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EL-C1600N100013-B
ELISA, or Enzyme-Linked Immunosorbent Assay, is a widely used technique in immunology for detecting and quantifying antibodies, antigens, and other molecules. It was first introduced by Engvall and Perlmann in 1971 as a method to measure IgG levels. The basic principle of ELISA involves immobilizing an antigen or antibody on a solid surface, such as a microplate, and then using enzyme-labeled detection reagents to produce a measurable signal—typically a color change—after the reaction with the sample.
The process generally includes several key steps: binding the target molecule to the plate, adding the enzyme-conjugated reagent, washing away unbound components, and finally adding a substrate that reacts with the enzyme to generate a detectable signal. This signal is proportional to the concentration of the analyte in the sample, allowing for both qualitative and quantitative analysis.
Despite its widespread use, ELISA is not without challenges. False positives and false negatives can occur due to various factors, including the quality of the specimen, the reagents used, and the technical execution of the assay. Among these, specimen-related issues are particularly common and can significantly impact results.
Serum is the most commonly used specimen in ELISA, though plasma is often treated similarly. However, interference from endogenous and exogenous substances can lead to inaccurate outcomes. Endogenous factors include rheumatoid factor (RF), complement proteins, heterophilic antibodies, autoantibodies, and iatrogenic anti-mouse antibodies. These substances can bind non-specifically to antibodies or enzymes in the assay, causing false readings.
For example, RF can interfere by binding to both capture antibodies and enzyme-labeled secondary antibodies, leading to false positives. Solutions include using F(ab)2 fragments instead of whole IgG, heat inactivation of samples, or pre-treating with proteases to degrade RF.
Complement activation can also cause false positives by cross-linking antibodies through C1q binding. Inactivating complement via heat treatment or dilution with EDTA can help reduce this effect.
Heterophilic antibodies, which react with rodent-derived antibodies, can similarly cause false signals. Adding excess animal Ig to the sample may help block this interaction, but it depends on the antibody subclass and concentration.
Autoantibodies against target antigens, such as thyroglobulin or insulin, may form complexes that interfere with the detection process. Pre-treatment with physical or chemical methods can help dissociate these complexes before testing.
Iatrogenic anti-mouse antibodies, often seen in patients receiving monoclonal antibody therapies, can also lead to false positives. Adding normal mouse Ig to the sample can neutralize this interference.
Cross-reactive substances, like digoxin or AFP-like compounds, may mimic the target antigen, especially when using monoclonal antibodies. Careful selection of antibody pairs can help minimize this risk.
Exogenous factors, such as hemolysis, bacterial contamination, improper storage, and incomplete clotting, can also affect ELISA performance. Hemolyzed samples release peroxidase-like activity, which may cause non-specific color development. Similarly, bacterial contamination can introduce interfering enzymes.
Long-term storage at improper temperatures may lead to protein aggregation or degradation, resulting in false negatives or high background signals. Proper handling and storage—such as refrigeration or freezing—can mitigate these issues.
Incomplete clotting of blood samples can leave fibrinogen in the serum, leading to false positives in indirect assays. Ensuring complete coagulation or using tubes with separation gels can prevent this.
Finally, additives in collection tubes, such as anticoagulants or enzyme inhibitors, may interfere with the ELISA reaction. Choosing appropriate collection tubes and following standard operating procedures are essential for reliable results.
In conclusion, while ELISA is a powerful tool in diagnostic and research settings, careful attention must be paid to specimen preparation, reagent quality, and procedural accuracy. By addressing potential sources of error, laboratories can ensure more accurate and consistent test results, ultimately supporting better clinical decision-making.
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