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| Product Name | Alanine Aminotransferase (ALT/GPT) Activity Colorimetric Assay Reagent |
| Catalog No. | ETR-HMM-0061 |
| Description | A colorimetric enzymatic reagent set for measuring alanine aminotransferase activity in serum, plasma, and cell lysates. ALT catalyzes the transfer of an amino group from alanine to alpha-ketoglutarate, producing pyruvate; in the coupled indicator reaction, pyruvate is reduced by NADH via lactate dehydrogenase, allowing kinetic measurement of NADH consumption at 340 nm. |
| Intended Use | Quantitative ALT activity measurement as a primary serum biomarker for liver cell damage and hepatotoxicity in clinical research, drug safety assessment, preclinical toxicology studies, and hepatocyte culture viability monitoring. |
| Principle / Technology | ALT (alanine aminotransferase, EC 2.6.1.2) catalyzes the reversible transamination: L-Alanine + α-Ketoglutarate ⇌ Pyruvate + L-Glutamate; the pyruvate produced is reduced to lactate by coupled lactate dehydrogenase (LDH) with simultaneous oxidation of NADH to NAD+; the resulting decrease in absorbance at 340 nm (kinetic rate of NADH consumption) is directly proportional to ALT activity. |
| Detection Method | UV absorbance kinetic measurement at 340 nm using a spectrophotometer or UV-capable microplate reader |
| Sample Type | Human and animal serum and plasma, culture medium conditioned by hepatocytes, liver tissue homogenates, liver-derived cell line lysates (HepG2, primary human hepatocytes) |
| Performance Range / Specifications | ALT activity range: 1–1,000 U/L with standard dilution; typical serum reference range 7–56 U/L; one unit defined as the amount oxidizing 1 μmol NADH per minute at 37°C; linear kinetic rate measurable over 2–5 minutes |
| Sensitivity / LOD | Lower detection limit: approximately 1 U/L ALT activity in diluted serum (1:10 dilution) with standard sensitivity UV plate reader |
| Specificity | LDH-coupled assay is specific for pyruvate generated by the ALT reaction; pyridoxal-5'-phosphate (P-5'-P) is included as cofactor to activate apoenzyme forms of ALT in stored sera; LDH co-substrate NADH concentration is non-rate-limiting to ensure ALT activity is the measured limiting factor |
| Reaction Conditions / Protocol | Equilibrate Reagent 1 (Tris buffer, alanine substrate, alpha-ketoglutarate, LDH, P-5'-P) to 37°C for 5 minutes; add 10 μL serum sample; add Reagent 2 (NADH in Tris buffer) to initiate reaction; allow 1 minute pre-incubation at 37°C for blank stabilization; begin kinetic readings at 340 nm every 30 seconds for 2–3 minutes; calculate mean ΔA340/min from the linear portion; multiply by the conversion factor from kit instructions to obtain ALT activity in U/L |
| Components / Formulation | Reagent 1 (Tris-HCl buffer pH 7.5, L-alanine 500 mM, α-ketoglutarate 15 mM, LDH ≥1,200 U/L, pyridoxal phosphate), Reagent 2 (NADH 0.18 mM in Tris buffer), ALT Calibrator (lyophilized human serum traceable reference), reconstitution buffer |
| Storage Conditions | Reagent 1 and Reagent 2 at 2–8°C protected from light; NADH-containing reagent sensitive to light and oxidation; ALT calibrator lyophilized at -20°C; reconstituted calibrator aliquoted at -20°C stable 4 weeks |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 assays, 500 assays, 1,000 assays (cuvette or 96-well format) |
| Product Form | Liquid IFCC-formulated reagents with lyophilized reference calibrator |
| Quality Control | Each lot validated against certified ALT calibrators; within-run CV ≤2%; between-run CV ≤3%; pyridoxal phosphate activation efficiency confirmed in apoenzyme-enriched sample; NADH reagent stability confirmed at 340 nm blank <0.001 OD/min |
| Key Features | IFCC reference method formulation providing international method harmonization; pyridoxal-5'-phosphate activation ensures complete measurement of total ALT including apo-form; validated for both serum and hepatocyte culture monitoring applications; ready-to-use liquid format without lyophilized reagent reconstitution |
For research use only, not for clinical use.
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