Lactate Dehydrogenase (LDH) Activity Assay Kit (LD-L, Colorimetric Method)

Cat. No.: L1510342

GRADE & PURITY BioReagent

Overview

Lactate dehydrogenase (LDH or LD) is an oxidoreductase that catalyzes the transfer of hydrogen atoms or electrons from one substrate to another. LDH is a crucial enzyme in glycolysis and gluconeogenesis, contains zinc ions, and is widely distributed in human and animal tissues, plants, and microorganisms. It catalyzes the reversible oxidation‑reduction reaction between lactate (L) and pyruvate (P). The reaction formula is: Lactate + NAD⁺ → Pyruvate + NADH + H⁺. The direction L → P is the forward reaction; P → L is the reverse reaction.

Detection Principle

This assay utilizes LDH to catalyze the forward reaction: L‑Lactate + NAD⁺ → Pyruvate + NADH + H⁺. During this reaction, lactate is oxidized to pyruvate while NAD⁺ is reduced to NADH, causing an increase in absorbance at 340 nm. The rate of increase is proportional to the LDH activity in the sample. The LDH activity is obtained by measuring the rate of absorbance increase at 340 nm using a spectrophotometer or automatic analyzer and performing calculations. Advantages of this LD‑L method include: 1. Lactate and NAD⁺ substrate solutions are more stable than the pyruvate and NADH substrate solutions used in the LD‑P method. 2. A wider linear rate‑reaction time range. 3. Better reproducibility compared to the LD‑P method and the dinitrophenylhydrazine method. 4. Better accuracy compared to the dinitrophenylhydrazine method. 5. Suitable for automatic analyzers. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.

Reagents, consumables and Equipments not provided

• UV spectrophotometer or automatic analyzer (capable of measuring absorbance at 340 nm)
• Centrifuge tubes or small test tubes, water bath, graduated cylinder, precision balance, 1 mL quartz cuvette
• Deionized water, physiological saline

Operating Steps (For Reference Only)

1. Sample Preparation

1.1 Plasma and Serum Samples

Plasma and serum prepared by conventional methods can be used directly for assay with this kit. Store at room temperature for up to 3 days for LDH detection.

1.2 Cell or Tissue Samples

Homogenize appropriate amounts of cells or tissue, centrifuge at low speed, and collect the supernatant. Store at room temperature for up to 3 days for LDH detection.

1.3 Long‑Term Sample Storage

If extracted samples cannot be assayed promptly and require longer storage, proceed as follows: Reconstitute one vial of LDH Protectant with 1 mL of deionized water to prepare the LDH Protectant Working Solution. Store at –20°C protected from light. Mix the sample (e.g., serum) with the LDH Protectant Working Solution at a ratio of 9:1. Store at 4°C protected from light.

Note: After sample preparation, protein concentration can be measured using a BCA Protein Assay Kit to facilitate subsequent calculation of LDH content per unit protein weight in tissues or cells. Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready‑to‑Use BCA Protein Quantification Kit is recommended.

2. Preparation of LDH Assay Working Solution

Weigh 42 mg of NAD using a precision balance, add 10 mL of LD‑L Assay Buffer, mix to dissolve. This is the LDH Assay Working Solution. Prepare fresh for each use.

3. Spectrophotometric Measurement

Set up the test tube according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the LDH concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.

Substance Added (mL)	Test Tube
Sample (serum, plasma, body fluid, etc.)	0.05
LDH Assay Working Solution (pre‑warmed to 37°C)	1

Mix well and incubate at 37°C for 30 s.

Using a 1 cm pathlength 1 mL quartz cuvette, immediately read the absorbance of the test tube at 340 nm with a UV spectrophotometer. Record as A_Test1. After t minutes, read the absorbance again and record as A_Test2. 

Note: Because the enzymatic reaction time is short, it is recommended to keep the sample addition time as brief as possible. The reaction essentially occurs within 1–3 minutes, after which it levels off. Reference ranges may vary due to differences in instruments, operational techniques, and sample enzyme activity levels.

4. Automatic Analyzer Measurement

If the sample concentration is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate samples. Set parameters according to the performance of the laboratory's automatic analyzer. The following parameters are for reference only. Record the rate of absorbance increase (ΔA/min) for the test sample tube.

Parameter	Setting
Temperature	37°C
Wavelength	340 nm
Delay time	90 s
Measurement time	180 s
Sample volume	15 μL
LDH Assay Working Solution volume	300 μL

5. Result Calculation

5.1 Manual Colorimetric Calculation Formula

LDH (U/L) = ΔA/min × (10⁶ / 6220) × (1.05 / 0.05) = ΔA/min × 3376

Parameter Explanation:

ΔA/min = (A_Test2 − A_Test1) / t 

t= Reaction time (min)

6220 = Molar absorptivity of NADH (L·mol⁻¹·cm⁻¹)

1.05 = Total reaction volume (mL)

0.05 = Sample volume (mL)

5.2 Automatic Analyzer Calculation Formula

LDH (U/L) = ΔA/min × (10⁶ / 6220) × (315 / 15) = ΔA/min × 3376

Parameter Explanation:

ΔA/min = Measured rate of absorbance increase at 340 nm

6220 = Molar absorptivity of NADH (L·mol⁻¹·cm⁻¹)

315 = Total reaction volume (μL)

15 = Sample volume (μL)

Note: If the sample was mixed with LDH Protectant Working Solution, divide the result by 0.9.

Precautions

1. Processed samples should be assayed promptly; otherwise, LD₄ and LD₅ may lose activity.

2. Serum or heparin‑anticoagulated plasma is preferred for detection. Oxalate and EDTA anticoagulants inhibit LDH activity.

3. Avoid using hemolyzed samples.

4. The enzymatic reaction time is generally 1–3 minutes. Prolonged time leads to a decrease in reaction rate.

5. This reaction must be measured at 340 nm, requiring a UV spectrophotometer or full‑wavelength microplate reader, quartz cuvettes, or a UV‑compatible microplate.

6. For your safety and health, please wear a lab coat and disposable gloves during operation.

7. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

L1510342	Component	100T	Storage
L1510342A	NAD	1EA	2-8℃. Store in the dark.
L1510342B	LD-L Assay Buffer	100 mL	2-8℃. Store in the dark.
L1510342C	LDH Protectant	1EA	2-8℃. Store in the dark.