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  • The basic principles of ELISA and the main reasons for the error results are determined.

    ELISA, enzyme-linked immunosorbent assay, is a common method for solid-phase enzyme immunoassay.

    Engvall and Perlmann in 1971 was the first application of the method of quantitative determination of IgG, and was named "enzyme linked immunosorbent assay (ELISA)". The basic principle of ELISA is:

    ①the antigen (or antibody) binds to the surface of some solid support, and to maintain its immune activity;

    ②Antigen (or antibody) with the enzyme to form some kind of enzyme-linked antigen (or antibody), and that this enzyme antigen (or antibody) both retain their immune activity, retaining its enzymatic activity;

    ③The subject specimen (antigen or antibody) ELISA and antigen (or antibody) when measured by the different steps of the reaction with the antigen or antibody, the solid support surface, washed forming method on a solid support and the antigen-antibody complex separated from other substances, and finally bound to a solid support and the amount of enzyme antibody or antigen in the sample amount is proportional subjects; the enzyme was added after the reaction substrate, the substrate is changed to a colored product after the enzyme-catalyzed, according to their color the reaction depth qualitative or quantitative analysis to understand the test specimen antibody or antigen content.

    ELISA method is widely used in a variety of antigen and antibody assay. However, many factors affect the ELISA assay, and its operations have a certain technical requirements, in addition to the normal clinical testing in the reaction, there often can see some incorrect results (ie false positive or false negative results). The main cause of ELISA assay erroneous results are:

    ①Factors specimens

    ②Reagents factors

    ③Operational factors

    This article follows the discussion on the impact of factors on the ELISA assay samples

    ELISA Serum samples are the most commonly used, may generally be considered equivalent to plasma and serum specimens,

    False positive and false negative results due to sample interfering substances are mainly caused by the material is divided into two kinds of exogenous and endogenous substances:

    1.Endogenous substances was considered about 40% of human serum samples contained non-specific interfering substances can affect the test results to varying degrees. Common interfering substances include: rheumatoid factor, complement, heterophile antibodies, addicted to the target antigen of autoantibodies, iatrogenic induced anti-mouse Ig (s) antibodies cross-reacting substances and other substances.

    (1)rheumatoid factor

    Human serum IgM, IgG rheumatoid factor (RF) can capture ELISA system antibodies and enzyme-labeled secondary antibody is directly bonded FC segment, leading to a false positive.

    Address this situation would be to:

    ① with F (ab) 2 substitute full IgG;

    ②Samples with associated thermal denaturation (63 ℃, 10 min) IgG solid adsorbent (heat-denatured IgG was added to the sample dilution equally valid);

    ③When detection of antigen, 2-mercaptoethanol may be added to the specimen diluent, etc., and the RF degradation.

    (2)complement ELISA Systems solid phase antibody and labeled secondary antibodies process, the antibody molecules allosteric complement its FC segment C1q molecule binding sites are exposed, so that C1q can connect the two together, resulting in false positive. The solution is: ① dilute samples with EDTA; ② with 53 ℃, 10 min or 56 ℃, 30 min heat-inactivated serum make C1q.

    (3)heterophile antibodies in human serum with rodents (such as rats, etc.) Ig (s) comprising a combination of natural heterophile antibodies, ELISA system can be a primary and secondary antibodies to link up can also cause a false positive. The solution is: can an excess of animal Ig (s) in the sample diluent, but insufficient amount or subclass are not valid simultaneously.

    (4)addicted target antigen autoantibodies anti-thyroglobulin, anti-insulin autoantibodies target antigen addicted, sometimes capable of binding to the target antigen to form a complex, and can interfere with the ELISA method, an antigen-antibody assay results. To avoid the above situation, the solution is: determination of physical and chemical methods required before dissociation measured after.

    (5)Using murine CD3 monoclonal antibody therapy and other iatrogenic induced anti-mouse Ig (s) to carry out clinical antibody, diagnostic imaging and targeted therapy with new technologies such as radiolabeled murine antibodies, are likely to make these the patient's body to produce anti-mouse antibody; in addition, bitten by rats and other rodents patient can produce anti-mouse Ig (s) antibodies. These patients can produce false-positive ELISA measured. The solution is: the determination of an antigen, was added a sufficient amount of normal mouse Ig (s) in the specimen, thereby overcoming the above causes of false positives.

    (6)Cross-reactive substances type digoxin, class AFP-like substances, the target antigen is cross-reactive substance. When measured with a polyclonal antibody antigen assay results have little effect, but in the determination of the antigen with monoclonal antibodies, if the cross-antigenic determinant is exactly when monoclonal antibodies corresponding to the target epitopes, also false positive results.

    (7)Effect of specimens of other ingredients high serum lipids, bilirubin, hemoglobin and blood viscosity is too large, are the results of the ELISA assay interference effect.

    2 . Xenobiotics

    Xenobiotics is often due to improper ELISA assays for blood specimen collection, storage and so on. Such as hemolysis, specimens were contaminated with bacteria samples stored for too long, and specimen collection tube agglutination incomplete additives and other effects.

    (1)due to various anthropogenic causes hemolysis hemolysis caused by the destruction of red blood cells may be due to the release of large amounts of dissolved with peroxidase activity of hemoglobin, the horseradish peroxidase marked ELISA assay, will resulting in non-specific color, interference measurement results. To overcome these interference effects, samples must be taken to avoid hemolysis acquisition.

    (2)Bacterial contamination of the specimen due to the bacterial cell may contain endogenous horseradish peroxidase, therefore, it specimens with bacterial contamination as hemolysis, can generate non-specific interference color and the measurement results.

    (3)Improperly stored specimens kept in the refrigerator too long specimens, serum IgG can be polymerized into polymers, AFP may form a dimer in an ELISA assay indirect method will result in the end is too deep, or even result in false positives; specimens standing time too long (eg more than one day), sometimes weakened immune activity of antigen or antibody can also be a false negative. To overcome such interference, serum samples should ELISA assay for the freshly collected; if not immediately determined serum samples measured 5 days can be stored at 4 ℃, serum samples should be measured 1 week after cryopreservation; after thawing frozen specimens , local protein concentrate, uneven distribution, should be thoroughly mixed before measuring, mix gently but should not be shaken vigorously.

    (4)Specimens agglutination incomplete without coagulant and anticoagulant presence, the normal blood collection 1/2 ~ 2h begins to solidify, 18 ~ 24h completely solidified. Clinical laboratory work, sometimes in order to gain time for rapid detection, that is often forced to separate serum by centrifugation at yet started clotting, at this time still remains part of the serum fibrinogen in an ELISA assay process can form visible fibrin block, could easily lead to false positive results; in such cases because the next day when the review is complete coagulation, serum fibrinogen no longer exists, so review the results become negative. To avoid this interference, the best solution is to have it fully coagulated blood samples were collected after separation of serum, or blood collection time by specimen collection with separating gel or adding a suitable coagulant in blood collection tubes.

    (5)Sample tube was added substances affect anticoagulant (such as heparin, EDTA), enzyme inhibitors (such as NaN3 ELISA system can inhibit horseradish peroxidase activity) and rapid separation of serum separating gel and so on the ELISA assay has certain interference. In summary, clinical laboratory ELISA assay appears false positive or false negative results, taking into account factors and operational factors reagent addition, more factors were analyzed specimens should respect and shall take appropriate measures to eliminate interference, thereby provide accurate and reliable test results for clinical practice.

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