From: Handbook of Drug Monitoring Methods Edited by: A. Dasgupta © Humana Press Inc., Totowa, NJ

immunoassays, the analyte is detected by its interaction with a specific antibody (or a pair of specific antibodies). This reaction is further utilized in various formats and labels. The common formats of immunoassays have been described in Chapter 3.

In addition to the common sources of interference, such as exogenous factors, immunoassays additionally may also suffer from interference of endogenous human antibodies to the analyte or one (or more) components of the assay reagents. Such antibody interference can be categorized in four groups.

a. Autoantibody (human endogenous antibody to the analyte).

b. Heterophilic antibody (human endogenous, non-specific antibody that interact with assay antibodies).

c. Anti-animal antibody (human endogenous, specific antibody that interacts with assay antibodies).

d. Therapeutic antibody (antibody or its fragments used therapeutically).

Because it is difficult to distinguish between the effects of the group b and c, many investigators term them in general as heterophilic antibodies. Although many of the immunoglobulin (Ig) clones in normal human serum may display anti-animal antibody properties, only those antibodies with sufficient titer and affinity toward the reagent antibody used in an assay may cause interference.

Interference from such antibodies has been observed in all kinds of immunoassays, competition and non-competition (sandwich) types. In general, competition immunoassays, using a single primary antibody, are less affected by such interference than the sandwich immunoassays (which use separate capture and label antibodies). Furthermore, types of label used (enzyme, fluorescent, or chemiluminescent tag) have lesser effect with respect to such interference. The commonly used assay formats can be summarized as below.

1. Competition (limited binding site or primary antibody):

a. Homogeneous (no separation; bound label has different signal than unbound ones):

a. Immunoturbidimetric: regular or latex (latex-antibody or latex-antigen).

b. Fluorescence: fluoroimmunoassay or fluorescence polarization immunoassay (FPIA).

c. Label can be conjugated to the antigen or the antibody.

b. Heterogeneous (bound label is separated from unbound ones):

a. Capture reagent may incorporate the primary antibody with the label conjugated to the antigen or vice versa.

b. A secondary antibody may be used to isolate the immune complex or to amplify signal.

2. Immunometric ("Sandwich") assays are mostly heterogeneous and more susceptible to interference from heterophilic antibody.

a. Both primary antibodies (capture and label) from same species or different species.

b. There is no report of any interference from heterophilic antibody in assay using avian (chicken) primary antibodies. However, few commercial assays use chicken primary antibody.

c. Use of Fab or Fab' fragments or chimeric antibody may reduce interference from heterophilic antibody.

Because heterophilic antibodies are found mainly in serum, plasma, or whole blood, but not in urine, tests involving urine as a specimen are not subjected to interference from heterophilic antibodies. Therefore, comparison of values of an analyte in serum and urine (provided the analyte is present in both biological matrixes) provides a clue of the presence of heterophilic antibody in serum. For example, many case studies with false-positive human chorionic gonadotropin (hCG) in serum/plasma have been described in the literature where such interferences were not observed when urine specimens were analyzed (1).

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