Glossary - interference in immunoassays


Human anti-animal antibodies (HAAA) are generated after contact with animal immunglobulins. Therefore HAAAs can be found in blood of human patients. They can be IgG, IgA, IgM or IgE antibodies. HAAAs are important interfering substances in many diagnostical immunoassays. Up to 80% of all samples from patients can contain HAAAs (depending on published study). Some antibodies can bind the Fc fragment as well as the Fab fragment of the used antibody. Thus analyte binding is reduced or prevented. The consequence is a false negative result. HAAAs can also cause false positive results. One example is the cross linking of assay antibodies by HAAAs in the absence of analyte. The results are false positive signals. If a HAAAs binds to the Fc fragment of an antibody it is called an anti-isotypic antibody. An anti-idiotypic antibody binds directly to the highly variable region of the Fab fragment.

Anti-idiotypic antibodies

Anti- idiotypic antibodies can bind directly to the highly variable region of the Fab fragment of another antibody.
They bind directly to the antigen binding site of the antibody.
Anti-idiotypic antibodies can therefore compete with the original antigens of other antibodies.
So they can cause different interference effects in immunoassays.

HAMA with anti-idiotypic binding properties to the capture antibody. The interfering antibody binds to the highly variable region of the Fab fragment. This prevents analyte binding with the consequence of false negative results.
HAMA with anti-idiotypic binding properties to the detection antibody. The interfering antibody binds to the highly variable region of the Fab fragment. This prevents analyte binding with the consequence of false neagtive results.

Anti-isotypic antibodies

Anti-isotypic antibodies are antibodies which bind to the Fc fragment of another antibody. If they bind to assay antibodies, they can cause interference in immunoassays.


Cross-reactivity is the ability of the antibody to bind other structures than the target analyte. These structures have often a high similarity or homology to the analyte. The term cross-reactivity is used if the cross-reacting substance is known and the ability of cross-reacting has been proved e.g. by testing the competitive concentration of the cross-reacting substance. Cross-reacting substances are known for many clinical parameters and have to be mentioned in diagnostical findings. This can be tested by parallel measurements of the known cross-reacting substances. Due to financial reasons it’s often done without the reasonable additional tests. There is a smooth transition between nonspecific binding and cross reactivities. If the used antibodies have a high affinity, these problems can be avoided. Cross-reactivity can be prevented by using LowCross-Buffer®.

Cross linking caused by heterophilic antibodies or by HAMAs. The capture antibody is linked with the detection antibody.
Cross reactivity of an interfering substance with the capture antibody. The results are fals negative signals.
Cross reactivity of an interfering substance with the detection antibody. The results are false negative signals.


Human anti-mouse antibodies (HAMA) are human antibodies which can bind specifically mouse antibodies. They originate from long-lasting contact to pets as well as from medication of therapeutic antibodies. HAMAs interfere with immunological methods working with mouse antibodies. Due to high sequence homology between different species, HAMA containing sera can also interfere with assays containing antibodies from other species.

Cross linking caused by HAMAs. The capture antibody is linked with the detection antibody. The results are false positive signals.

Heterophilic antibodies

Heterophilic antibodies are antibodies which can cross-react with several antigens. They can bind not only to the specific antigen but also to many other antigens too. They are multispecific antibodies of the early immune response or interfering antibodies with unknown origin. They can be IgG, IgA, IgM or IgE antibodies. Especially IgM play a key role in sera from rheumatic patients. These antibodies can bind Fc fragments of human antibodies. Thus they can also bind Fc fragments of the antibodies used in an assay. Rheumatic sera can connect capture and detector antibody with the result of false positive signals. The interfering heterophilic antibodies have normally low binding affinity. They interfere mainly with assays with low dilution of serum or plasma specimens due to low concentration of analyte.

Cross linking caused by heterophilic antibodies. The capture antibody is linked with the detection antibody. The results are false positive signals.

Hook effect

The Hook effect or high dose Hook effect describes a wrong low measurement of analytes which are present in the specimen in a very high concentration.
If the analyte concentration is too high, all antibody binding sites are fully occupied.
Additional analyte molecules can not be measured within the limit of the binding curve.
This fact leads to wrong low results. Parallel measurements of different sample dilutions can detect a high dose hook effect and so you can adjust the measurement.
Well-known clinical parameters which are subject to high dose hook effect are: e.g. CRP, AFP, CA 125, PSA, ferritin, prolactin and TSH.

Hook effect. High analyte concentration can simulate under specific conditions false low signals.


Interference is a generic term for effects leading to bad results. Interference leads to bad trueness, precision and reproducibility of immunoassays. There are many different kinds of interference. In many cases the decision, which kind of interference effect occurs, is not clear due to unknown molecular reasons for interference. Interference can be matrix effects, if the matrix is the source of the effect. It can be sample interference, if one or a few constituents of one sample generate the problem. Also cross-reactivities are a kind of interference, when the cross-reactant is known. Different kinds of interference are explained in the glossary.

Interference caused by endogenous components of the sample

Also natural existing proteins of the specimens can cause interference.
Well-known interfering substances in human sera are: albumins, complement, lysozyme and fibrinogen. Endogenous proteins can bind assay antibodies or target analyte and so mask them. Substances can be demasked by using LowCross-Buffer® as assay buffer.

Cross reactivity with capture and detection antibody. In practice it is an uncommon phenomenon, but possible with antibodies of low specificity. Such interference can be caused by antibodies which are targeted on conserved amino acid sequences of a protein. These conserved amino acid sequences can also be found in other (therefore interfering) proteins.
Masking of the analyte by a specimen protein. The capture antibody can not bind the epitope. Binding of analyte is not possible or bad (in case of sterical hindrance). The results are false negative signals.

Matrix effect

Matrix effects are a kind of interference with an unknown cause. Matrix effects lead to poor accuracy of immunoassays. The molecular reason for the problem is not known, but the composition of the sample causes the interference. The transition to other interfering effects is fluent. Responsible for matrix effects are for example: Anti-animal antibodies, heterophilic antibodies, endogenous interfering substances or influence of viscosity, pH value or salt concentration. In some cases one can detect the molecular reason for such matrix effects. Then an effect, which was firstly described just as a matrix effect can be called a sample interference or a cross-reactivity, if the cross-reactant has been detected and described.

Nonspecific binding

Nonspecific binding is binding of the assay antibodies which is not correlated with the specificity of the antibodies. Also analytes can bind non-specifically.
There are two kinds of nonspecific binding which normally occurs in the lab and which can not be distinguished from each other easily.
On the one hand there is nonspecific binding to surfaces like ELISA wells or Western blotting membranes. The results are e.g. high background or false positive results in ELISA. The second reason for nonspecific binding also can lead to high background in ELISA. Reason is binding to substances in solution which are present in high concentration e.g. albumin or immunoglobulin. This effect can be prevented by using modern buffer like LowCross-Buffer®. Even nonspecific binding of analytes can also cause wrong low results instead of false positive results. This happens if the analyte is partially and nonspecifically bound to albumin or other constituents of the sample. The so masked analyte can not be detected by the assay antibodies. In this case LowCross-Buffer® can help as well.

Nonspecific binding of a labeled detection antibody on a not saturated surface. The results are false positive signals.
Nonspecific binding of al labeled detection antibody to a blocked surface. The surface is blocked, but the antibody binds to the blocking proteins itself.
An interfering protein binds to the Fc fragment of the detection antibody. Thus binding of the analyte is inhibited sterically. The results are false negative signals.
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