Analysis of erroneous results by ELISA - Database & Sql Blog Articles

ELISA, an enzyme-linked immunosorbent assay, is a commonly used method for solid phase enzyme immunoassay. Engvall and Perlmann first applied this method to IgG quantitative determination in 1971 and named it "enzyme linked immunosorbent assay (ELISA)". The basic principle of ELISA is:
1 binding an antigen (or antibody) to the surface of a solid support and maintaining its immunological activity;
2 linking an antigen (or antibody) to an enzyme to form an enzyme-labeled antigen (or antibody), and the enzyme-labeled antigen (or antibody) retains both its immunological activity and its enzymatic activity;
(3) The test specimen (antibody or antigen) and the 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 by the method. Separate from other substances, the amount of enzyme finally bound to the solid phase carrier is proportional to the amount of antibody or antigen to be detected in the specimen; after the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to become a colored product, according to Qualitative or quantitative analysis of the depth of the color reaction to understand the antibody or antigen content of the sample being tested.
The elisa method is widely used in various antigen and antibody assays. However, there are many influencing factors in the ELISA assay, and there are certain technical requirements in the operation. In addition to the normal reaction, some wrong results (ie, false positive or false negative results) are often seen in the clinical test. The main reasons for the ELISA measurement error are:
1 specimen factor;
2 reagent factors;
3 operational factors.
The effects of specimen factors on ELISA assays are discussed below.
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 specimens are mainly caused by interfering substances, and 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 are: rheumatoid factor, complement, heterophilic antibody, target antigen autoantibodies, iatrogenic induced anti-mouse Ig (s) antibodies, cross-reactive substances and other substances.
(1) Rheumatoid factor Human serum IgM, IgG type rheumatoid factor (RF) can directly bind to the capture antibody and the FC segment of the enzyme-labeled secondary antibody in the ELISA system, resulting in false positive.
The solution to this situation is:
1 replace the complete IgG with F(ab)2;
2 specimens with thermal denaturation 63 ° C , 10 min) IgG solid phase adsorbent treatment (addition of heat-denatured IgG to the sample dilution is also effective); 3 When detecting the antigen, it can be added to the sample dilution to degrade the RF.
(2) In the process of solid phase primary antibody and labeled secondary antibody in the complement ELISA system, the antibody molecule is allosteric, and the complement C1q molecular binding site of the FC segment is exposed, so that C1q can connect the two, thereby causing false Positive. The solution is: 1 dilute the specimen with EDTA; 2 53°C , 10 min or 56 ° C The serum was heated for 30 min to inactivate C1q.
(3) Heterophilic antibodies Human serum contains natural heterophilic antibodies that bind to rodent (such as mice) Ig(s), which can link primary and secondary antibodies in an ELISA system and can also cause false positives. The solution is to add an excess of animal Ig (s) to the sample dilution, but it is not effective when the amount is insufficient or the subclass is different.
(4) Autoantibodies against target antigens Autoantibodies against target antigens such as thyroglobulin and anti-insulin may sometimes form a complex with a target antigen, and may interfere with antigen-antibody measurement results in an ELISA method. In order to avoid the above situation, the solution is: before the measurement, it needs to be dissociated by physical and chemical methods before measurement.
(5) iatrogenic induced anti-mouse Ig (s) antibody clinically carried out with monoclonal antibodies such as murine CD3, new techniques such as imaging diagnosis and targeted therapy using radiolabeled murine antibodies It is possible to produce anti-mouse antibodies in these patients; in addition, anti-mouse Ig (s) antibodies can also be produced in patients who are bitten by rodents such as rats. These patients can produce false positives in ELISA assays. The solution is to add a sufficient amount of normal mouse Ig (s) to the specimen to determine the false positive caused by the above reasons.
(6) A cross-reactive substance such as digoxin or an AFP-like substance, which is a substance that cross-reacts with a target antigen. When the antigen is measured by the polyclonal antibody, the measurement results are not greatly affected, but when the antigen is measured by the monoclonal antibody, a false positive result may occur if the cross-antigenic determinant is exactly the target determinant corresponding to the monoclonal antibody used.
(7) Effects of other components in the specimens Serum hyperlipidemia, bilirubin, hemoglobin and excessive blood viscosity all have an interference effect on the ELISA results.
2. Exogenous substances Exogenous substances are often caused by improper collection, storage, etc. of blood samples for ELISA. Such as specimen hemolysis, specimen contamination by bacteria, specimen storage for too long, specimen agglutination and additives in blood collection tubes.
(1) Specimen hemolysis Due to various human causes, hemolysis of specimens can release a large amount of hemoglobin with peroxidase activity when erythrocyte destruction is dissolved. In the ELISA assay using horseradish peroxidase as a marker, Causes non-specific color development and interferes with the measurement results. In order to overcome the above interference effects, the specimen must be collected to avoid hemolysis.
(2) The specimen is contaminated by bacteria. The bacteria may contain endogenous horseradish peroxidase. Therefore, the specimen contaminated by bacteria may produce non-specific coloration and interfere with the measurement result, just like the hemolyzed specimen.
(3) Specimens that are not preserved in the refrigerator for a long time, the IgG in the serum can be polymerized into a multimer, and the AFP can form a dimer, which may cause the background to be too deep or even cause a false positive in an indirect ELISA assay; Specimens are placed for too long (such as more than one day), sometimes antigen or antibody immunoreactivity is weakened, and false negatives may occur. In order to overcome the above interference, the serum sample determined by ELISA should be freshly collected; if it cannot be measured immediately, the serum sample measured within 5 days can be stored in 4 ° C The serum samples measured after 1 week should be cryopreserved at low temperature; the specimens melted after cryopreservation, the protein is partially concentrated, and the distribution is uneven. It should be mixed thoroughly and then measured, but it should be gentle when mixing, and should not be strongly oscillated.
(4) Incomplete agglutination of the specimen In the absence of a coagulant and an anticoagulant, the normal blood begins to solidify after 1/2 to 2 hours, and completely solidifies at 18 to 24 hours. In the clinical laboratory work, sometimes in order to obtain rapid detection of time, the serum is forcibly centrifuged when the blood has not begun to coagulate. At this time, some fibrinogen remains in the serum, and fibrin can be formed by macroscopic observation during ELISA. Blocks are prone to false positive results; in this case, the blood coagulation is complete at the next day, and fibrinogen is no longer present in the serum, so the review results become negative. In order to avoid the above interference, the solution is preferably that the blood sample must be fully coagulated after the blood sample is collected, and then the serum is separated, or the blood collection tube with the separation gel or the appropriate coagulant is added to the blood collection tube.
(5) Influence of added substances in the specimen tube Anticoagulant (such as heparin, EDTA), enzyme inhibitor (such as NaN3 can inhibit horseradish peroxidase activity in ELISA system) and separation gel 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 the reagent factors and operational factors, more should be analyzed from the specimen factors, and corresponding measures should be taken to eliminate the interference, thus Provide accurate and reliable test results for the clinic.