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Cat. No.: TLDAKW8-100 (for 100T)

Description

Total Lactate Dehydrogenase Assay Kit with WST-8, also known as the Total LDH Assay Kit with WST-8, is a colorimetric assay kit based on the WST-8 reaction. It rapidly and sensitively detects total lactate dehydrogenase activity in biological fluids such as serum, plasma, urine, tissues, cells, and tissue or cell culture supernatants. Typically, 0.1-1µl of serum or plasma is sufficient for detection with this kit. It specifically detects NAD+-dependent total lactate dehydrogenase.

Lactate dehydrogenase (LDH) is an enzyme present in most active mammalian cells, widely distributed across various tissues, with the heart, skeletal muscles, and kidneys being the most abundant. It is an important indicator in clinical myocardial enzyme spectrum tests and can aid in diagnosing myocardial diseases. LDH catalyzes the conversion of lactate to pyruvate, accompanied by the transformation of NAD+ to NADH. Since it is often released during tissue injury (typically due to the loss of cell membrane integrity and release of intracellular LDH), it is also considered a biomarker for cell necrosis and related diseases. LDH can be categorized into NAD+-dependent lactate dehydrogenase and NAD+-independent lactate dehydrogenase based on its coenzyme binding. NAD+-dependent LDH is more widely distributed and common in humans, animals, and microorganisms. According to substrate specificity, LDH can be classified into D-lactate dehydrogenase (D-LDH) and L-lactate dehydrogenase (L-LDH).

D-lactate dehydrogenase catalyzes the interconversion of D-lactate and pyruvate. D-LDH is an important enzyme in the methylglyoxal (MGO) pathway, responsible for converting the end product D-lactate into pyruvate. Excessive accumulation of D-lactate can lead to D-lactic acidosis or D-lactate encephalopathy. D-LDH is significantly overexpressed in tumor stem cells of esophageal squamous cell carcinoma (ESCC), and high levels of D-LDH are associated with poor prognosis in ESCC patients.

L-Lactate Dehydrogenase (NAD+ oxidoreductase, EC 1.1.1.27) is a type of NAD+-dependent isoenzyme that catalyzes the interconversion of L-lactate and pyruvate, accompanied by the oxidation/reduction of NAD+ to NADH. Active L-LDH is a tetramer, typically composed of homogenous or heterogeneous combinations of LDHA and LDHB subunits, or homogenous combinations of LDHC subunits specific to sperm and testes. Serum L-LDH is a commonly used biomarker in oncology, with the normal physiological range in serum being 140-245 IU/L. High serum L-LDH levels are associated with a broad spectrum of cancers, including pancreatic, prostate, and breast cancers, malignant melanoma, and hematologic malignancies. Serum L-LDH levels are also closely linked to poor prognosis in various cancers and have significant prognostic value. Additionally, serum L-LDH levels can serve as markers for anticancer therapy, with potential for predicting treatment outcomes and/or dynamically monitoring treatment response.

Components

• Buffer A for Metabolic Assay
• Total LDH Assay Buffer
• Substrate
• DL-Lactate Solution (50X)
• WST-8
• NADH

Principle

Lactate dehydrogenase catalyzes the oxidation of DL-lactate to pyruvate, during which NAD+ is reduced to NADH. The resulting NADH, in the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate), reduces WST-8 to orange-colored formazan, which has an absorption peak around 450nm. The amount of formazan produced is proportional to the activity of total lactate dehydrogenase. By measuring the absorbance at 450nm, the total lactate dehydrogenase activity in the sample can be calculated.

Features

• This kit offers high sensitivity, a wide linear range, and low sample usage. With a sample volume of 50µl, it can detect total lactate dehydrogenase activity as low as approximately 0.3mU/ml, maintaining a good linear relationship within the activity range of about 0.3-30mU/ml. The kit includes a NADH standard solution, the product of the lactate dehydrogenase reaction. By plotting a standard curve, the total lactate dehydrogenase activity in the sample can be calculated. For specific detection of D-lactate dehydrogenase and L-lactate dehydrogenase activities, the D-Lactate Dehydrogenase Assay Kit with WST-8 and the L-Lactate Dehydrogenase Assay Kit with WST-8 are recommended.
• This kit offers versatile lysis buffer for detection. The Buffer A for Metabolic Assay provided with this kit can be used to lyse cells or tissue samples, which can then be analyzed using other metabolic assay kits from our range that also use Buffer A for Metabolic Assay. This provides strong compatibility and can also be used for protein concentration measurement, SDS-PAGE, or Western blotting for easily soluble proteins.
• The kit is flexible, fast, and widely applicable. It can be used to analyze biological fluids such as serum, plasma, and urine from mice, rats, humans, as well as cell culture supernatants, tissue, or cell samples. The entire process takes approximately 0.5-1 hour to complete. The kit is suitable for both small sample analyses and high-throughput screening automation systems.

Storage

Store at -20ºC. Valid for one year. WST-8 should be protected from light.

Precautions

• Buffer A for Metabolic Assay and Total LDH Assay Buffer need to be completely thawed and equilibrated to room temperature before use; otherwise, the detection results may be affected. Other solutions should be used on ice.
• NADH is not very stable; please use it as soon as possible after removing it. If the standard curve is not satisfactory, it is likely that the standard has degraded.
• When removing the substrate from -20ºC, some precipitation may occur during thawing. Once equilibrated to room temperature, the precipitates will dissolve and will not affect the detection results.
• Serum, plasma, and other samples stored at 4ºC should not be kept for more than 2 weeks, as this may affect the accuracy of the detection results. Typically, serum samples should be stored at -20ºC or -80ºC for best results.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, food or drug use, or to be stored in a regular residence.
• For your safety and health, please wear a lab coat and disposable gloves during operation.