Lactate dehydrogenase (LDH) is a crucial enzyme in the anaerobic metabolic pathway, catalyzing the reversible conversion of lactate to pyruvate while reducing NAD+ to NADH and vice versa. LDH is ubiquitously present in various body tissues. Elevated LDH levels in the bloodstream can result from diverse conditions, including liver disease, anemia, heart attack, bone fracture, muscle trauma, cancer, and infection. LDH is also a general marker of tissue turnover, reflecting normal metabolic processes.

The RayBio® Lactate Dehydrogenase Activity Assay Kit offers a user-friendly, reliable, and highly sensitive method for quantifying LDH activity in plasma, serum, cell lysates, and other biological fluid samples. In this assay, LDH facilitates the oxidation of lactate into pyruvate while concurrently reducing NAD to NADH. The rate of NAD reduction can be quantified by monitoring the increase in absorbance at 340nm. This rate of absorbance change is directly proportional to the LDH activity within the sample.

• Microplate reader capable of measuring absorbance at 340 nm at 37°C
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Multi-channel pipettes to deliver 20 μl to 200 μl volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C.
• 50 ml conical tubes

Positive control, sample and Sample Buffer (used as a blank) should be assayed in duplicate or triplicate. A freshly prepared positive control should be used each time the assay is performed.

1. Set up a microplate reader or a microplate incubator at 37°C.
2. Prepare Working Solution (See Reagent Preparation, section A), and incubate it at 37°C for five minutes.
3. Prepare Positive Control (See Reagent Preparation, section B).
4. Pipette 10 μL of sample, Sample Buffer (as the blank) and Positive Control into each well of the 96-well plate.
5. Transfer 200 μL of pre-warmed Working Solution into each well (it is recommended to use a multi-channel pipette), mix and incubate at 37°C for 30 seconds.
6. After 30 seconds, read and record absorbance at 340nm (A1 Reading). Continue Incubating at 37°C for one minute.
7. After exactly one minute, read and record absorbance at 340nm (A2 Reading).