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| Product Name | Urease Activity Detection Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0072 |
| Description | A colorimetric assay for quantifying urease enzyme activity in biological samples using urea as the natural substrate. Urease cleaves urea to ammonia and carbon dioxide; the ammonia produced reacts with the Berthelot reagent (phenol-hypochlorite) to form a blue indophenol chromophore measured at 630 nm. |
| Intended Use | Measurement of urease activity in H. pylori bacterial cultures and biopsies for ulcer disease research, soil microbiology, measurement of urease contamination in industrial biological processes, and assessment of urease inhibitor efficacy for drug development. |
| Principle / Technology | Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea: (NH2)2CO + H2O → 2 NH3 + CO2; the ammonia generated reacts with sodium salicylate and sodium hypochlorite (Berthelot reagent) in the presence of sodium nitroprusside (catalyst) at alkaline pH to form indophenol blue, with maximum absorbance at 630 nm; the reaction is highly sensitive because each mole of urea generates two moles of ammonia, both contributing to the colorimetric signal. |
| Detection Method | Colorimetric endpoint absorbance at 630 nm using a microplate reader or standard spectrophotometer |
| Sample Type | Bacterial lysates and culture supernatants (H. pylori, Proteus mirabilis, other urease-producing organisms), soil and plant tissue extracts, purified recombinant or native jack bean urease, gastric biopsy homogenates |
| Performance Range / Specifications | Ammonia standard curve: 1–100 nmol per well; urease activity range: 0.001–100 U/mL; one unit defined as amount hydrolyzing 1 μmol urea per minute at 25°C pH 7.0; incubation time 15–60 minutes at 37°C |
| Sensitivity / LOD | Detection limit: approximately 1 nmol ammonia per well, equivalent to urease activity of approximately 0.01 mU/mL in 50 μL sample input |
| Specificity | The Berthelot reaction for ammonia detection is highly sensitive and selective; urea itself does not produce color; ammonium ions from other nitrogen sources can contribute to background and should be assessed with a no-substrate control; acetohydroxamic acid (AHA) at 50 μM specifically inhibits urease and serves as a negative control to verify urease-specific signal |
| Reaction Conditions / Protocol | Add 50 μL sample or urea-free negative control to microplate; initiate reaction by adding 50 μL Urea Substrate Solution (400 mM urea in phosphate buffer pH 7.0); incubate at 37°C for 15–60 minutes; stop reaction with 50 μL Stop Reagent (containing NaOH to halt enzyme activity); add 100 μL Berthelot Reagent A (sodium salicylate, sodium nitroprusside) and 50 μL Berthelot Reagent B (sodium hypochlorite solution); incubate 15 minutes at 37°C protected from light; measure absorbance at 630 nm against ammonia standard curve |
| Components / Formulation | Urea Substrate Solution (400 mM urea in phosphate buffer pH 7.0), Berthelot Reagent A (sodium salicylate, sodium nitroprusside in NaOH), Berthelot Reagent B (sodium hypochlorite, NaOH), Ammonium Chloride Standard (for NH3 calibration curve), Stop Reagent, Urease Positive Control (jack bean urease, lyophilized, type VI), AHA inhibitor control solution |
| Storage Conditions | All liquid reagents at 2–8°C protected from light; Berthelot reagents sensitive to light and should be stored opaque; jack bean urease at -20°C in single-use aliquots; stable 12 months |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 assays, 500 assays (96-well format) |
| Product Form | Liquid Berthelot reagent components with lyophilized urease standard and ammonia calibration set |
| Quality Control | Each lot validated with ammonia standard curve (R2 ≥0.999); jack bean urease positive control activity within ±15% of nominal value; AHA inhibition ≥98% of positive control urease signal; Berthelot reagent blank <0.02 OD at 630 nm |
| Key Features | Sensitive Berthelot reaction approach provides high-throughput detection capability; AHA inhibitor specificity control distinguishes urease from other ammonia-producing enzymes; compatible with complex biological matrices including tissue homogenates and gastric biopsies; ammonia standard curve ensures accurate absolute quantification |
For research use only, not for clinical use.
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