Homocysteine Assay Kit (Fluorometric) (ab228559)
Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Sample type: Plasma, Serum
- Sensitivity: 5 µM
Overview
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Product name
Homocysteine Assay Kit (Fluorometric) -
Detection method
Fluorescent -
Sample type
Serum, Plasma -
Assay type
Quantitative -
Sensitivity
5 µM -
Product overview
Homocysteine Assay Kit (Fluorometric) (ab228559) allows for quantification of total homocysteine in biological fluids such as plasma and serum. The assay is based on the reduction of homocysteine disulfides to free homocysteine, which is cleaved by a homocysteine-selective enzyme, generating an intermediate product. The intermediate reacts with a probe solution to form a stable fluorophore that emits in the far-red spectrum (Ex/Em = 658/708 nm). The assay is not affected by physiological concentrations of other biological thiols (such as cysteine, methionine and glutathione), is high-throughput adaptable and can detect as low as 5 μM homocysteine.
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Notes
Homocysteine is a non-proteogenic amino acid synthesized intracellularly by removal of the N-methyl group from the essential amino acid methionine. Homocysteine is exported from cells into the blood, where it exists mainly as an oxidized disulfide species, either as a dimer or bound to cysteine residues of serum proteins. The reduced form of homocysteine (‘free’ homocysteine) can be metabolized into cysteine via the transsulfuration pathway; however, it can also undergo intramolecular cyclization, forming the highly reactive pro-oxidant homocysteine thiolactone. Subsequent N-homocysteinylation of protein lysine residues by the reactive thiolactone disrupts protein conformation, leading to formation of cytotoxic protein aggregates. Homocysteinylated proteins may also act as autoantigens, triggering arterial inflammation and atherosclerosis. Elevated plasma homocysteine concentration is a clinical biomarker for increased risk of cardiovascular disease, ischemic stroke and myocardial infarction. Severely elevated homocysteine levels (hyperhomocysteinemia) are correlated with a 4-fold increase in mortality due to heart attack and a 16-fold increase in the likelihood of recurrent stroke.
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Platform
Microplate reader
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Developer Solution 1 x 5ml Disulfide Reducing Agent (DTT) 1 x 300µl Fluorogenic Probe Solution 1 x 5ml Homocysteine Assay Buffer 1 x 25ml Homocysteine Disulfide Standard 1 vial Homocysteine Enzyme Mix 1 vial -
Research areas
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Relevance
Homocysteine is a chemical compound with the formula HSCH2CH2CH(NH2)CO2H. It is a homologue of the naturally-occurring amino acid cysteine, differing in that its side-chain contains an additional methylene (-CH2-) group before the thiol (-SH) group. Alternatively, Homocysteine can be derived from methionine by removing the latter's terminal C methyl group. Elevations of Homocysteine also occur in the rare hereditary disease homocystinuria and in methylene-tetrahydrofolate-reductase deficiency. The latter is quite common and usually goes unnoticed, although there are reports that thrombosis and cardiovascular disease occurs more often in people with elevated Homocysteine. -
Alternative names
- (2S) 2 amino 4 sulfanyl butanoic acid
Images
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Typical Homocysteine standard curve and assay data
(a) Homocysteine standard curve. (b) Specificity for detection of Homocysteine (HCY) over other thiols. At an 8-fold molar excess versus HCY, cysteine (CYS) contributes ≤15% interference, while methionine (MET) and glutathione (GSH) contribute ≤2%. (c) Estimation of total HCY in single-donor human plasma and serum (10 µl), spiked with 50 pmol Homocysteine Disulphide Standard (equivalent to 100 pmol or 10 µM free HCY). Total HCY concentrations for plasma and serum samples were 10.1 ± 0.28 µM and 15.9 ± 0.99 µM, with respective spike recoveries of 95.1% and 103.4%. Data are mean ± SEM of 3 replicates, assayed according to the kit protocol.
