25-OH Vitamin D3, HRP Conjugated – Catalog #B2026710

25-OH Vitamin D3, HRP Conjugated (Catalog #B2026710) combines 25-hydroxyvitamin D3 (calcifediol), the primary circulating form of vitamin D produced by hepatic 25-hydroxylation of vitamin D3, with horseradish peroxidase (HRP). Supplied as 100 μg in solution, this conjugate is used in enzyme immunoassays and chemiluminescence assays to measure serum 25-hydroxyvitamin D concentration, the standard biomarker of vitamin D status.

Scientific Overview

Vitamin D metabolism begins with ergocalciferol (vitamin D2) or cholecalciferol (vitamin D3) ingestion or skin synthesis. In the liver, both forms are hydroxylated by 25-hydroxylase (CYP2R1) to form 25-hydroxyvitamin D [25(OH)D or calcifediol], which circulates at the highest concentration (typically 20–100 ng/mL). This 25-hydroxy metabolite is the best indicator of total body vitamin D stores and is the standard clinical marker of vitamin D status. Further hydroxylation by 1α-hydroxylase in the kidney produces 1,25-dihydroxyvitamin D, the most biologically active form, which regulates calcium homeostasis and immune function.

Horseradish peroxidase is a widely used enzyme label in immunoassays, catalyzing the oxidation of chromogenic or chemiluminescent substrates to produce colored or luminescent products proportional to analyte concentration. HRP conjugation to 25-OH D3 enables sensitive detection in competitive immunoassays where the conjugate competes with sample 25-OH D3 for antibody binding.

Key applications include:

• Enzyme immunoassay (EIA) and ELISA for 25-hydroxyvitamin D measurement
• Chemiluminescence immunoassay for high-sensitivity vitamin D quantification
• Development and optimization of vitamin D diagnostic assays
• Research on vitamin D metabolism and physiological roles

Usage & Handling Guidance

Store at -20°C and protect from light. Before use, allow the vial to come to room temperature and mix gently. Aliquot to avoid repeated freeze-thaw cycles.

• Optimization: Titrate the conjugate concentration in your assay to achieve optimal signal-to-background ratio before running samples.
• Substrate selection: Choose HRP substrates appropriate for your detection method (colorimetric, chemiluminescent, or electrochemical).
• Buffer compatibility: Use assay buffers free of strong reducing agents that may inactivate the HRP enzyme.

What You Get

• 100 μg of 25-OH Vitamin D3 conjugated to horseradish peroxidase, supplied in solution
• A ready-to-use enzyme immunoassay tracer for vitamin D quantification
• For research use only (RUO)

Why Researchers Choose It

• Specific for 25-hydroxyvitamin D, the clinically relevant vitamin D biomarker
• HRP enzyme label enables sensitive detection by multiple methods
• Suitable for developing competitive immunoassays
• Well-characterized substrate chemistry with established kinetics

Frequently Asked Questions (FAQ)

• What does this conjugate measure?
  It is used to quantify 25-hydroxyvitamin D, the primary circulating form of vitamin D and the standard biomarker for vitamin D status.
• What HRP substrates can I use?
  Any HRP substrate, including TMB (3,3',5,5'-tetramethylbenzidine), OPD (o-phenylenediamine), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and chemiluminescent substrates.
• Is this suitable for clinical diagnostics?
  This product is for research use only (RUO). Clinical assays require validation and regulatory approval; consult available documentation on the lot.
• How should I optimize my assay?
  Titrate the conjugate concentration and antibody concentration to maximize signal-to-background ratio using positive and negative controls.
• Can I get a COA or TDS?
  Yes. Request a quote or contact us and we will provide available lot documentation.

References

• Han S, Qiu W, Zhang J, Bai Z, Tong X. Development of a Chemiluminescence Immunoassay for Quantification of 25-Hydroxyvitamin D in Human Serum. J Anal Methods Chem. 2020;2020:9039270.
• Shrivastav TG. Preparation of horseradish peroxidase hydrazide and its use in immunoassay. J Immunoassay Immunochem. 2003;24(3):301-9.
• Lin AV. Direct ELISA. Methods Mol Biol. 2015;1318:61-7.