Blockers Practical Guide

Interference in Sandwich Immunoassays

A sandwich immunoassay uses two antibodies (either monoclonal or polyclonal) that bind to different sites on the antigen or analyte of interest. The capture antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. However, human anti-animal immunoglobulin antibodies (HA antibodies) can interfere with this interaction and reduce assay sensitivity and specificity.

Mixed Species Assays

Double Mouse Monoclonal Assays

e.g. goat, sheep, rabbit, rat, chicken, mouse

Detection Ab

Mouse MAb Antigen Mouse MAb

Antigen Capture Ab

Blocking Buffer

e.g. mouse, chicken, rabbit, rabbit, goat

For mixed species sandwich assays, the IgG blocker used must be the same as the host of the capture and detection antibodies. More than one species of IgG is required when two different antibody species are used in a sandwich assay.

For double mouse monoclonal antibody sandwich assays, mouse IgG in addition to an active HAMA blocker (TRU Block) should be used to remove a specific type of HA interference called human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF).

interference in IgM Capture Assays

IgM antibodies are the first type of antibodies produced by the immune system in response to an infection. Consequently, lgM detection assays have proven to be valuable diagnostic tools that assist in identifying early and recent infections. However, lgM antibodies only comprise 5% to 10% of all the antibodies in the body. In contrast, lgG antibodies are the most abundant immunoglobulin and comprise about 75% to 80%. In order to ensure an lgM assay is both sensitive and specific, it is necessary to reduce assay interference, especially from the more plentiful lgG antibodies and other non-specific proteins, such as rheumatoid factors (RF).

Goat Anti-human IgG or IgM Diluent

Detection Antibodies

Patient IgG

Detector Label

Patient’s IgM

Antigen

IgM Capture Assay