Monitoring the concentration of TNF antagonist drugs and detecting the development of anti-drug antibodies (ADA) enables physicians to optimize patient treatment over time. The test results help physicians understand underlying causes of suboptimal outcomes, make informed therapy choices, and provide more effective treatment to their patients. The use of TNF antagonist has revolutionized the treatment of patients with several non-infectious inflammatory disorders, including Crohn disease and ulcerative colitis.
There are different approaches to manage patients with treatment failure to TNF antagonists; one approach is to monitor drug levels and anti-drug antibodies. A new guideline from the American Gastroenterological Association on therapeutic drug monitoring in inflammatory bowel disease recommends that physicians should perform reactive therapeutic drug monitoring to guide changes in TNF antagonist therapy.
Current methods for ADA detection are complicated by the fact that most TNF antagonist are antibodies and by the complexity of measuring antibodies against antibodies in non-functional binding assays. More importantly, all non-ARUP methods fail to differentiate binding from neutralizing ADA.
ARUP’s TNF antagonist activity and neutralizing antibody assays are cell-based bioassays that measure the ability of a drug to inhibit TNF. The assays also detect the presence of antibodies that neutralize drug activity. Emergence of these neutralizing antibodies in a patient leads to treatment failure. Other methods detect anti-drug antibodies that bind to the drug, but unlike ARUP’s assays, these methods are not able to distinguish whether the antibodies neutralize drug activity or not.
How ARUP’s Test Works
- TNF antagonist activity and its inhibition by TNF antagonists are measured by stimulation of cells carrying a TNF-responding luciferase gene, and quantifying luciferase expression by luminometry. The assay uses the principles of iLite™ technology (reference).
- View the method brochure (download).
- The following cutoff values are used: 0.65 ug/mL (for TNF antagonists), and 1:20 dilution (for anti-drug antibodies). Values below these cutoffs will be reported as "Not Detected."
This functional reporter gene assay uses the principles of iLite technology (licensed by Euro Diagnostica).
Reporter cells carry a TNF inducible, NFκB-regulated firefly luciferase reporter-gene construct. When TNF is added to the cells, the reporter gene turns on and generates firefly luciferase, which is measured by a luminometer. Results of firefly luciferase expression are normalized relative to the expression of the renilla luciferase gene, which is carried by the same reporter cell and under the control of a constitutive promoter.
Serum of a patient taking a TNF antagonist drug is mixed with TNF and added to the cells. If the drug is present, it will block the activity of TNF, decreasing luminescence. Serum concentration of biologically active TNF drug can be calculated using a calibration curve.
Some patients develop antibodies to the drug. In the presence of neutralizing antibodies, the reporter gene is turned on despite the presence of exogenous drug in the assay. The antibody titer is obtained by identifying the dilution point of a patient’s serum where blocking of the drug activity is no longer observed.
The functional reporter gene assays were clinically validated for diagnosing and monitoring TNF antagonist treatment failure.
Currently, the ARUP assays are the only clinical assays available for the detection of biologically active TNF drugs and ADA with drug-neutralizing function, as recommended by the FDA.
The ARUP cell-based assay is inherently more reflective of the in vivo situation in tissue and circulation, under which TNF antagonists drugs are believed to function, and can easily be adapted for all known anti-TNF drugs.
How It Compares with Other Tests
Methods to monitor anti-drug antibody (ADA) levels in serum The main difference is that ARUP's test measures bioactivity of the drug and of the neutralizing antibody.
|ARUP Infliximab assay
(reporter gene assay)
|Fluid-phase RIA||Homogeneous mobility-shift assay (size exclusion HPLC)|
|What the test measures
|in vivo relevance to the drug's capacity to inhibit TNF in patient||High||Low||Moderate||Moderate|
|Specifically detects ADAs with drug-neutralizing function||
|Detection of IgG4 antibodies (major isotype of ADAs)||Yes||No||Yes||Yes|
|Possibility of false negative ADA due to drug in sample||Low||High||Low||Low|
|Possibility of false positives due to neoepitopes, nonspecific binding, rheumatoid factors, complement components||Low||High||Low||Low|
ADA = anti-drug antibodies; ELISA= enzyme-linked immunosorbent assay; RIA = radioimmunoassay
How does ARUP's Infliximab test compare with other tests?
- The main difference is that ARUP's test measures bioactivity of the drug and of the neutralizing antibody. See table below for comparison of methods to monitor anti-drug antibody (ADA) levels in serum.
ARUP Infliximab/Adalimumab Assay
ARUP assy (reporter gene assay) ELISA (solid-phase bridging) Fluid-phase RIA Homogeneous mobility-shift assay (size exclusion HPLC) What the test measures Bioactivity Binding Binding Binding in vivo relevance to the drug's capacity to inhibit TNF in patient High Low Moderate Moderate Specifically detects ADAs with drug-neutralizing function Yes No No No Detection of IgG4 antibodies (major isotype of ADAs) Yes No Yes Yes Possibility of false negative ADA due to drug in sample Low High Low Low Possibility of false positives due to neoepitopes, nonspecific binding, rheumatoid factors, complement components Low High Low Low
What is the precision of measurements of drug and drug-neutralizing antibody in this test?
- Based on ARUP's internal validation data, the CVs are less than 20% for both the drug and the drug-neutralizing antibody measurements.
The instruction says to test the patient before infliximab or adalimumab treatment. Why? How soon can the test be done after the last infusion?
- The most effective timing for the test is just prior to the next treatment when the drug level is lowest in the patient (at trough). This is the time when information about drug metabolism can best be determined. Accurate levels of anti-drug antibody (ADA) cannot be measured if the patient is tested immediately after infliximab treatment. The overwhelming amount of drug will absorb the ADAs and will interfere with its detection. This is true with all tests regardless of the methodology used. The minimum amount of time required after the last infusion for testing will depend on the pharmacokinectics and pharmacodynamics of the drug in each patient. In general, it should be at least 72 hours after treatment and we strongly encourage testing right before the next treatment. The bioactivity measurements of the iLite assay used in ARUP's test has a lower possibility of false negative results for ADA measurements compared to traditional methods using solid phase ELISA.
- Spotlight on Testing Video Test-Guided Management of Treatment Failure to TNF-alpha antagonists by Julio Delgado, MD, MS
- TNF-Inhibitor Testing Information Brochure
- Clinical Laboratory Validation of a Functional Assay for Monitoring Infliximab Biological Activity and Neutralizing Anti-infliximab Antibodies Presented by Julio Delgado, MD, Igor Pavlov, PhD, Noriko Kusukawa, PhD, and Lisa A. Skodack Jones
- Comparison between ARUP and Prometheus Infliximab Assays ARUP’s Infliximab Activity and Neutralizing Antibody assay and Prometheus’ Anser IFX test
- ARUP Consult Inflammatory Bowel Disease - IBD
- ARUP Consult Rheumatoid Arthritis - RA
- Adalimumab Activity and Neutralizing Antibody
- Adalimumab Activity with Reflex to Antibody
- Infliximab or Biosimilar Activity and Neutralizing Antibody
- Infliximab or Biosimilar Activity with Reflex to Antibody
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- Velayos FS, et al. A test-based strategy is more cost effective than empiric dose escalation for patients with Crohn's disease who lose responsiveness to infliximab. Clin Gastroenterol Hepatol. 2013 Jun;11(6):654–66.
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- Radstake TR, et al. Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis. Ann Rheum Dis. 2009 Nov;68(11):1739–45.
- Lazar-Molnar E, Delgado JC. Immunogenicity assessment of tumor necrosis factor antagonists in the clinical laboratory. Clinical Chemistry. 2016 Sep;62(9):1186–98.
- Pavlov IY, et al. Clinical laboratory application of a reporter-gene assay for measurement of functional activity and neutralizing antibody response to infliximab. Clinica Chimica Acta. 2016 Jan 30;453:147–53.