Sirolimus (Sirolimus) ELISA Kit

Cat. No.: EJ1515331

GRADE & PURITY BioReagent

Overview

This kit employs the competitive enzyme-linked immunosorbent assay (ELISA) principle. Microplate wells are pre-coated with Sirolimus 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 Sirolimus 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

cell culture supernatants, Cell Lysate, Plasma, Serum, tissue homogenates

Detection Range

3.12ng/mL~200ng/mL

Specificity

Detecting Sirolimus 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 facts

• Tested applications, recommended dilutions, plate format, and incubation schedules are Not specified for this item; refer to the kit insert.

General protocol outline for a competitive ELISA (not item-specific)

1. Equilibrate all components to room temperature. Prepare standards by serial dilution in the recommended diluent.
2. Add standards, controls, and samples to wells according to plate map. Add enzyme conjugate and/or antibody as specified by the format.
3. Incubate for the prescribed time and temperature to reach competitive binding equilibrium. Avoid edge effects by uniform handling.
4. Wash wells thoroughly (e.g., 3–5 times) to remove unbound material.
5. Add substrate (e.g., TMB for HRP systems); incubate until adequate color develops while monitoring timing across the plate.
6. Add stop solution if required and read absorbance at the recommended wavelength (commonly 450 nm) within the specified time.
7. Generate a standard curve (4PL recommended) and interpolate sample concentrations; apply dilution factors.

Practical tips (general)

• Run duplicates or triplicates for all standards and samples.
• Include matrix-matched QCs to monitor assay performance.
• Avoid strong organic solvents and sodium azide in contact with HRP conjugates.
• Validate intra-/inter-assay CV% within acceptable research limits.

Note

• For exact reagent volumes, incubation times, and plate configuration, consult the kit’s insert; these details are not provided in this listing.

Biological Roles

Scope note

• The information below describes the analyte, sirolimus (rapamycin), in a biochemical research context. It does not imply any diagnostic or therapeutic use of this kit.

General/literature knowledge about sirolimus

• Biochemical function: Binds FKBP12 to form a complex that inhibits mTOR complex 1 (mTORC1), modulating protein synthesis, autophagy, and cellular metabolism.
• Pathway context: Impacts PI3K/AKT/mTOR signaling; influences translational control via S6K and 4E-BP1, and can alter lipid and nucleotide biosynthesis pathways.
• Physicochemical behavior in biology: Highly lipophilic and extensively protein-bound; partitions into cellular membranes; may associate with lipoproteins in blood.
• Metabolism (general): Extensively metabolized by CYP3A isoforms; subject to transporter interactions (e.g., P-glycoprotein) in cellular systems (context for research matrices).

Relevance for assay design

• Matrix effects: Protein binding and lipoprotein association can affect free vs. total concentrations in serum/plasma, influencing recovery.
• Selectivity concerns: Structural analogs or metabolites can exhibit cross-reactivity depending on antibody design; kit-specific data should be reviewed in the insert.

Item-specific note

• This ELISA kit is intended for research quantification of sirolimus; detailed selectivity/specificity metrics are Not specified for this item; refer to CoA/Spec Sheet.

Buffer Applications

Applicability to this product

• ELISA performance is tightly coupled to buffer composition. While this kit’s exact buffers are unspecified, the following reflects standard practice for competitive small-molecule ELISA.

General/literature guidance

• Coating buffer: Carbonate–bicarbonate, pH ~9.4–9.6 (for plate coating steps when applicable). Not item-specific.
• Blocking buffer: Protein-based (e.g., BSA, casein) in PBS or TBS; minimizes nonspecific adsorption. Composition should be compatible with small-molecule hapten conjugates.
• Sample/assay diluent: Near-neutral pH (PBS or TBS, pH 7.2–7.6) with carrier protein and detergent (e.g., 0.05% Tween 20) to stabilize proteins and reduce nonspecific binding.
• Wash buffer: PBS/TBS with 0.05% Tween 20; rigorous washing between steps to lower background.
• Substrate buffer: If HRP/TMB is used, maintain low metal ion content and protect from light; stop with dilute acid when signaled in protocol.

Practical tips

• pH and ionic strength affect antibody–hapten binding; keep them consistent across standards and samples.
• Matrix matching: Prepare standards in a matrix-matched diluent to minimize matrix effects.
• Preservatives: Sodium azide can inhibit HRP; avoid azide in buffers contacting HRP conjugates unless validated.

Item-specific note

• Exact buffer recipes, pH, and preservatives for this kit are Not specified for this item; refer to kit insert/CoA.

Green Alternatives

Context

• ELISA is inherently resource-efficient compared to many analytical methods, requiring microliter-scale reagents and generating relatively low chemical waste. Still, greener choices can further reduce environmental impact.

Greener practice suggestions (general)

• Minimize single-use plastics by selecting recyclable plate seals and consolidating wash steps when allowed by the protocol.
• Prepare only the volumes needed of working solutions (substrate, conjugate) to reduce discard.
• Prefer aqueous buffers and avoid unnecessary organic solvents in sample prep; if extraction is needed, choose lower-toxicity solvents and recover by evaporation with proper capture.
• Manage hazardous constituents: Many wash buffers contain preservatives (e.g., ProClin, sodium azide); segregate waste from bleach to prevent azide-related hazards.
• Energy use: Store at 2–8°C as specified; avoid repeated door openings and batch assays to reduce cold-chain burden.

Comparison (general)

• ELISA vs. LC–MS for routine screening: ELISA offers lower solvent consumption and simpler waste streams but may have cross-reactivity. LC–MS provides higher specificity with higher solvent use and energy footprint. Selection should balance environmental impact with analytical requirements.

Item-specific data

• Specific component compositions and preservatives are Not specified for this item; refer to CoA/Spec Sheet.

Pharmaceutical Uses

Scope and limitations

• This product is for research use only and not for diagnostic, clinical, or therapeutic applications.

Research/formulation contexts where such a kit may be used (general)

• Preclinical PK/PD research: Quantitative monitoring of sirolimus exposure in non-clinical studies to support mechanism or formulation screening.
• Formulation R&D: Comparative assessments of release profiles or stability indicating changes in sirolimus concentration in in vitro systems.
• Manufacturing research support: Process or stability studies in development labs to track analyte levels in model matrices.

Notes on analytical strategy (general)

• ELISA can offer high throughput and low cost per sample for screening; consider orthogonal confirmation (e.g., LC–MS) when structural specificity is critical.
• Establish matrix-specific validation (accuracy, precision, selectivity, dilution linearity, stability) per your organization’s research SOPs; these are not provided by this listing.

Item-specific data

• Any claims of compliance with pharmacopeial methods, clinical validation, or diagnostic performance are not applicable. Specific performance characteristics are Not specified for this item; refer to CoA/Spec Sheet.

Physical Properties

Item-specific facts (from Product Data)

• The product is a multi-component ELISA kit. Physical constants (BP, MP, density, refractive index, logP, solubility) are not applicable to the kit as a whole.
• Appearance of individual components: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values (for context on the analyte, sirolimus)

• Typical properties (literature): Hydrophobic macrolide; sparingly soluble in water; soluble in organic solvents (e.g., ethanol, methanol, DMSO). Exact numerical values vary by source and are not specifications of this kit.

Practical notes for users (general)

• Assay matrices: Common research matrices include serum, plasma, tissue homogenate, or cell culture supernatant; matrix composition can influence recovery and apparent values via protein binding and interference.
• Buffer considerations: ELISA buffers are typically aqueous, near-neutral pH; detergents and organic solvents above low percentages may disrupt binding interactions in competitive assays.

Reminder

• Do not treat literature analyte properties as item specs. For kit component concentrations, preservatives, or buffer compositions, consult the kit insert and CoA/Spec Sheet.

Quality & Grades

Item-specific facts (from Product Data)

• Grade: BioReagent (from Product Data). This denotes suitability for laboratory research workflows and biochemical assays.

What BioReagent grade implies (general)

• Intended use: Research use only; not for diagnostic or clinical use. Components are prepared for reproducibility in common lab assays.
• Typical controls in ELISA kits: Calibrators/standards, quality controls, coated plate or capture reagents, enzyme conjugate, substrate, stop solution, sample/assay buffers, and wash concentrate. Exact inclusions, formulations, and concentrations are lot-specific and not provided here.

Analytical performance considerations (general; not item-specific specs)

• Key metrics often reported: Sensitivity (LOD/LOQ), dynamic range, accuracy/recovery, linearity on dilution, matrix compatibility, and precision (intra-/inter-assay CV%).
• Cross-reactivity: Competitive small-molecule ELISAs require careful characterization of antibody cross-reactivity to structural analogs and metabolites; consult the kit insert for specific data.
• Traceability: Calibrators may be traceable to internal reference standards; confirm calibration hierarchy in the CoA.

What is not specified for this item

• Plates, well format, calibrator concentrations, CV targets, LOD/LOQ, and cross-reactivity: Not specified for this item; refer to CoA/Spec Sheet and kit insert.

Reaction & Applications

Applicability to this product

• This section addresses analytical applications rather than chemical reactions, as the product is an ELISA kit.

Research applications (general; expand on manufacturer’s “For research use only” note)

• Quantitative measurement of sirolimus in research samples such as serum, plasma, tissue homogenates, and cell culture supernatants to support pharmacology, ADME, and mechanistic studies in a research setting.
• Method validation in-house: Establish matrix-specific calibration, precision, accuracy, and dilution linearity per institutional SOPs.

Practical assay tips (general)

• Standard curve: Prepare fresh serial dilutions using the provided or recommended diluent; run duplicates or triplicates.
• Plate layout: Include blanks, zero standard, full-range standards, QCs (low/mid/high), and matrix-matched spikes to assess recovery.
• Competitive format nuances: Incubation times/temps critically affect equilibrium; maintain consistent timing across the plate. Avoid high organic content in samples.
• Interferences: Hemolysis, lipemia, and high bilirubin can affect colorimetric readings; use matrix-matched controls and consider sample cleanup where needed.
• Washing: Validate plate washer settings to prevent under- or over-washing; residual wash buffer can dilute reagents, while inadequate washing elevates background.

What remains item-specific

• Exact incubation times, temperatures, well coating chemistry, conjugate identity, substrate type, and stop solution: Not specified for this item; refer to kit insert.

Reaction Conditions

Not directly applicable

• As an immunoassay kit, there are no chemical reaction conditions in the traditional synthetic sense.

General assay conditions for competitive ELISA (literature/practice; not item-specific)

• Temperature: Commonly ambient (20–25°C) incubations; some protocols recommend 37°C for specific steps to accelerate binding—follow the kit insert if provided.
• Timing: Competitive binding steps typically range 30–120 minutes depending on format; substrate development often 10–20 minutes until the target OD is reached.
• Wash cycles: 3–5 cycles between steps, ensuring complete removal of residual liquids.
• Detection: Colorimetric (e.g., HRP/TMB) read at 450 nm with reference 570–620 nm, or alternative enzymatic systems.
• Calibration: 6–8 standards spanning the dynamic range; duplicates or triplicates advised.

Item-specific parameters

• Incubation times, temperatures, wash volumes, substrate identity, and stopping conditions are Not specified for this item; refer to the kit insert for exact instructions.

Safety & Handling

Item-specific facts (from Product Data)

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to the product SDS.
• Storage conditions: Store at 2–8°C (from Product Data).
• Shipping: Shipped on wet ice (from Product Data).

General safety guidance for ELISA kits (literature/practice)

• PPE: Wear lab coat, gloves (nitrile), and safety glasses. Avoid skin/eye contact with buffers, standards, and enzyme conjugates.
• Incompatibilities: Avoid mixing oxidizers with HRP-containing reagents; keep acids/bases and sodium azide away from bleach-containing waste to prevent hazardous gas formation.
• Peroxidase substrates: If the kit uses HRP/TMB, protect substrate from light and metal contamination; avoid contact with strong acids (TMB can form colored cations).
• Biological samples: Treat all human/animal specimens as potentially biohazardous; follow BSL-2 practices where appropriate.
• Spill/first aid (overview): Rinse affected skin/eyes with water for ≥15 minutes; remove contaminated clothing. If inhaled or ingested, seek medical attention and show SDS.
• Waste: Collect HRP/TMB residues and azide-preserved buffers for compatible chemical waste streams; decontaminate biohazardous liquids by validated methods (e.g., chemical or autoclave) per institutional policy.

Authoritative source

• For exact hazards, composition, and first-aid measures of this specific kit lot, defer to the manufacturer’s SDS and kit insert.

Solvent Selection

Applicability to this product

• This is an aqueous immunoassay kit; organic solvent selection is generally not applicable. Use provided buffers or those recommended in the kit insert.

General guidance for small-molecule ELISA matrices (literature/practice)

• Sample compatibility: Excess organic solvent (e.g., >1–2% v/v DMSO, methanol, acetonitrile) may disrupt antibody–hapten interactions. If extraction is required, evaporate and reconstitute into assay buffer.
• Protein binding: Sirolimus exhibits strong protein binding in biological matrices; dilution into appropriate buffer and, when recommended, sample pretreatment can improve apparent recovery and linearity.
• Detergents/surfactants: Low levels of nonionic detergents (e.g., 0.05% Tween 20) are typical in wash buffers. Avoid introducing unvalidated surfactants into sample or conjugate solutions.

Comparison (general)

• Native matrix vs. extracted: Direct assay of diluted plasma/serum is higher throughput but more prone to matrix effects; SPE or protein precipitation (followed by reconstitution in buffer) can improve specificity but adds steps. Choose based on your study’s tolerance for matrix interference and throughput needs.

Always follow the specific kit instructions

• Use only the diluent and wash solutions specified in the kit insert to maintain performance characteristics.

Storage & Reconstitution

Item-specific facts (from Product Data)

• Storage temperature: Store at 2–8°C.
• Shipping: Shipped on wet ice.

General guidance for ELISA kit components (not item-specific)

• Upon receipt: Inspect for any signs of leakage or damage. Record lot numbers and expiration dates. Store all components at 2–8°C unless the insert specifies freezing for particular items.
• Reconstitution: Some components may arrive concentrated or lyophilized (e.g., standards, wash buffer concentrates). Reconstitute/dilute with the specified volume of deionized water or diluent immediately before use. Exact volumes and solvents are Not specified for this item; refer to the kit insert.
• Handling: Bring reagents to room temperature before use to reduce edge effects. Mix gently; avoid foaming, which can trap proteins and affect volumes.
• Light sensitivity: Protect enzyme substrates and conjugates from light. Do not leave plates or reagents uncovered longer than necessary.
• Stability: Working solutions (e.g., diluted wash buffer, prepared standards) are typically stable for the working day; prepare fresh each run unless the insert states otherwise.
• Freeze–thaw: Avoid repeated freeze–thaw of protein reagents. Aliquot reconstituted standards or controls if freezing is permitted by the insert.

Not specified for this item

• Exact component list, volumes, preservative content, and post-reconstitution stability: Not specified for this item; refer to CoA/Spec Sheet and kit insert.

Compliance note

• For research use only.

Structure & Identity

Item-specific facts (from Product Data)

• Product type: ELISA Kit targeting sirolimus (rapamycin) in research samples
• Grade: BioReagent
• Kit-format identifiers (SMILES, InChIKey, CAS, CID, MF, MW): Not specified for this item; refer to CoA/Spec Sheet.

General/literature context (for the small-molecule analyte “sirolimus” measured by this kit)

• Common name(s): Sirolimus; Rapamycin
• Functional class: Macrolide lactone; small-molecule mTOR pathway modulator (biochemical context)
• Structural features (descriptive, literature): Large 31-membered macrolactone ring with multiple hydroxyls, a hemiketal, conjugated triene segment, and a pipecolate moiety; overall amphipathic with significant hydrophobic surface.
• 2D structure (in words): A macrocyclic polyketide backbone forming a lactone ring; internal ketal/hemiketal; several secondary/tertiary alcohols and a cyclohexyl-derived pipecolate attached via amide-like linkage.

Notes

• This is an immunoassay kit. Structural identifiers above pertain to the analyte (sirolimus) for context only and are not item specifications. Always consult the kit insert and CoA for item-specific identity details (e.g., antibody clone, hapten conjugate chemistry, calibrator identity).

Synthetic Utility

Not typically applicable

• This is an ELISA kit used for analytical quantification of sirolimus in research samples. It is not a chemical building block or reagent for synthesis.

Where relevant background may help (general)

• For researchers synthesizing sirolimus analogs or conjugates for immunogen/hapten work, separate synthetic literature addresses macrolide derivatization and linker installation, but such chemistry is outside the scope of this product.

Item-specific note

• No synthetic transformations or reagent roles are associated with this kit. Details about any conjugate chemistry used on the plate or in the tracer are Not specified for this item; refer to the kit insert if disclosed.

Target Specificity

Item-specific facts (from Product Data)

• Target/analyte: Sirolimus (rapamycin) in research samples.
• Antibody type, clone, host species, isotype, and epitope mapping: Not specified for this item; refer to CoA/Spec Sheet.
• Cross-reactivity profile to related macrolides/metabolites: Not specified for this item; refer to kit insert.

General information (literature/practice)

• Competitive ELISA principle for small molecules: The analyte in the sample competes with a plate-bound hapten (sirolimus analog conjugate) for binding to a limited amount of anti-sirolimus antibody, or vice versa using a labeled tracer. Signal is inversely proportional to analyte concentration.
• Specificity drivers: Antibody raised against a sirolimus–carrier conjugate; selectivity depends on hapten design (exposed epitope), linker position, and immunization strategy.
• Potential interferences: Structurally related macrolides or sirolimus metabolites may show cross-reactivity; serum components (lipoproteins) can alter apparent free concentration.

Recommendation

• Review the kit insert for specificity, cross-reactivity tables, and matrix validation relevant to your study. Without these item-specific data, users should perform spike-and-recovery and dilution linearity in their matrices.