±15–20%.
  • Acceptable CV: ≤10% intra-assay, ≤15% inter-assay (general targets).

These parameters are general guidance only. Optimize empirically with your matrix and strictly follow the kit-specific instructions when available.

Safety and Handling

• GHS classification, signal word, H-statements, pictograms: Not specified for this item; defer to the SDS for each component.
• Potential hazards (general/literature for ELISA kits):
  • Substrate solutions (e.g., TMB) and stop solutions (often dilute mineral acid) can be irritants/corrosive.
  • Preservatives (e.g., isothiazolinones) may cause skin/eye irritation or sensitization.
  • HRP conjugates and proteinaceous materials can cause allergic reactions in sensitized individuals.
• Recommended PPE: laboratory coat, nitrile gloves, safety glasses; use in a clean area away from food/drink. Handle acids/bases with appropriate splash protection.
• Handling practices:
  • Avoid microbial contamination of reagents; use sterile tips and dedicated reservoirs.
  • Do not mix components from different lots unless the protocol explicitly permits.
  • Bring cold components to room temperature before use to reduce edge effects (general best practice).
• Incompatibilities (general):
  • Strong oxidizers/reducers near chromogenic substrates; azide can inactivate HRP—avoid azide-containing buffers when using HRP detection.
• First-aid overview (general):
  • Skin/eye contact: rinse with water for ≥15 minutes; remove contaminated clothing.
  • Ingestion/inhalation: move to fresh air; rinse mouth; seek medical advice.

Always consult the product SDS and the kit insert for authoritative safety and waste-disposal instructions. For research use only.

Solvent Selection

This product is an ELISA kit supplied in aqueous buffers; solvent selection in the classical sense (for organic synthesis) is not typically applicable.

Practical guidance (general/literature):

• Use only the provided diluents and wash buffers or validated equivalents to avoid altering antibody affinity or enzyme activity.
• Avoid sodium azide or other peroxidase inhibitors in sample or wash buffers when using HRP-based detection.
• For sample preparation:
  • Plasma/serum: follow recommended anticoagulants and dilution buffers (e.g., PBS/BSA/Tween systems). Exact recipes for this SKU are not specified; refer to the kit insert.
  • Cell culture supernatants: clarify by centrifugation and use the provided sample diluent to minimize matrix effects.
• Cleaning/readout:
  • If microplate washers are used, validate aspiration/dispense volumes with aqueous buffer and avoid solvent carryover.

If non-aqueous solvents are present in samples (e.g., DMSO), keep final concentrations low (commonly ≤1–2% v/v, literature) to prevent enzyme inhibition or altered binding. The precise tolerance for this kit is not specified; confirm experimentally.

Storage and Reconstitution

• Item-specific storage: Store at 2–8°C.
• Shipping condition: Wet ice.
• Research Use Note: For research use only.

General guidance for ELISA kits (literature/general):

• Upon receipt: Inspect components; record lot numbers and expiration dates. Do not freeze unless the insert explicitly allows freezing of specific vials (e.g., lyophilized standards).
• Stability: Keep reagents tightly capped; avoid prolonged exposure to room temperature and light (especially substrates and HRP conjugates).
• Reconstitution:
  • If standards or controls are lyophilized, reconstitute with the specified diluent and volume provided in the insert. For this SKU, exact diluents/volumes are not specified; refer to CoA/Spec Sheet.
  • Mix gently; avoid foaming. Allow complete dissolution before use.
• Aliquoting and reuse:
  • Prepare only the volumes needed for the immediate run; avoid repeated freeze–thaw of protein components.
  • If the kit supports multiple uses, store opened components at 2–8°C and use within the period stated by the insert.
• Plate handling:
  • If the plate is strip-based, return unused strips to the foil pouch with desiccant; reseal tightly and store at 2–8°C.
• Instrumentation: Calibrate the microplate reader for 450 nm (and reference wavelength if applicable) per the kit’s instructions.

For authoritative stability and reconstitution details, follow the kit insert and the CoA/Spec Sheet.

Structure and Identity

This product is a multi-component immunoassay kit designed for quantitative detection of human thrombin antigen by ELISA format.

• Item-specific identifiers:
  • SKU: H1510083
  • Product name: Human Thrombin ELISA Kit
  • Grade/Purity: BioReagent (kit components for laboratory research use)
  • CAS, CID, InChIKey, SMILES: Not applicable to a kit; not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula / molecular weight (item-level): Not specified for this item; refer to CoA/Spec Sheet.
• Kit nature:
  • Typically includes capture/detection antibodies, standards, sample diluent, wash buffer, substrate (e.g., TMB), stop solution, and a coated microplate (literature/general). Exact contents for this SKU: Not specified; refer to the product insert.
• Analyte identity (general/literature):
  • Human thrombin is a trypsin-like serine protease generated from prothrombin; it contains a catalytic triad (His–Asp–Ser) and features exosite I/II binding regions pertinent to fibrinogen and cofactors.
• 2D description (general/literature):
  • The kit’s functional architecture comprises an immobilized capture layer recognizing thrombin epitopes and a secondary detection layer producing an optical signal proportional to antigen amount.

Note: This is an ELISA kit, not a single defined small molecule; structural identifiers (SMILES, InChI) are inherently not applicable.

Synthetic Utility

Not typically applicable. This product is an ELISA kit for protein quantification, not a chemical building block or reagent for synthetic transformations.

If your workflow involves chemical steps (general):

• Upstream sample treatments (e.g., protease inhibitors, denaturants, reducing agents) can impact immunoreactivity and enzyme labels; avoid or keep at validated low concentrations.
• Detergents or solvents used in sample prep (e.g., DMSO, methanol) should be minimized to maintain antibody binding and HRP activity.

For chemical synthesis needs, refer to Aladdin Scientific’s catalog of small-molecule reagents and solvents. For this item, focus on assay design and sample handling rather than synthetic transformations.

Target Specificity

• Target antigen: Human thrombin (general context). Item-specific antigen form (active, zymogen-derived epitopes, complexed vs free) and epitope mapping for SKU H1510083 are not specified for this item; refer to CoA/Spec Sheet.
• Cross-reactivity: Not specified for this item. Literature cautions that closely related serine proteases or thrombin–inhibitor complexes may cross-react depending on antibody design.
• Species reactivity: Intended for human analyte based on the product name. Validation for other species is not specified; empirical verification is recommended.
• Calibration material: Not specified (recombinant vs native standard not indicated).
• Antibody details (clone, isotype): Not specified for this item.

For definitive specificity, cross-reactivity, and interference data, consult the kit insert and validation report. Perform spike-recovery and dilution linearity studies in your sample matrix to confirm suitability.

Preguntas frecuentes

How is this product shipped?

This product ships chilled on wet ice. Unpack on arrival and transfer it to the storage condition stated above.

How should this product be stored?

Store at 2–8 °C. Refrigerated storage is required to maintain the specified shelf life.