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EL-C1600N100013-B
ELISA, or Enzyme-Linked Immunosorbent Assay, is a widely used technique in immunology for detecting and quantifying specific antibodies or antigens. First introduced by Engvall and Perlmann in 1971, it revolutionized the way scientists could measure immune responses. The basic principle of ELISA involves several key steps:
1. An antigen (or antibody) is immobilized on a solid surface, such as a microplate, while retaining its biological activity.
2. An enzyme-labeled version of the corresponding antibody (or antigen) is added, which retains both its binding and enzymatic activity.
3. The sample, containing either the target antibody or antigen, is incubated with the immobilized molecule. After washing to remove unbound components, the enzyme's activity is measured by adding a substrate that produces a detectable signal, usually a color change.
This method allows for both qualitative and quantitative analysis based on the intensity of the reaction. Despite its widespread use, ELISA is not without challenges. False positives or false negatives are common in clinical settings, often due to various factors affecting the accuracy of the results.
One major source of error comes from the specimen itself. Serum is the most commonly used sample, but it may contain interfering substances. These can be endogenous (from within the body) or exogenous (introduced externally). Common endogenous interferents include rheumatoid factor, complement, heterophilic antibodies, autoantibodies, and cross-reactive molecules. Each of these can lead to non-specific binding, causing inaccurate results.
For example, rheumatoid factor (RF) can bind to both the capture antibody and the enzyme-labeled secondary antibody, leading to false positives. Solutions include using F(ab)2 fragments instead of whole IgG, heat-inactivating the sample, or adding proteolytic enzymes to degrade RF.
Complement proteins can also interfere by binding to the FC region of antibodies, causing false signals. This can be mitigated by diluting the sample with EDTA or heat inactivation. Similarly, heterophilic antibodies—naturally occurring antibodies that react with animal IgGs—can cause similar issues. Adding excess animal IgG to the sample can help block this interaction.
Autoantibodies against target antigens may form complexes that interfere with detection. In such cases, pre-treatment with physical or chemical methods can dissociate these complexes before testing. Iatrogenic anti-mouse antibodies, often seen in patients receiving monoclonal antibodies or exposed to rodents, can also cause false positives. Adding normal mouse IgG to the sample can neutralize this effect.
Cross-reactive substances like digoxin or AFP-like molecules may mimic the target antigen, especially when using monoclonal antibodies. Careful selection of reagents is essential to avoid such issues.
Exogenous factors, such as hemolysis, bacterial contamination, improper storage, and additives in blood collection tubes, can also impact ELISA results. Hemolysis releases hemoglobin with peroxidase activity, which can interfere with color development in HRP-based assays. Bacterial contamination may similarly introduce non-specific signals. Proper handling, storage at 4°C, and avoiding long-term storage are critical to maintaining sample integrity.
Incomplete coagulation of blood samples can lead to fibrin formation, which may block wells and cause false positives. Ensuring full clotting before serum separation or using tubes with gel separators can prevent this issue.
Lastly, anticoagulants like heparin or EDTA, and enzyme inhibitors like NaN3, may interfere with the ELISA reaction. Understanding these potential sources of error helps in optimizing the assay and ensuring reliable results. By addressing these factors, laboratories can improve the accuracy and consistency of their ELISA tests.
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