119ELISA-based assays are not currently employed in most commercial tests for
anti-TNF levels and antibodies.
An alternative to the sandwich-based ELISA is the fluid-phase-based radioactive
immunoassay (RIA). This assay is not typically employed in the United States [ 42 ]
but has been employed in some parts of Europe. In the RIA, serum is incubated with
radiolabeled soluble antigen, followed by the addition of an anti-Fc fragment. These
complexes then precipitate out of solution and are collected via centrifugation. The
RIA can measure anti-drug antibodies in the presence of the drug and can also mea-
sure the drug in the presence of the antibody. The use of radioactive compounds has
limited the utilization of this assay, however.
Another liquid-phase assay that has been developed to measure anti-TNF drug
levels and antibodies is the high-pressure liquid chromatography mobility shift
assay (HMSA). With an initial acid dissociation phase that allows for separation
of drug and anti-drug antibody complexes, this assay is capable of measuring both
drug levels and anti-drug antibody levels when each is present. After dissolution,
fluorescent-labeled drug or anti-drug antibody is used to measure anti-drug anti-
bodies and drug levels, respectively. Liquid chromatography can also be combined
with mass spectrometry (LC-MS/MS). These unique approaches allow for
increased sensitivity compared to ELISA, without requiring the radiolabeled
markers needed for RIA.
One of the most recently developed assays is the electrochemiluminescence
immunoassay (ECLIA), a solid-phase test that can measure both anti-TNF levels
and antibodies directed against anti-TNFs. Although anti-drug antibodies can be
measured in the presence of drug, their presence may increase the inaccuracy of
determining the levels of the drug itself. This assay still requires clinical valida-
tion [ 42 ].
Several of these assays are available commercially. Prometheus has developed
two HMSAs: the ANSER IFX assay for the measurement of infliximab and ATI
levels and the ANSER ADA assays for the measurement of adalimumab and anti-
adalimumab antibodies. As noted, HMSAs are capable of measuring both drug
and anti-drug antibodies independently of each other. The ANSER assays have
also been evaluated in several clinical studies and have been validated. One sig-
nificant limitation of preventing widespread utilization of these assays is their
cost [ 42 ]. LabCorp has also developed a commercially available assay for the
measurement of both infliximab and adalimumab drug levels and anti-drug anti-
bodies, specifically ECLIAs. As mentioned, these assays are also capable of mea-
suring anti-drug antibodies in the presence of the drug and are generally less
expensive. However, there remains a paucity of data regarding the clinical valid-
ity and application of this commercial assay. Lastly, the Mayo Clinic has also
developed a new assay combining both mass spectrometry and liquid chromatog-
raphy. Unlike other assays that determine both antibody levels and drug levels
automatically, this two-step assay first measures serum drug concentrations and
then reflexively measures antibodies only when the anti-TNF level is below
5.1 μg/mL. This assay is currently available commercially for infliximab, and
validation is still required.
8 Therapeutic Drug Monitoring of Biologic Agents