Melatonin (MT) ELISA Kit

Cat. No.: EJ1515330

GRADE & PURITY BioReagent

Overview

This kit employs the competitive enzyme-linked immunosorbent assay (ELISA) principle. Microplate wells are pre-coated with Melatonin‌ (MT) antigen. The sample, standards, a biotin-labeled antibody, and an HRP enzyme conjugate are then added sequentially to the wells. The procedure includes incubation and wash steps between each addition. TMB substrate is used for color development. In the presence of the peroxidase (HRP) enzyme, TMB is catalyzed to produce a blue color, which is then converted into a final stable yellow color upon the addition of an acid stop solution. The intensity of the resulting yellow color is inversely proportional to the concentration of Melatonin‌ (MT) in the sample. The optical density (OD) is measured at a wavelength of 450 nm using a microplate reader, and the concentration of the target analyte in the sample is calculated based on the standard curve.

Specifications

Specifications & Purity

BioReagent

Grade

BioReagent

Application

ELISA

Sample type

Plasma, Serum

Detection Range

7.8pg/mL~500pg/mL

Specificity

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

Detection instrument

Microplate reader

Detection wavelength

450nm

Repeatability

The intra- and inter-assay coefficients of variation (CV) are both <10%.

Detection duration

3.5 hours, not including equilibration and sample preparation time.

Storage and Shipping

Storage

Store at 2-8°C

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ for 6 months.

Application Protocols

Item-specific validated protocols, recommended dilutions, and incubation times are not specified for this item; refer to the kit insert for authoritative instructions.

General competitive ELISA workflow for small molecules like melatonin (informational guidance):

1. Equilibrate all reagents to room temperature; prepare standards by serial dilution in the supplied sample diluent.
2. Add standards, samples, and (if applicable) enzyme-labeled melatonin tracer to antibody-coated wells or wells coated with hapten–carrier conjugate (format-dependent).
3. Incubate for the specified time to reach competitive binding equilibrium; protect from strong light.
4. Wash thoroughly (e.g., 3–5 cycles) to remove unbound materials.
5. Add enzyme substrate (e.g., TMB for HRP); incubate for a controlled time.
6. Add stop solution (if required) and read absorbance at the recommended wavelength (commonly 450 nm with reference 620–650 nm for HRP/TMB systems; exact values not specified for this item).
7. Fit a 4PL/5PL curve to standards; interpolate sample concentrations, applying dilution factors. Include QC samples to monitor assay performance.

Notes:

• Validate matrix effects via spike–recovery and dilution linearity.
• Avoid residual organic solvents in wells; they can impair binding and enzyme activity.

Biological Roles

Content in this section concerns the analyte melatonin, not the kit components.

Melatonin overview (literature/general):

• Endogenous indoleamine produced primarily in the vertebrate pineal gland through the serotonin → N-acetylserotonin → melatonin pathway, with key enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT).
• Exhibits circadian and circannual rhythms in biosynthesis, typically peaking during the dark phase; secretion is entrained by the light–dark cycle via the suprachiasmatic nucleus and sympathetic signaling to the pineal.
• Binds to G protein–coupled receptors MT1 (MTNR1A) and MT2 (MTNR1B) to modulate intracellular signaling cascades (e.g., cAMP pathways) in various tissues (literature/general). Also acts as a redox-active small molecule in chemical systems; avoid inferring therapeutic effects.
• Distribution: Found in many organisms and matrices (blood, saliva, urine), and in plants and foods at varying levels (literature reports).

Research implications (general):

• Quantifying melatonin supports studies in chronobiology, endocrine physiology, light exposure effects, and environmental/food chemistry. Assay-based readouts should be validated for the specific matrix used (spike–recovery, dilution linearity, and parallelism). No clinical interpretation is implied for this research-use-only kit.

Buffer Applications

Applicability: This ELISA kit relies on aqueous buffers for coating, blocking, washing, sample/standard dilution, and substrate development. Item-specific buffer identities, compositions, and pH are not specified for this item; refer to the kit insert.

General ELISA buffer systems (literature/practice):

• Coating buffer: carbonate–bicarbonate, pH ~9.4–9.6, promotes passive adsorption of antigens/haptens–carrier conjugates to polystyrene plates.
• Blocking buffer: proteins such as BSA, casein, or fish gelatin in PBS/TBS to occupy nonspecific sites; choice affects background for small-molecule competitive ELISAs.
• Wash buffer: PBS-T or TBS-T (0.05% Tween 20 typical) to reduce nonspecific binding.
• Sample/standard diluent: protein-containing buffer (BSA/casein) to stabilize low-MW analytes and minimize matrix effects.
• Substrate buffer: depends on enzyme system (e.g., citrate–phosphate for HRP/TMB), stop solution often sulfuric acid to fix the endpoint (only if specified by the kit).

Practical tips (general):

• Match ionic strength and protein content of diluent across standards and samples to ensure parallelism.
• Prepare fresh buffers or use pre-formulated concentrates supplied in the kit; filter to remove particulates when necessary.
• Maintain consistent wash cycles and dwell times; residual wash buffer volume can affect competitive assay precision.

Green Alternatives

Analytical context: Immunoassays such as ELISA can offer greener alternatives to chromatographic quantification by reducing organic solvent consumption and energy use.

Comparison (general; literature-based):

• ELISA vs. LC–MS/MS for melatonin quantification:
  • Solvent use: ELISA predominantly aqueous buffers; LC–MS/MS often requires organic mobile phases (MeCN/MeOH) and solid-phase extraction solvents.
  • Energy and footprint: ELISA has lower instrument energy demand compared with high-vacuum mass spectrometers.
  • Waste: ELISA generates mainly aqueous, low-toxicity waste; LC–MS/MS produces mixed solvent waste requiring specialized disposal.
  • Specificity/linearity: LC–MS/MS can provide higher chemical specificity and wider dynamic range; ELISA depends on antibody specificity and may be susceptible to matrix cross-reactivity.

Greener practice within ELISA (general guidance):

• Right-size experiments to minimize reagent waste; use strip plates if available.
• Opt for reusable reservoir boats and calibrated multichannel pipettes to reduce plastics.
• Segregate acidic stop solution waste if used; neutralize before disposal per EHS policy.

Item-specific notes: Component compositions and any eco-label attributes are not specified for this item; refer to the kit documentation.

Pharmaceutical Uses

Not applicable to this product. The Melatonin (MT) ELISA Kit is supplied for laboratory research use only and is not intended for manufacturing, clinical diagnostics, or therapeutic applications.

General context (informational only):

• Melatonin is an established active pharmaceutical ingredient (API) in some regulatory jurisdictions; however, this kit is not an API and is not for any clinical or formulation use.
• In pharmaceutical research settings, melatonin quantification by ELISA may be used for exploratory studies, pre-formulation research, or comparative method development alongside chromatographic assays. Any GMP/GLP or pharmacopeial compliance requirements would necessitate item-specific validation data, which are not provided here.

Physical Properties

Item-specific physical specifications for the kit components (e.g., standard concentration, buffer compositions, preservatives, pH, protein content) are not specified for this item; refer to CoA/Spec Sheet and the kit insert.

Analyte reference values (literature; melatonin, for context only):

• Physical state: crystalline solid (pure compound).
• Melting point: typical literature range ~116–118 °C (compound-level; not a kit specification).
• Solubility: readily soluble in organic solvents such as DMSO, ethanol, methanol; limited solubility in water (literature, qualitative).
• Acid/base: indole NH very weakly acidic; amide is non-basic; melatonin is largely neutral at neutral pH (literature).
• Partitioning: moderately lipophilic with polar functionality; typical logP reported in literature ~1–2 (qualitative range; exact value varies by method).

Practical notes (general guidance):

• ELISA standards for small molecules like melatonin are typically supplied lyophilized or in stabilized solution; reconstitute/dilute using the kit’s specified assay buffer only (not provided here).
• For calibration curves, prepare serial dilutions immediately before use to minimize degradation and adsorption; protect analyte solutions from strong light.

Quality & Grades

From Product Data (item-specific):

• Grade: BioReagent

Interpretation and implications (general):

• BioReagent grade indicates reagents and components suitable for biological/biochemical research workflows (e.g., immunoassays), emphasizing low background, consistency, and performance in bioanalytical methods. It does not imply clinical or diagnostic use.
• For ELISA kits, quality is typically reflected in parameters such as sensitivity, dynamic range, precision (intra-/inter-assay CV%), specificity/cross-reactivity, and lot-to-lot consistency. These performance metrics are not specified for this item; refer to the kit insert/CoA.
• Stabilizers/preservatives: Many ELISA kits include protein stabilizers, detergents (e.g., Tween 20), and antimicrobial agents; presence and concentrations are not specified for this item; refer to CoA/Spec Sheet.
• Documentation: For regulated laboratories, retain CoA, SDS, and any performance validation data (e.g., recovery, linearity, spike–recovery in matrix) with the lot number for traceability.

What is specific here:

• Storage at 2–8 °C and shipment on wet ice supports preservation of antibody activity and enzyme conjugates during transit and storage, consistent with BioReagent-grade handling.

Reaction & Applications

Not applicable in the classical synthetic-chemistry sense. This product is an immunoassay kit intended for quantitative measurement of melatonin in research samples (research use only). No chemical transformations are performed as an end in themselves.

Relevant analytical application (general guidance):

• ELISA format for small molecules like melatonin is typically competitive. Sample melatonin competes with a labeled melatonin tracer for a limited number of antibody binding sites. Signal is inversely proportional to analyte concentration.
• Enzyme reporter systems commonly employ HRP/TMB or alkaline phosphatase/pNPP substrates; the specific system for this kit is not specified; refer to the kit insert.
• Use cases (research): circadian biology studies, endocrine signaling investigations, environmental/food research involving melatonin content. Avoid any clinical/diagnostic interpretation unless specifically validated and authorized, which is not claimed for this item.

Practical tips (general):

• Adhere strictly to incubation times and temperatures; competitive assays are sensitive to deviations.
• Prepare fresh standard curves for each run; include blanks and quality controls.
• Protect melatonin-containing standards/samples from strong light; indole derivatives may photodegrade.

Reaction Conditions

Not applicable in the context of chemical synthesis. However, controlled assay conditions are crucial for ELISA performance.

Assay condition guidance (general; not item-specific):

• Temperature: perform incubations at room temperature (20–25 °C) or as directed by the kit insert; maintain consistency across plates/runs.
• Timing: competitive ELISAs require strictly timed incubations for standards and samples to ensure comparability. Start timing immediately after adding reagents to the first wells; use multichannel pipettes.
• Mixing: gentle agitation during incubations can improve equilibrium; avoid splashing/cross-contamination.
• Wash cycles: typically 3–5 washes per step with adequate soak time; ensure complete removal of residual wash buffer.
• Detection: follow the specified substrate incubation time, then stop the reaction uniformly across the plate before reading absorbance at the recommended wavelength(s). The exact enzyme/substrate and wavelengths are not specified for this item; refer to the kit insert.
• Calibration/fit: generate a full standard curve for each plate; competitive ELISAs are commonly fit with 4PL/5PL logistic models.

Safety & Handling

Item-specific hazard classification (GHS, pictograms, H-statements) is not specified for this item; refer to the SDS for the Melatonin (MT) ELISA Kit and for each included component.

General laboratory safety guidance (professional best practice):

• PPE: wear lab coat, safety glasses, and appropriate chemical-resistant gloves. Avoid skin/eye contact and ingestion/inhalation of reagents and standards.
• Engineering controls: perform reagent preparation and sample handling in a clean area; use a biosafety cabinet if processing potentially biohazardous samples (e.g., serum, plasma).
• Incompatibilities: avoid mixing assay buffers with oxidizing agents or strong acids/bases unless specified. Do not use organic solvents in the assay unless the kit protocol permits (may disrupt antigen–antibody binding).
• Preservatives: ELISA buffers may contain biocides (e.g., isothiazolinones) or detergents; handle accordingly and dispose of waste per institutional policy (details not specified for this item).
• Light sensitivity: small indole derivatives like melatonin can be light sensitive; protect standards and samples from intense light to prevent degradation (general guidance).
• First aid (overview; defer to SDS): rinse skin/eyes with water upon contact, remove contaminated clothing, and seek medical attention if irritation persists; if inhaled/ingested, move to fresh air and follow institutional incident procedures.

Waste: collect assay waste, including stop solutions and sample residues, according to local regulations and institutional EHS guidance.

Solvent Selection

Applicability to this product: The Melatonin (MT) ELISA Kit is an aqueous immunoassay system; you should use only the buffers and diluents provided with the kit or explicitly recommended in the kit insert. Organic solvent selection for synthetic chemistry is not applicable.

General guidance (ELISA context):

• Polarity: Assay buffers are water-based, often PBS or TBS with surfactant (e.g., 0.05% Tween 20) to minimize nonspecific binding.
• Sample compatibility: If samples are extracted using organic solvents (e.g., for melatonin from biological matrices), ensure complete evaporation and reconstitution into the assay buffer, or follow any kit-specific extraction/dilution instructions. Residual organic solvent can disrupt antibody–antigen interactions and enzyme activity.
• pH: Buffers typically near neutral pH for binding/washing; stop solutions (e.g., 1–2 N H2SO4) are acidic for endpoint development in HRP/TMB systems. Use only if specified by the kit documentation (not specified for this item).

When to choose alternatives: If matrix effects persist, consider additional dilution in assay buffer, matrix-matched calibration, or validated extraction protocols rather than changing solvents, as non-specified solvents may invalidate results.

Storage & Reconstitution

From Product Data (item-specific):

• Storage conditions: Store at 2–8 °C.
• Shipped in: Wet ice.

Item-specific details not provided: Component list, lyophilized vs liquid status, reconstitution volumes, preservative content, working stability, and on-board stability are not specified for this item; refer to the kit insert/CoA.

General guidance for ELISA kits (informational):

• Upon receipt: Verify contents against the packing list; record the lot number and expiration date. Immediately store all components at 2–8 °C unless the insert specifies freezing for select components.
• Reconstitution: If standards or controls are lyophilized, equilibrate to room temperature in a desiccator, then reconstitute with the exact volume of the designated diluent. Mix gently (do not vortex vigorously). Allow complete dissolution before use.
• Aliquoting: Prepare single-use aliquots of reconstituted standards/controls to avoid repeated freeze–thaw; store as directed in the kit insert (conditions not specified here).
• Working solutions: Prepare wash buffer, substrate, and stop solution on the day of use as instructed. Do not mix components from different lots.
• Stability: Follow the kit’s stated on-board (room temperature) and post-reconstitution stability limits to maintain performance; not specified for this item—consult the kit documentation.

Structure & Identity

Item-specific identity: This listing is for an immunoassay kit (Melatonin (MT) ELISA Kit), not a single chemical substance. As such, no single CAS, SMILES, or InChIKey applies to the kit as a whole.

From Product Data (item-specific):

• SKU: EJ1515330
• Product name: Melatonin (MT) ELISA Kit
• Grade: BioReagent
• Storage: 2–8°C
• Shipped on: Wet ice
• Research use: For research use only

Analyte reference (literature; for context only, not the kit’s identity):

• Target analyte: Melatonin (N-acetyl-5-methoxytryptamine)
• Molecular formula: C13H16N2O2 (literature)
• Molecular weight: 232.28 g/mol (literature)
• SMILES: COc1cc2[nH]ccc2cc1C(=O)NCC (literature; common depiction for melatonin)
• InChIKey: DRLFMBDRBRZALE-UHFFFAOYSA-N (literature)

Structural features of melatonin (literature):

• Indole ring (benzene fused to pyrrole) bearing a 5-methoxy substituent.
• Terminal acetamide side chain at C3 of the indole (an amide: weakly basic/neutral under physiological conditions).
• No stereocenters; planar indole core with rotatable amide/alkyl bonds.

2D description (literature):

• A bicyclic indole scaffold with a methoxy group para to the indolic nitrogen (on the benzene portion) and an acetamide side chain at the 3-position of the indole. The amide carbonyl is conjugated with the side chain; the indole N is weakly acidic and typically non-ionized in neutral water.

Synthetic Utility

Not applicable. This product is an ELISA kit designed for analytical quantification of melatonin in research samples, not a synthetic building block or reagent.

Informational note (general chemistry):

• While melatonin itself is an indole derivative, the kit does not provide reagents intended for chemical synthesis or transformation. Do not repurpose kit components (e.g., antibodies, enzyme conjugates, buffers) for synthetic reactions.

Target Specificity

Only item-specific information may be listed here. The detailed antigen/epitope, antibody clone/isotype, tracer identity, and species cross-reactivity are not specified for this item; refer to the kit insert or CoA/Spec Sheet.

From product naming (informational):

• Intended analyte: melatonin (MT), based on the product name. No further specificity claims (e.g., cross-reactivity with related indoleamines) are provided here.