Blockers Practical Guide

Blocking Assay Interference

Immunoassay interference is a general term for substances that can change the outcome of an assay by causing a false positive or false negative test result. Examples of potentially interfering particles include endogenous antibodies or other binding proteins present in a patient sample, polyreactive antibodies or autoantibodies (heterophiles), and human anti-animal antibodies. Background information on Immunoassay interference

TRUE Positive Result

Interfering substances can have non-specific reactions that disrupt the reaction between the analyte and reagent antibodies in an immunoassay by either: (1) out-competing the analyte of interest for binding to the assay antibodies (false negative) or (2) simulta- neously binding to the assay capture and detection antibodies in the absence of any analyte (false positive). Assays that are inherently vulnerable to interference include double- sandwich antigen detection assays, competitive assays and lgM capture assays. Specific examples which have been reported in the literature are assays for ToRCH, CEA, CA-125, CK-MB, LH, FSH, prolactin, TSH, AFP, cardiac troponin I (cTnl) and hCG. The most common type of interference in sandwich and competitive assays is heterophilic antibodies (HA) which are naturally occurring human antibodies with low affinities that can react with immunoglobulins from different species, including mouse, goat, rabbit, sheep and chicken. In diagnostic assays, HAs are able to bind to multiple and seemingly unrelated epitopes to disrupt the assay’s specific antigen-antibody interaction. Human anti-mouse antibodies (HAMA) is one type of HA interference that specifically binds to mouse antibodies. Due to the high use of mouse monoclonal antibodies in commercial diagnostic immunoassays, HAMA interference is the most widely experienced type. Rheumatoid factor (RF), an autoantibody that reacts with the patient’s own immunoglobulin (lg), may also cross-react with animal lg resulting in “RF interference”, which is similar to HA/HAMA interference. Generally, isotype (Fc region)-specific interfering HA is more common than idiotype (Fab or binding site)-specific HA. lgM capture assays generally experience two types of interference. The first is from high levels of patient lgG antibodies that can compete with lgM for antigen binding sites on the solid phase. Since lgG antibodies are highly abundant, representing approximately 75% of serum antibodies in humans, they can outcompete lgM due to their sheer quantity. The second type of interference is caused by lgM RF which can produce false-positive signals by reacting with the Fc fragment on immunoglobulins. RF are found in 1% to 4% of the general population and in 75% of adult patients over 65 years of age.

Detection Antibody Capture Antibody Antigen

Blocking Buffer

FALSE Negative Result

Blocking Buffer

Interfering antibody blocks the binding site on the capture or detection antibody. Occurs in both sandwich and competitive assays.

FALSE Positive Result

Interfering Antibody (HA interference)

Blocking Buffer

Interfering antibody binds to both the capture and detection antibody even in the absence of the antigen. Occurs in sandwich assays only.


Blockers Practical Guide

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