Chemiluminescent immunoassay kits
1.Add 100 l of standards or samples to each well and incubate at 37°C for 90 min.
2.Pour off the liquid from the wells, pat dry, add 100 l of biotinylated antibody working solution, and incubate at 37°C for 60 min.
3.Wash 3 times.
4.Add 100 l of enzyme conjugate working solution and incubate at 37°C for 30 min.
5.Wash 5 times.
6.Add 100 l of the luminescent substrate mixture and incubate at 37 °C for about 5 min.
7.The chemiluminescence value of each well is determined immediately.
8.Result calculation.
Experimental considerations.
1.Storage of antigens and antibodies: Antigens and antibodies should be stored in strict accordance with the requirements of the instructions, and repeated freezing and long-term exposure to high temperatures should be avoided. At the same time, it is important to ensure that the kit has not expired to obtain reliable experimental results.
2.Sampling technology: When adding, the volume added should be accurate and no bubbles should be generated, and it should be avoided on the edge of the hole wall, so as not to affect the contact with the solid surface. A dosing control well should be placed next to each well to monitor the accuracy and reproducibility of the dosing.
3.Incubation: Incubation is an important step in ELISA experiments, and the control of incubation time and temperature is critical to the conduct and outcome of antigen-antibody reactions. Incubation must be carried out in strict accordance with the requirements of the instructions, and attention must be paid to maintaining a constant temperature state.
4.Washing: Washing is an indispensable step in ELISA experiments to remove unbound material and improve the specificity and sensitivity of the assay. When washing, ensure that the washing time is sufficient for each wash, and replace the washing liquid to ensure the washing effect. At the same time, it is necessary to avoid the formation of bubbles during the washing process, so as not to affect the washing effect.
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