Influencing factors of wrong results of ELISA

ELISA, or enzyme-linked adsorption test, is a commonly used solid-phase enzyme immunoassay method. Engvall and Perlmann first applied this method to quantify IgG in 1971, and named it “enzyme linked immunosorbent assay (ELISA)“.

The Basic Principle of ELISA:

① Make the antigen (or antibody) bind to the surface of a certain solid phase carrier and maintain its immunological activity;

② Link the antigen (or antibody) with a certain enzyme to form an enzyme-labeled antigen (or antibody), and this enzyme-labeled antigen (or antibody) retains its immune activity and its enzyme activity;

③In 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 with Separate from other substances, the amount of enzyme finally bound to the solid phase carrier is proportional to the amount of antibody or antigen tested in the specimen; after adding the enzyme reaction substrate, the substrate becomes a colored product after being catalyzed by the enzyme. Perform qualitative or quantitative analysis on the intensity of the color response to understand the content of antibodies or antigens in the test specimen.

The ELISA method is widely used in the determination of various antigens and antibodies. However, there are many influencing factors in the ELISA measurement, and there are certain technical requirements in its operation. In addition to the normal reaction, sometimes some wrong results (ie false positive or false negative results) are often seen. The main reasons for the erroneous results of ELISA determination are: ①specimen factors; ②reagent factors; ③operation factors. This article discusses the influence of specimen factors on ELISA determination as follows.

Serum is the most commonly used ELISA specimen. Plasma can generally be 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 antibodies, target antigen autoantibodies, iatrogenic induced anti-mouse Ig (s) antibodies, cross-reactive substances and other substances.

(1) Rheumatoid factor IgM, IgG type rheumatoid factor (RF) in human serum 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 intact IgG with F(ab) 2; ② Treat the specimen with a solid phase adsorbent coupled with heat-denatured (63°C, 10 min) IgG (add the heat-denatured IgG to the specimen diluent It is also effective in medium); ③When detecting antigen, 2-mercaptoethanol can be added to the sample diluent to degrade RF.

(2) During the process of the solid-phase primary antibody and the labeled secondary antibody in the complement ELISA system, the antibody molecule undergoes allosteric, and the complement C1q molecule binding site of its FC segment is exposed, so that C1q can connect the two, thus causing false Positive. The solution is: ①dilute the specimen with EDTA; ②heat the serum at 53℃, 10 min or 56℃, 30 min to inactivate C1q.

(3) Heterophile antibodies Human serum contains natural heterophile antibodies that can bind to Ig (s) of rodents (such as mice, etc.), which can connect the primary and secondary antibodies in the ELISA system, and can also cause false positives. The solution is: you can add an excessive amount of animal Ig (s) to the specimen diluent, but the addition is insufficient or the subtype is not simultaneously invalid.

(4) Autoantibodies against target antigens Autoantibodies against target antigens such as thyroglobulin and insulin can sometimes bind to the target antigen to form a complex, which can interfere with the results of the antigen-antibody determination in the ELISA method. In order to avoid the above situation, the solution is to use physical and chemical methods to dissociate it before measurement.

(5) Iatrogenically induced anti-mouse Ig (s) antibody clinical development of treatment with murine-derived CD3 and other monoclonal antibodies, radioisotope-labeled murine-derived antibodies, imaging diagnosis and targeted therapy, and other new technologies, have It may make these patients produce anti-mouse antibodies; in addition, patients bitten by rodents such as mice can also produce anti-mouse Ig (s) antibodies. These patients can produce false positives in the ELISA test. The solution is to add a sufficient amount of normal mouse Ig (s) to the specimen when determining the antigen to overcome the false positive caused by the above-mentioned reasons.

(6) Cross-reactive substances Digoxin-like, AFP-like substances, etc., are substances that cross-react with the target antigen. The use of polyclonal antibodies to determine antigens has little effect on the results of the determination, but when using monoclonal antibodies to determine antigens, if the cross-epitope determinants happen to be the target determinants corresponding to the monoclonal antibody used, false positive results will also occur.

(7) The influence of other components in the specimen. Excessive serum lipids, bilirubin, hemoglobin and excessive viscosity, etc., all interfere with the ELISA measurement results.

2. Exogenous Substances

Exogenous substances are often caused by improper collection and storage of blood samples for ELISA determination. Such as hemolysis of the specimen, contamination of the specimen, excessive storage of the specimen, incomplete specimen agglutination, and additives in the blood collection tube.

(1) Specimen hemolysis. Specimen hemolysis caused by various man-made causes can release a large amount of hemoglobin with peroxidase activity when red blood cells are destroyed and lysed. In the ELISA assay marked with horseradish peroxidase, it will Causes non-specific color development and interferes with the measurement results. In order to overcome the above interference effects, attention must be paid to avoid hemolysis when collecting specimens.

(2) The specimen is contaminated by bacteria. The bacteria may contain endogenous horseradish peroxidase. Therefore, the specimen contaminated by bacteria, like the hemolyzed specimen, may also produce non-specific color development and interfere with the measurement result.

(3) Improper specimen storage. Specimens stored in the refrigerator for a long time, IgG in the serum can polymerize into multimers, and AFP can form dimers, which may cause excessive background and even false positives in the indirect ELISA assay; If the specimen is placed for too long (such as more than one day), sometimes the antigen or antibody immune activity is weakened, and false negatives may also appear. In order to overcome the interference mentioned above, the serum samples measured by ELISA should be freshly collected; if the measurement cannot be done immediately, the serum samples measured within 5 days can be stored at 4℃, and the serum samples measured after 1 week should be frozen at low temperature; the samples thawed after freezing , The protein is locally concentrated and unevenly distributed. It should be mixed thoroughly before measurement, but it should be gentle and not vigorously shaken when mixing.

(4) Specimen incomplete agglutination In the absence of coagulants and anticoagulants, normal blood begins to coagulate 1/2 to 2 hours after collection, and complete coagulation in 18 to 24 hours. In clinical laboratory work, sometimes in order to gain time for rapid detection, the serum is often forcibly centrifuged before the blood has begun to coagulate. At this time, there is still some fibrinogen in the serum, which can form visible fibrin during the ELISA measurement process. Block, it is easy to cause false positive results; in this case, the blood coagulation has been completed in the next day’s re-examination and there is no longer fibrinogen in the serum, so the re-examination result becomes negative. In order to avoid the above interference effects, the best solution is to separate the blood after the blood sample is fully coagulated, or use a blood collection tube with a separating gel or add an appropriate coagulant to the blood collection tube when the sample is collected.

(5) The influence of the substances added to the specimen tube Anticoagulants (such as heparin, EDTA), enzyme inhibitors (such as NaN3 can inhibit horseradish peroxidase activity in the ELISA system), and the separation gel for rapid serum separation, etc., all affect the ELISA The measurement has a certain interference effect.

To sum up, for the false positive or false negative results in the clinical test ELISA determination, in addition to the reagent factors and operating factors, more analysis should be carried out from the aspect of specimen factors, and corresponding measures should be taken to eliminate interference effects. Provide correct and reliable test results for the clinic.