The ELISA detection system is one of the most widely used and flexible technologies in immunological research. I’ve been working with this technique for a long time, and over the years, I've gained more confidence and experience. I now have a good grasp of the ELISA system, and it's become second nature to me. Doing ELISA has become a routine part of my work, and I can usually complete a full test within a single day. However, even with all this experience, there are still challenges when dealing with new reagents or procedures. When I first started, I faced many issues—like false positives, non-specific binding, and inconsistent color development. These problems were frustrating, but they also helped me learn and improve. One of the key factors in successful ELISA is the coating process. The quality of the antigen, its concentration, and whether it’s degraded all play a role in how well the antibody recognizes it. That’s why it's so important to preserve the integrity of the antigen. When I was working with recombinant proteins, my colleagues always reminded me to handle them carefully, especially when thawing them on ice. Blocking is another critical step—it involves filling the empty spaces on the plate with unrelated proteins to prevent non-specific binding. Sometimes, depending on the experiment, we might use a neutral buffer instead of a blocking solution, but that’s less common. Blocking is essential to reduce background noise and improve specificity. Common blocking agents include BSA (0.05%–0.5%), gelatin (10% or 1%), skim milk (5%–10%), and other animal sera. Each has its own advantages and limitations, and the choice depends on the specific needs of the experiment. It’s crucial to select the right one based on your samples and antibodies. Washing is one of the most critical steps in ELISA. Since proteins tend to adsorb onto plastic surfaces like polystyrene, thorough washing is necessary to remove unbound enzymes, non-specific substances, and any residual reagents. Even small mistakes during washing—like incomplete rinsing or missed wells—can significantly affect the results. While automated washers help, manual techniques still rely heavily on the operator’s skill and attention to detail. When adding the primary and secondary antibodies, it's important to be precise. Using the correct pipette tip and ensuring proper dilution of the sample is vital. If you're using two antibodies, pay close attention to their concentrations—too high or too low can lead to poor signal or background noise. Color development is another area that requires careful control. There are various color systems available, and choosing the right one depends on the enzyme-substrate combination. For example, some systems use thiomersal, while others use sodium azide. It's important to prepare fresh solutions each time and maintain consistency. Any deviation in timing or reagent preparation can lead to inaccurate results. Keeping track of all variables ensures reliable and reproducible data.
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