Reasons of ELISA test failure

1. Samples

2. Reagents

3. Operation

Serum is mostly common used samples for ELISA test, plasma is normally regarded same as serum. False positive or false negative of samples are mostly resulted by disruptors which is divided into endogenous substances and exogenous substances.
1. Endogenous substances
About 40% of human serum contains nonspecific disruptors which will affect testing result at some degree. Some common disruptors are: rheumatoid factors (RF), complement, heterophil antibodies, target antigen autoantibodies, Iatrogenic induced anti-rat ig(s)antibodies, cross reacting material and others.
(1) Rheumatoid factors (RF)
The IgM and IgG rheumatoid factors in human serum can combine with capture antibodies and FC fragment of DON-HRP-Conjugated in the ELISA system, its result will be false positive.
Solution: a. Replace intact IgG by F(ab)2 b. Process samples by solid adsorbent combined thermal denaturation IgG(Adding thermal denaturation IgG into sample diluent will work as well) c. while antigen testing, add 2-ME into sample diluent to degrade rheumatoid factors.
(2) Complement
During solid detection antibody and labeling conjugated antibody, antibody molecules may change, its C1q molecules binding site of FC fragment will be revealed which allows C1q to connect both, and result false positive.
Solution: a. Use EDTA to dilute samples, b. use 53°C，10 min or 56°C，30 min to heat serum and inactivate C1q.
(3) Heterophil antibodies
Human serum contains natural heterophil antibodies which can combine with Ig (s) of rodents(like rat). It can connect detection antibody and conjugated antibody, resulting false positive.
Solution: Adding excessive animal Ig (s) into sample diluent. But if not enough adding or different subtype, it will not work.
(4) Target antigen autoantibody
Some target antigen autoantibodies like antithyroglobulin, anti-insulin, can combine with target antigen and form complex. It will affect the antigen, antibody testing result while ELISA assay.
Solution: Dissociate the target antigen autoantibody by physicochemical method before testing.
(5) Iatrogenic induced anti-rat Ig(s) antibodies
Some new technologies, like using mouse CD3 etc monoclonal antibodies for treatment, imaging diagnosis and targeted treatment by radioisotope labeled murine antibodies, may generate anti-rat antibodies in bodies of patients. Moreover, patients which are bitten by rodents may have anti-rat Ig (s) antibodies. While making ELISA assay on these patients, result may be false positive.
Solution: Adding enough normal mouse Ig (s) into samples to overcome false positive caused by the above reasons.
(6) Cross reacting material
Some substance may make cross reaction with targeted antigens. When testing antigens by polyclonal antibodies, it will affect result too much, but when monoclonal antibodies, if determinant of crossing antigens is just the corresponding targeted determinant of monoclonal antibodies used, it will cause false positive.
(7) Others
High serum lipid, bilirubin, hemoglobin and high blood viscosity etc will interfere the ELISA testing result.

2. Exogenous substances
Exogenous substances are usually caused by improper blood sample collection and storage.
(1) Sample hemolysis
Sample hemolysis caused by human factors will give out tremendous hemoglobin with peroxidase activity while red cells are damaged and lyse. In ELISA testing labeled by horseradish peroxidase, when collecting samples, hemolysis should be prevented to overcome the above problem.
(2) Polluted samples
Thallus may have endogenous horseradish peroxidase, so samples polluted by gems, as well as hemolysis samples, will have nonspecific staining which will interfere testing result.
(3) Improper sample storage
Samples stored in fridge for long time, IgG of its serum can polymerize to polymer, AFP may form dipolymer. For indirect ELISA assay, it will result deep background, or even false positive.
Samples are placed for long time(more than 1 day), immunoactivity of its antigens or antibodies will reduce, and false negative may occur. To overcome the above interference, the serum samples should be freshly collected. If ELISA assay can not be made instantly, serum samples should be kept in 4°C for 5 days, or lower temperature for one week. The protein of melted samples may be concentrated partially, and unevenly distributed. It should be fully mixed up before testing. While mixture, keep soft and avoid strong vibration.
(4) Incomplete agglutination
Blood will start to be coagulated after 1/2-2 hours without coagulant or anticoagulant. After 18-24 hours, it will be completed coagulated. To save time for fast test, serum is separated centrifugally before starting coagulation. In this case, serum may be still left some fibrin. While ELISA assay, it may form some white fibrin block which can be detected by eyes, and results false positive. When double check in following day, as blood is completely coagulated, and no fibrin in serum, its result will become negative. To overcome the above problem, it is better to separate serum after blood is completely coagulated. Or, collect samples by blood vessel with separation gel or add some coagulant into blood vessel.
(5) Additions in sample vessel
Anticoagulant (heparin, EDTA), enzyme inhibitor(NaN3 can inhibit the activity of horseradish peroxidase in ELISA system), separation gel which can rapidly separate serum will interfere the ELISA assay. Totally speaking, if false positive or false negative occurs, except reagents and operation problem, we should consider more about samples, take corresponding actions to clear away interference and provide accurate, reliable result for testing.