Lactate dehydrogenase assay method

We know that there are various enzymes in real life. These enzymes can act alone or interact with each other and have a great impact on our human body. However, I believe that everyone is not very familiar with the determination method of enzymes. Different enzymes have different methods of determination, and the methods are also different. Nowadays, the determination method of lactate dehydrogenase has become a topic of research for many people. So how to determine it? Let us learn about the determination method of lactate dehydrogenase.

method:

Before starting the experiment, please prepare all reagents in advance. When diluting reagents or samples, they must be mixed evenly, and try to avoid foaming during mixing. A standard curve should be prepared for each test. If the sample concentration is too high, dilute it with sample diluent to make the sample fit the detection range of the kit.

1. Sample addition: Set up blank wells, standard wells, and wells for samples to be tested. Add 100 μl of sample diluent to the blank well and 100 μl of standard or test sample to the remaining wells. Be careful not to have bubbles. Add the sample to the bottom of the well of the ELISA plate, try not to touch the well wall, shake gently to mix, cover or cover the ELISA plate, and react at 37°C for 120 minutes.

To ensure the validity of the experimental results, please use a new standard solution for each experiment.

2. Discard the liquid, spin dry, and do not wash. Add 100 μl of biotin-labeled antibody working solution to each well (prepare 1 μl of biotin-labeled antibody with 99 μl of biotin-labeled antibody diluent, mix gently, and prepare within one hour before use) and incubate at 37°C for 60 minutes.

3. After incubation for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 3 times, soaking for 1-2 minutes each time, 350μl/well, and spin dry.

4. Add 100 μl of horseradish peroxidase-labeled avidin working solution (same as biotin-labeled antibody working solution) to each well and incubate at 37°C for 60 minutes.

5. After incubation for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 5 times, soaking for 1-2 minutes each time, 350μl/well, and spin dry.

6. Add 90 μl of substrate solution to each well in sequence and develop color at 37°C in the dark (within 30 minutes, the first 3-4 wells of the standard will have a clear blue gradient visible to the naked eye, while the gradient in the last 3-4 wells is not obvious, and the process can be terminated).

7. Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow). The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. In order to ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time is up.

8. Use an enzyme-linked immunosorbent assay (ELISA) to measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm. Perform the assay within 15 minutes after adding the stop solution.

The above content introduces us to the lactate dehydrogenase determination method. I believe that these contents can arouse everyone's interest. I believe that everyone can effectively, quickly and directly determine the lactate dehydrogenase. Maybe you are also a research enthusiast, so hurry up and practice it!