ELISA, or enzyme-linked immunosorbent assay, is a commonly used solid-phase enzyme immunoassay method. Engvall and Perlmann first applied this method for quantitative IgG determination in 1971 and named it "enzyme linked immunosorbent assay (ELISA)". The basic principle of ELISA is:
â‘ Make the antigen (or antibody) bind to the surface of a certain solid carrier and maintain its immunological activity;
â‘¡ The antigen (or antibody) is linked to an enzyme to form an enzyme-labeled antigen (or antibody), and the enzyme-labeled antigen (or antibody) retains both its immune activity and its enzyme activity;
â‘¢During the measurement, the test specimen (antibody or antigen) and enzyme-labeled antigen (or antibody) are reacted with the antigen or antibody on the surface of the solid phase carrier in different steps, and then the antigen-antibody complex formed on the solid phase carrier is washed Separated from other substances, the amount of enzyme finally bound to the solid phase carrier is proportional to the amount of antibody or antigen detected in the specimen; after adding the enzyme reaction substrate, the substrate becomes a colored product after being catalyzed by the enzyme, according to its Perform a qualitative or quantitative analysis of the depth of the color reaction to understand the antibody or antigen content in the tested specimen.
The ELISA method is widely used in the determination of various antigens and antibodies. However, there are many influencing factors in ELISA measurement, and there are certain technical requirements in its operation. In addition to normal reactions in clinical testing, sometimes some erroneous results (ie, false positive or false negative results) are often seen. The main causes of ELISA measurement error results are:
â‘ Sample factor;
â‘¡Reagent factors;
â‘¢Operating factors.
This article discusses the influence of specimen factors on ELISA measurement as follows.
Serum is the most commonly used ELISA specimen. Plasma is generally regarded as the same specimen as serum. The false positive and false negative results caused by the specimen are mainly caused by interfering substances, which are divided into endogenous substances and exogenous substances:
1. Endogenous substances Some people think that about 40% of human serum samples contain non-specific interfering substances, which can affect the test results to varying degrees. Common interfering substances include: rheumatoid factor, complement, heterophile antibody, target antigen autoantibody, iatrogenic induced anti-mouse Ig (s) antibody, cross-reactive substances and other substances.
(1) Rheumatoid factor
Human serum IgM and IgG rheumatoid factor (RF) can directly bind to the FC segment of the capture antibody and enzyme-labeled secondary antibody in the ELISA system, resulting in false positives.
The solution to this situation is:
â‘ Use F (ab) 2 to replace complete IgG;
②Samples are treated with solid-phase adsorbent combined with heat-denatured (63 ℃, 10 min) IgG (adding heat-denatured IgG to the specimen diluent is also effective); ③When detecting antigen, 2-mercaptoethanol etc. In the sample dilution, RF is degraded.
(2) During the solid phase primary antibody and the labeled secondary antibody in the complement ELISA system, the antibody molecule undergoes allosteric structure, and the binding site of the complement C1q molecule in the FC segment is exposed, so that C1q can link the two, resulting in false Positive. The solution is: ①Dilute the specimen with EDTA; ② Heat the serum at 53 ℃, 10 min or 56 ℃, 30 min to inactivate C1q.
(3) Heterotropic antibodies Human serum contains natural heterophilic antibodies that can bind to Ig (s) of rodents (such as mice, etc.). It can connect the primary and secondary antibodies in the ELISA system and can also cause false positives. The solution is: excess animal Ig (s) can be added to the sample dilution, but it is not effective when the amount is insufficient or the subclass is not the same.
(4) Autoantibodies against target antigens Autoantibodies against target antigens such as thyroglobulin, anti-insulin, etc., can sometimes bind to the target antigen to form a complex, which can interfere with the results of antigen-antibody measurements in ELISA methods. In order to avoid the above situation, the solution is: before measurement, it needs to be dissociated by physical and chemical methods and then measured.
(5) Iatrogenic induced anti-mouse Ig (s) antibodies are clinically developed using monoclonal antibodies such as murine-derived CD3, radioactive isotope-labeled mouse-derived antibodies, imaging diagnosis, and targeted therapy. Anti-mouse antibodies may be produced in these patients; in addition, anti-mouse Ig (s) antibodies can also be produced in patients bitten by rodents such as rats. These patients can produce false positives when tested by ELISA. The solution is: when determining the antigen, add a sufficient amount of normal mouse Ig (s) to the specimen to overcome the false positives caused by the above reasons.
(6) Cross-reactive substances such as digoxin and AFP-like substances are substances that cross-react with target antigens. When using multiple antibodies to determine antigen, it has little effect on the measurement results, but when using monoclonal antibodies to determine antigen, if the cross-antigenic determinant happens to be the corresponding target determinant of the monoclonal antibody used, false positive results will also appear.
(7) The influence of other components in the specimen is too high in serum lipids, bilirubin, hemoglobin and blood viscosity, etc., all have interference effects on the results of ELISA measurement.
2. Exogenous substances
Foreign substances are often caused by improper collection and storage of blood samples used for ELISA. Such as hemolysis of specimens, contamination of specimens with bacteria, storage of specimens for too long, incomplete agglutination of specimens, and additives in blood collection tubes.
(1) Specimen hemolysis Specimen hemolysis caused by various human causes can release a large amount of hemoglobin with peroxidase activity when erythrocytes are destroyed and lysed. In the ELISA measurement labeled with horseradish peroxidase, Causes non-specific color development and interferes with the measurement results. In order to overcome the above interference, care must be taken to avoid hemolysis during specimen collection.
(2) The specimen is contaminated with bacteria. The bacteria may contain endogenous horseradish peroxidase. Therefore, the specimen contaminated with bacteria, like the hemolytic specimen, can also produce non-specific color and interfere with the measurement results.
(3) Improper storage of specimens Specimens that have been stored in the refrigerator for too long, IgG in the serum can be polymerized into multimers, and AFP can form dimers, which will cause the background to be too deep and even cause false positives in the indirect ELISA assay; If the specimen is left for a long time (such as more than one day), sometimes the immune activity of the antigen or antibody is weakened, and false negatives may also occur. In order to overcome the above interference, the serum samples measured by ELISA should be freshly collected; if it cannot be measured immediately, the serum samples measured within 5 days can be stored at 4 ℃, serum samples measured after 1 week should be cryopreserved; thawed samples after freezing The protein is locally concentrated and unevenly distributed. It should be thoroughly mixed before measuring, but it should be gentle when mixing, and it should not be shaken vigorously.
(4) Incomplete agglutination of specimens In the absence of coagulants and anticoagulants, normal blood begins to coagulate 1/2 to 2 hours after blood collection and completely coagulates 18 to 24 hours. In clinical testing, sometimes in order to obtain time for rapid detection, the serum is often forcibly centrifuged when the blood has not started to coagulate. At this time, some fibrinogen remains in the serum, and fibrin can be formed during the ELISA measurement process. Block, easy to cause false positive results; this type of situation is completed when the blood coagulation is complete when the next day is reviewed, and there is no longer fibrinogen in the serum, so the review results become negative. In order to avoid the above interference, the best solution is to separate the serum after collecting the blood sample, or to use a blood collection tube with a separation gel or add an appropriate coagulant to the blood collection tube.
(5) The effect of added substances in the sample tube Anticoagulants (such as heparin, EDTA), enzyme inhibitors (such as NaN3 can inhibit horseradish peroxidase activity in the ELISA system), and separation gels for rapid separation of serum, etc. The measurement has a certain interference effect. In summary, for the false positive or false negative results in the clinical test ELISA, in addition to reagent factors and operational factors, more should be analyzed from the sample factor, and appropriate measures should be taken to eliminate interference, so Provide accurate and reliable test results for the clinic.
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