The development of immunoassays has revolutionized laboratory medicine, especially during the last 30 years. The implementation of new endpoint tracers, new assay formats, automatization, reproducibility, duration time of assay and availability of analyses have contributed for immunoassays to become everyday practice.
The main characteristic of all immunoassays – from immunoprecipitation to biochip assays – is that the reagent that discovers or quantifies the target analyte (antigen) contains the antibody. Interferences may be defined as the effect of substances present in an analytical system which causes deviation of the measured value from the true value.
In immunoassays, an antibody used as a reagent, detects an analyte (antigen) of interest. Although the noncovalent bound between the analyte and complementary antibody is specific, false-positive and false-negative interferences are possible. Some interferences are similar to those in chemical analyses and some are typical only for immunoassays. One should suspect interferences in following cases: a) upon receiving an unacceptable result, if there is non-linearity during dilution b) if there is no agreement with other test results or clinical data, c) if different immunoassays in determination of the same analyte provide significantly different results. These possible interferences can be described as:
1. Cross-reactivity with endogenous and exogenous non antibody-structured substances.
Cross-reactivity is the most common interference in immunoassays, but mostly in competitive ones. It is non-specific influence of these substances in a sample that structurally resembles the analyte (carries similar or the same epitopes like analyte) and competes for binding site on the antibody.
2. Cross-reactivity with endogenous and exogenous antibody-structure substances.
Immunoreactions can be influenced by antibodies present in the biological sample sof a patient or antibodies from the reagent. Biological samples can contain both exogenous and endogenous antibodies.
There are two types of endogenous antibodies in patients’ serum. Heterophilic antibodies (natural antibodies and autoantibodies) and anti-animal antibodies (human anti-animal antibodies; HAAAs). Although endogenous antibodies differ in some of these characteristics they interfere according to identical mechanism in saturating (sandwich) analyses – they “bridge” or simultaneously create complexes with capture and tracer antibodies of the reagent. Heterophilic antibodies are multi-specific antibodies synthesized to very poorly defined antigens. Human anti-animal antibodies are antibodies of high avidity and are synthesized to well defined antigens
3) The “Hook Effect”.
The hook effect is based on the saturation curve of an antibody with an antigen. Primarily, the hook effect depends on the concentration of the analyte. It implies the presence of a huge excess of analyte which saturates all binding sites on antibody. The effect occurs mostly (but not exclusively) in assays where all three components (antigen, antibody and marker) incubate simultaneously in a single step assay. The hook effect does not occur in competitive immunoassays. That means that in reaction there is a surplus on analytes that did not penetrate to analyte-antibody complex compound. This results in falsely decreased value of the measured analyte which could even lie in the reference interval.
4) The “Matrix” effect.
By knowing and recognizing interferences in immunoassays, one can avoid possible undesired consequences: diagnostic errors, treatment and monitoring of its efficacy, unnecessary additional laboratory testing, unnecessary therapy.
Serum or plasma samples are a complex compound of lipids, proteins, carbohydrates, salt and water. The sum of interferences of all sample components (with exception of analytes), which affect the target analyte to be measured is known as “the matrix effect”. The serum components that cause the most so called matrix effect have low binding affinity to the analyte or antibody. This component usually disguises the analyte or the antibody causing the absence of the binding reaction of analyte to antibody.
With the exception of endogenous elements which cause inter- and intra-individual variability of results, the concept of the matrix effect could be widened to exogenous components that relate to the impact of anticoagulant during plasma sampling or the impact of coagulation activator and separator during serum sampling. For example, Heparin therapy in patients with acute myocardial infraction (AMI) affects the result of determination of troponin I concentration. In addition, heparin binds with different affinity to some troponin forms present in patient’s blood in different phases after myocardial infraction. Further, EDTA can act upon release of free cTnl from calcium ion dependent cTnI-troponin C complex that causes falsely decrease of values in methods containing antibodies, directed to troponin complex.
Finally, Knowledge of numerous interferences is a prerequisite for their recognition which helps avoid possible undesirable consequences important for the Patient which may include: diagnostic errors, incorrect treatment plans and monitoring of unnecessary therapy.