D-Lactate Assay Kit

Catalog No. ABIN1000306

Product Details

Target: D-Lactate

Application: Biochemical Assay (BCA)

Sample Type: Serum, Plasma, Cell Culture Supernatant

Specificity: 0.05 mM

Characteristics: Sensitive and accurate. Detection limit of 0.05 mM and linearity up to 2 mM D-lactate in 96-well plate assay. For cell culture samples containing phenol red: detection limit of 0.1 mM and linearity up to 1 mM D-lactate in 96-well plate assay.
Convenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and at 20 min. Room temperature assay. No 37°C heater is needed.
High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Components: Assay Buffer: 10 mL. NAD Solution: 1 mL. Enzyme A: 120 µL. MTT Solution: 1.5 mL. Enzyme B: 120 µL. Standard: 1.0 mL 20 mM D-lactate.

Material not included: Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.

Application Details

Application Notes: Direct Assays: D-lactate in serum, plasma, and cell media samples.

Comment:

The following substances interfere and should be avoided in sample preparation: EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).

Protocol: Important: this assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended.
1. Standard Curve. Prepare 1000 µL 2.0 mM D-lactate Premix by mixing 100 µL 20 mM Standard and 900 µL distilled water. For cell culture samples containing phenol red, prepare 1000 µL 1.0 mM lactate Premix by mixing 50 µL 20 mM Standard and 950 µL culture medium without serum. Transfer 20 µL standards into wells of a clear bottom 96-well plate. Samples: Add 20 µL sample per well in separate wells. For samples with potential endogenous enzyme activity (i.e. serum, plasma, tissue extracts), two reactions should be run: one with added Enzyme A and a No Enzyme A control. Serum and Plasma should be diluted at least 2° with dH2O prior to assay.
2. Reagent Preparation. Spin the Enzyme tubes briefly before pipetting. For each Sample and Standard well, prepare Working Reagent by mixing 60 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 10 µL NAD and 14 µL MTT. Fresh reconstitution is recommended. For the No Enzyme A control, the Working Reagent includes 60 µL Assay Buffer, 1 µL Enzyme B, 10 µL NAD and 14 µL MTT.
3. Reaction. Add 80 µL Working Reagent per reaction well quickly. Tap plate to mix briefly and thoroughly.
4. Read optical density (OD0) for time zero at 565 nm (520-600nm) and OD20 after a 20-min incubation at room temperature.
5. Calculation. Subtract OD0 from OD20 for the standard and sample wells. Use the OD values to determine the sample D-lactate concentration from the standard curve. For samples requiring a No Enzyme A control, subtract the ODNoEnz value from the ODSample and use this OD value to determine the sample D-lactate concentration from the standard curve. Note: if the sample OD value is higher than OD for 2 mM D-lactate standard, dilute sample in water and repeat the assay. Multiply the results by the dilution factor.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Raykov, Grekova, Bour, Lehn, Giese, Nicolau, Aprahamian: "Myo-inositol trispyrophosphate-mediated hypoxia reversion controls pancreatic cancer in rodents and enhances gemcitabine efficacy." in: International journal of cancer. Journal international du cancer, Vol. 134, Issue 11, pp. 2572-82, (2014) (PubMed).

Guzmán-de la Garza, Ibarra-Hernández, Cordero-Pérez, Villegas-Quintero, Villarreal-Ovalle, Torres-González, Oliva-Sosa, Alarcón-Galván, Fernández-Garza, Muñoz-Espinosa, Cámara-Lemarroy, Carrillo-Arriaga: "Temporal relationship of serum markers and tissue damage during acute intestinal ischemia/reperfusion." in: Clinics (São Paulo, Brazil), Vol. 68, Issue 7, pp. 1034-8, (2013) (PubMed).

Witkin, Mendes-Soares, Linhares, Jayaram, Ledger, Forney: "Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections." in: mBio, Vol. 4, Issue 4, (2013) (PubMed).

Gigante, Sardo, Gasperini, Molinaro, Riggio, Laviano, Amoroso: "D-Lactic acidosis 25 years after bariatric surgery due to Salmonella enteritidis." in: Nutrition (Burbank, Los Angeles County, Calif.), Vol. 28, Issue 1, pp. 108-11, (2011) (PubMed).

Kurundkar, Killingsworth, McIlwain, Timpa, Hartman, He, Karnatak, Neel, Clancy, Anantharamaiah, Maheshwari: "Extracorporeal membrane oxygenation causes loss of intestinal epithelial barrier in the newborn piglet." in: Pediatric research, Vol. 68, Issue 2, pp. 128-33, (2010) (PubMed).

Target Details

Target: D-Lactate

Background: Quantitative determination of D-lactic acid by colorimetric (565nm) method.
Procedure: 20 min.

Lactate is generated by lactate dehydrogenase (LDH) under hypoxic or anaerobic conditions. Monitoring lactate levels is, therefore, a good indicator of the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. D-lactate is produced in only minor quantities in animals and measuring for D- lactate in animal samples is a means to determine the presence of bacterial infection. Simple, direct and automation-ready procedures for measuring lactate concentration are very desirable. This lactate assay kit is based on lactate dehydrogenase catalyzed oxidation of lactate, in which the formed NADH reduces a formazan (MTT) Reagent. The intensity of the product color, measured at 565 nm, is proportionate to the lactate concentration in the sample.