ELISA, or Enzyme-Linked Immunosorbent Assay, is a widely used technique in biomedical research and clinical diagnostics. However, it's not uncommon to encounter erroneous results during the process. These errors can be broadly categorized into three main areas: specimen-related issues, reagent-related problems, and operational mistakes.
The fundamental principle of ELISA involves immobilizing an antigen or antibody onto a solid-phase carrier while preserving its immunological activity. It also includes conjugating another antigen or antibody with an enzyme, creating an enzyme-labeled complex that retains both immunological and enzymatic properties. During testing, the sample (antibody or antigen) interacts with the enzyme-labeled reagent, forming complexes on the solid support. After washing to remove unbound components, the amount of enzyme bound to the plate correlates with the concentration of the target analyte in the sample. A substrate is then added, which is converted by the enzyme into a detectable color change. The intensity of this color is measured to determine the presence or quantity of the target molecule.
Despite its reliability, ELISA is sensitive to various factors that can lead to false positives or false negatives. One of the most common causes is the quality of the specimen. Serum is the most frequently used sample type, and plasma is often considered equivalent. However, several substances—both endogenous and exogenous—can interfere with the test results.
Endogenous substances, such as rheumatoid factor, complement proteins, heterophilic antibodies, autoantibodies against the target antigen, and iatrogenic anti-mouse Ig antibodies, can cause non-specific binding and lead to inaccurate readings. These are naturally present in the body and may affect the assay depending on the individual’s immune status.
On the other hand, exogenous contaminants arise from improper handling of samples. For instance, hemolysis, bacterial contamination, prolonged storage, or agglutination of the blood sample can all distort the results. Additionally, certain additives in blood collection tubes might interfere with the reaction, making the outcome unreliable.
To minimize errors, it's crucial to follow proper protocols for sample collection, storage, and handling. Using high-quality reagents and ensuring precise execution of each step in the ELISA procedure can significantly improve the accuracy of the results. Understanding these potential pitfalls helps researchers and lab technicians maintain the integrity of their data and avoid misleading conclusions.
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