Immunoassay Testing Post

Interferences in Immunoassays, Part 1

Substances that alter the measurable concentration of the analyte or antibody (Ab) binding result in immunoassay interference. Interferences can be of 2 types – analyte dependent and analyte independent.

Analyte independent interferences refer to common interferences like hemolysis, lipemia, effects of anti-coagulant and sample storage. The term pre-analytical variables are commonly used for these types of interferences.

Analyte dependent interferences refer to interaction between constituents in the sample with one or more reagent antibodies. They include compounds with chemical differences but structural similarities that cross react with the antibody i.e. heterophile antibodies, human anti-mouse antibodies (HAMA), rheumatoid factors and other proteins.

Interferences can cause false positive and false negative results. RF, HAMA and heterophile antibodies bind to the Fc portion of the IgG molecule and cause false positive results (Fig A). The interferences can bind to the capture Ab due to structural similarities and cause false negative results (Fig. B).

Immunoassay manufacturers take several steps to minimize interference (Fig. C).

1. Characterize epitypes to increase Ab specificity.
2. Use antibody coating methods that minimize the exposure of the Fc portion of the IgG.
3. HAMA blockers.

It is sometimes necessary for laboratories to confirm the presence of HAMA or other interfering substances and pre-treat the sample to neutralize interference.

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