How To Detect And Remove Antibody Interference

If a test result is unexpected and heterophilic antibody is suspected as the source of interference, several strategies can be adopted for investigation.

1. Dilution linearity study with the specimen is the simplest way to document interference when observed values after dilution deviate significantly from the target values. Figure 1 illustrates the effect of successive dilutions of a HAMA containing sample (spiked with 32 ^g/mL of theophylline but observed value was 59 ^g/mL) compared to a serum-based calibrator for the assay (60 ^g/mL) for a theophylline immunoassay that uses mouse anti-theophylline antibody. The HAMA in the specimen interfered with the assay and initial value observed was 59 ^g/mL. After successive dilutions (with the assay diluent), the interfering antibody was diluted enough and with high dilution the interference was minimal (Datta, unpublished data). However, dilutions do not always correct the analyte value in the sample because of increased imprecision in the low end of the assay and the "matrix effect" between the calibrator matrix and the matrix of the original specimen.

2. Careful examination of the patient history (exposure to immunogenic animals or animal products; history of hyperactive immune system) may provide an important clue.

3. The treatment of the sample to block the interference or remove the interfering antibody and then repeating the assay.

There are various types of commercial or home-brew blockers for heterophilic antibody (26). The blocker can be a non-immune animal serum, polyclonal antibody,

Hama Dilution

Expected Theophylline (^g/ml)

Fig. 1. Human anti-mouse antibody (HAMA) interference detected by sample serial dilution.

Expected Theophylline (^g/ml)

Fig. 1. Human anti-mouse antibody (HAMA) interference detected by sample serial dilution.

polymerized IgG, nonimmune mouse monoclonals, and a mixture of monoclonal antibodies or fragments of IgG [Fc, Fab, F(ab')2] preferably from the same species used to raise the reagent antibodies. Commercially available blocking agents are immunoglobulin inhibiting reagent (IIR; Bioreclamation), heterophililic blocking reagent (HBR; Scanti-bodies), heteroblock (Omega Biologicals), MAB 33 (monoclonal mouse IgG1) and poly MAB 33 (polymeric monoclonal IgG1/Fab; Boehringer Mannheim). IIR is a proprietary formulation of high-affinity anti-animal antibody, and HBR is monoclonal mouse antihuman IgM. A suspected discordant sample, for example, sample giving false-positive hCG, may be separately incubated with the blocker and then re-assayed (26). Most commercial assay reagents now include such blockers. However, because of the heterogeneous nature of the interfering antibodies, no blocker can guarantee that there should be no interference from heterophilic antibody in all specimens analyzed by that particular assay.

Other approach to remove antibody interference is selective removal of the antibodies from a specimen. This can be achieved by selective adsorption of human IgG by a solid phase containing protein A or protein G (6). However, this method does not work if majority of the interfering antibodies are of IgM type. Alternately, the antibody fraction in the sample may be precipitated out with polyethylene glycol (preferable PEG 6000) reagent (27).

Because most drugs routinely monitored in clinical laboratories have small molecular weight, a simple approach to eliminate interference of heterophilic antibody is to analyze drug concentration in protein-free ultrafiltrate (free drug monitoring). The centrifugal ultrafiltration is a fast and relatively easy method to prepare proteinfree ultrafiltrate of serum using ultra-centrifugal cartridges with 10,000- or 30,000-D molecular weight cutoff filter membrane (e.g., Centrifree Micropartition System). Assay kits are commercially available for determination of free concentrations of phenytoin, carbamazepine, and valproic acid. For a drug with poor protein binding, for example, digoxin, assay kit for determination of total digoxin can be used for the determination of free digoxin concentration.

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