[Protein research series]-13丨 ELISA true and false
ELISA is widely used in agriculture, aquaculture, food safety, clinical diagnosis and other fields. It is a detection method with high sensitivity, specificity and easy operation. In practical applications, all aspects of ELISA operations have an impact on the results, such as the use of pipettes, sample storage, sample loading, hemolysis, reagent temperature, and protection from light. In addition, some non-subjective factors can also cause large deviations from the results.
The false negative and false positive results in the ELISA experiment, let the experimental monarchs are big, right? Want to cry without tears? Today, Xiaobian and everyone share the reasons and solutions for false negatives and false positives.
False negative
1 specimens with anticoagulation treatment such as heparin or EDTA, easy to cause false negative HBsAg test results. Heparin anticoagulated plasma increases the OD value, which may have a strong negative charge with high concentrations of heparin, which is related to the difficulty in eluting the enzyme label. EDTA, an enzyme inhibitor such as NaN 3 , inhibits horseradish peroxidase activity in the ELISA system, causing false negatives.
heparin
2 When the antigen is determined by the double antibody sandwich method, if the concentration of the analyte is too high, the detection result may be negative due to the hook effect. To avoid this, you can either dilute the sample or directly switch to the two-antibody sandwich two-step method.
Double antibody sandwich method
3 The concentration of the analyte in the sample is low, which is also likely to cause false negative results. For example, in the early stage of development of certain diseases, the concentration of the test substance is lower than the detection limit.
4 In some cases, genetic mutations in individual individuals may also result in false negative results. In particular, in the case where the binding site of the antibody for detection is in the mutated region, this case is extremely rare, but may exist.
False positive
False positives usually occur when the sample is doped with homologous or isoforms, such as rheumatoid factors, complements, cross-reactive substances, and other substances.
Rheumatoid factor
1 When the blood sample is treated, if hemolysis occurs, the methionin catalytic substrate tetramethylbenzidine (TMB) having weak peroxidase activity in the sample will be colored, resulting in a false positive result. Therefore, when dealing with the serum samples most frequently examined by ELISA, attention should be paid to the details of the agglutination process, centrifugation process, etc., to avoid hemolysis and doping of blood cells.
2RF, a self-IgM antibody that binds to multiple animal IgG Fc, can bind to the enzyme-labeled secondary antibody Fc to cause false positives. To avoid this, RF can be degraded by adding 2-mercaptoethanol when detecting the antigen, or blocking with heat-denatured IgG (63 ° C, 10 min). In addition, the use of F(ab) 2 in place of intact IgG also achieves the goal. Some other target antigen autoantibodies, such as anti-thyroglobulin and anti-insulin globulin, may combine with the target antigen to form a complex when interrogating, which interferes with the assay results. Therefore, it needs to be dissociated by physical and chemical methods before measurement and then measured.
3 Complement on the one hand will cause the primary antibody and the enzyme-labeled secondary antibody molecule to undergo an allosteric exposure to the Fc-C1q molecule binding site, thereby linking the two together to cause a false positive. On the other hand, it is also possible to bind a solid phase antibody to block its binding epitope with an antigen to cause a false negative. Therefore, it is necessary to perform serum inactivation of serum and animal serum samples at 56 °C for 30 min. In addition, this effect can be reduced by diluting the sample with EDTA.
Complement C1q
4 The digoxin-like, AFP-like substances present in the sample can cross-react with the antigen. Especially when the antigen is determined using a monoclonal antibody, a false positive result occurs if the antigenic determinant that happens to cross-react is the monoclonal antibody binding site.
Digoxin
5 If the sample contains experimentally treated or contaminated certain bacteria, such as epidermis, the endogenous HRP released by the bacteria may cause false positive results.
In order to avoid false negatives or false positives in the ELISA test, it is necessary not only to strictly implement the standard operating procedures, but also to analyze the various factors related to the specimens, and to use corresponding measures to eliminate the interference effects of the interfering substances, so that the test results are accurate and consistent.
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