I. Introduction. Enzyme-linked immunosorbent assay (ELISA) is an immunoassay widely used in the biomedical field, which has the advantages of high sensitivity, high specificity and quantification. However, in practice, ELISA results often have false positives, which brings great problems to the accuracy and reliability of the experiment. In this paper, the culprit of false positives in the ELISA experiment is examined, in order to provide a reference for improving the quality and reliability of the experiment.
2. Common causes of false positives in ELISA experiments.
1.Poor sample quality.
Sample quality is one of the important factors influencing the results of ELISA experiments. If there are interfering substances in the sample, such as endogenous enzymes, impurities, proteins, etc., it will interfere with the experiment and lead to the appearance of false positives. Therefore, the samples should be adequately treated and cleaned up before the experiment to ensure that the sample quality meets the experimental requirements.
2.The quality of the reagents is poor.
The reagents used in ELISA experiments are diverse and of varying quality. Poor quality reagents can lead to biased and false positives in experimental results. Therefore, when selecting reagents, high-quality and reliable reagents should be selected, and the instructions for use of reagents should be followed to ensure the accuracy and standardization of experimental operations.
3.Improper operation.
Improper manipulation is another common cause of false positives in ELISA experiments. For example, if there is residue or contamination of the tip or pipette during the dispensing process, this can have an impact on the experimental results and lead to false positives. In addition, if the washing is not thorough, the color development is not sufficient, or the termination is not timely, the accuracy of the experimental results will also be affected. Therefore, in the process of experimentation, the operating procedures should be strictly followed to ensure the accuracy and standardization of each step of the operation.
4.Cross-reactivity.
Cross-reactivity refers to the cross-reactivity of an antibody with a non-target antigen, resulting in inaccurate and false-positive experimental results. For example, the antigens of certain bacteria or viruses may cross-react with certain antibodies, resulting in biased experimental results. Therefore, when selecting antibodies, it is important to choose antibodies with strong specificity and to fully understand the cross-reactivity of antibodies.
3. Ways to avoid false positives in ELISA experiments.
1.Strict control of sample quality.
Samples should be adequately treated and cleaned up prior to the experiment to remove impurities and interfering substances. In addition, sample dilution can be used to reduce the effects of endogenous enzymes.
2.Choose high-quality reagents.
When selecting reagents, high-quality and reliable reagents should be selected, and the instructions for use of reagents should be followed to ensure the accuracy and standardization of experimental operations. In addition, reagent validation methods can be employed to ensure the quality and reliability of reagents.
3.Standardize the operation process.
In the process of experiment, the operation procedures should be strictly followed to ensure the accuracy and standardization of each step of the operation. In addition, repeated experiments can be used to verify the accuracy and reproducibility of experimental results.
4.The blocking ELISA method is used.
Blocking ELISA is a method that eliminates cross-reactivity. By combining the specific antibody to the target antigen to form a complex, and then binding to the enzyme-labeled secondary antibody to form a whole antigen-antibody-enzyme-labeled secondary antibody complex, the cross-reactivity between the antibody and the non-target antigen is eliminated, and the accuracy and specificity of the experimental results are improved.
IV. Conclusions. False positives in ELISA experiments are a common phenomenon in the experimental process, which will greatly interfere with and affect the experimental results. In this paper, we analyze the common causes of false positives in ELISA experiments and the methods to avoid them, which provides a reference for improving the quality and reliability of ELISA experiments. In practice, corresponding measures should be taken according to the specific situation to avoid the occurrence of false positives, so as to ensure the accuracy and reliability of the experimental results.