Measurement of Infliximab and Anti-Infliximab Antibody Levels Can Help Distinguish Maintenance Versus Loss of Response
Steenholdt C, Bendtzen K, Brynskov J, Thomsen OØ, Ainsworth MA. Cut-off levels and diagnostic accuracy of infliximab trough levels and anti-infliximab antibodies in Crohn’s disease. Scand J Gastroenterol. 2011;46:310-318.
Inflammatory bowel disease (IBD), comprised primarily of Crohn’s disease (CD) and ulcerative colitis (UC), is estimated to affect approximately 1 million individuals in the United States alone, with approximately 30,000 new cases diagnosed annually.1 A hallmark of this disease is chronic inflammation of the intestinal mucosa, which results in part from production of the tumor necrosis factor–α (TNF-α) cytokine. Understanding of this pathogenesis has been exploited for treatment purposes, and antibodies directed against TNF-α have proven to be highly effective for the induction and maintenance of remission in both CD and UC. Infliximab (Remicade, Janssen Biotech) was the first anti–TNF-α biologic therapy approved for the treatment of both CD and UC, based on clinical trial data showing robust efficacy in these conditions.2-6
Despite its proven efficacy, a subset of patients do not respond to infliximab (and other anti–TNF-α agents).2 Additionally, some patients achieve an initial response to induction therapy but lose this response over time with maintenance treatment. The reasons for these therapeutic failures remain a matter of debate. One possibility is that loss of response is due to an immunologic mechanism, whereby the patient mounts an immune response to infliximab, thus forming anti-infliximab antibodies. Multiple studies in CD patients have linked the development of anti-infliximab antibodies with loss of treatment response and shorter duration of response.7-10 Another possibility is that loss of response to infliximab is pharmacologic in nature; under this mechanism, individuals’ differing pharmacokinetic or pharmacodynamic profiles may contribute to their inability to maintain a therapeutic serum level of infliximab. Indeed, low serum infliximab concentrations have been linked to a lack of clinical response in both CD and UC.7,11-14
Therapeutic failures with infliximab, and the underlying reasons for these failures, pose a significant challenge for clinicians who manage patients with IBD. There are no standard guidelines defining a therapeutic strategy among this patient subset, although treatment algorithms have been proposed.15,16 The lack of such guidance is primarily due to a paucity of data demonstrating clinically relevant threshold levels of infliximab and/or anti-infliximab antibodies. Further, clinicians do not yet know whether the use of such threshold levels, if identified, would aid in the discrimination of responding versus nonresponding patients. These knowledge gaps led to the design of the recent study by Steenholdt and colleagues, published in the Scandinavian Journal of Gastroenterology, which is the first study to establish threshold values for clinically relevant concentrations of circulating serum levels of both infliximab and anti-infliximab antibodies in IBD patients.17
Study Description
A total of 106 patients (85 with CD and 21 with UC) were identified over the course of 10 years (January 2001 to June 2010); these patients were all receiving care at a tertiary care center. All patients received infliximab treatment for IBD, as well as concurrent hydrocortisone, acetaminophen, and cetirizine to prevent acute infusion reactions and to limit the development of anti-infliximab antibodies.8
IBD patients who received infliximab maintenance therapy (defined as regular infliximab infusions every 4–12 weeks, with the first infusion occurring within 8 weeks following completion of induction therapy) were classified as having 1 of 2 responses: maintenance of response or loss of response. Patients who maintained response had a good clinical response to infliximab induction therapy and continued this response over the course of maintenance treatment. In contrast, patients with a loss of response initially experienced a good clinical response to infliximab induction therapy but subsequently lost this response during maintenance treatment, resulting in discontinuation of therapy. Classification of infliximab response was based on clinical assessment alone; investigators were blinded to the results of the serum trough level analyses.
Trough levels of infliximab and/or anti-infliximab antibodies were measured; these levels were defined as the serum concentration immediately prior to an infliximab infusion. Infliximab levels were measured using a fluid-phase radioimmunoassay, in which radiolabeled TNF-α (125I-TNF-α) was used as a tracer. When measuring infliximab concentrations, this assay selected for functional infliximab molecules by detecting only those infliximab antibodies that bound the radiolabeled TNF-α tracer. Anti-infliximab antibody concentrations were measured in a similarly designed assay, with radiolabeled infliximab (125I-infliximab) used as a tracer. A receiver operating characteristics (ROC) analysis was performed to identify optimal cutoff levels for each antibody (infliximab and anti-infliximab); the cutoff values that were selected displayed the least difference between sensitivity and specificity.
The majority of patients included in this study had CD (n=85). Of these patients, 69% maintained their response to infliximab, while the remaining 31% were characterized as having lost response to infliximab. The baseline characteristics of the 2 patient groups were well balanced, including patient age, sex, duration of disease, and disease type. Further, there were no significant differences in the total number of infliximab infusions administered to the 2 patient groups.
Infliximab trough levels were significantly increased among CD patients who maintained response to therapy compared to patients who lost response (median infliximab trough level, 2.8 µg/mL vs 0 µg/mL; P<.0001). Using data from these patients, a cutoff value of 0.5 µg/mL was defined as clinically relevant for infliximab trough concentrations. Infliximab trough concentrations less than 0.5 µg/mL were associated with a sensitivity of 86% (95% confidence interval [CI], 64–97) and a specificity of 85% (95% CI, 72–94) for identifying patients with a loss of response to infliximab maintenance therapy. In a ROC analysis, the cutoff value of 0.5 µg/mL for infliximab trough level was determined to have significant effectiveness (P<.0001), with an 87% accuracy rate. When this cutoff value was considered in a binary manner (<0.5 µg/mL vs ≥0.5 µg/mL), 73% (95% CI, 52–88) of CD patients with low infliximab trough levels showed a loss of response, while 95% (95% CI, 83–99) of CD patients with high trough levels maintained response.
Trough levels of anti-infliximab antibodies were equally revealing. Anti-infliximab antibody trough levels were significantly higher in CD patients who had lost response to infliximab maintenance therapy compared to patients who had maintained response (median anti-infliximab antibody trough level, 35 U/mL vs 0 U/mL; P<.0001). Using these data, a cutoff value of 10 U/mL was defined as clinically relevant for anti-infliximab antibody trough concentrations. Notably, this value corresponded to the detection limit of the radioimmunoassay used to measure antibody levels. Anti-infliximab antibody trough levels of 10 U/mL or higher were associated with a sensitivity of 81% (95% CI, 61–93) and a specificity of 90% (95% CI, 79–96) for the identification of CD patients who had lost response to infliximab maintenance therapy. In a ROC analysis, the cutoff value of 10 U/mL for anti-infliximab antibodies was found to have an 87% accuracy rate. This cutoff value was applied to define a binary threshold for anti-infliximab antibody trough levels (<10 U/mL vs ≥10 U/mL); using this binary threshold, 78% (95% CI, 57–91) of CD patients with high anti-infliximab levels had a loss of response to infliximab maintenance therapy, while 91% (95% CI, 80–97) of patients with low anti-infliximab antibody levels maintained their response.
Interestingly, patients who developed detectable (≥10 U/mL) trough levels of anti-infliximab antibodies were nearly 3 times more likely not to have received concomitant immunosuppressive therapy (azathioprine, 6-mercaptopurine, or methotrexate; odds ratio, 2.8; 95% CI, 1.2–6.8; P=.02). In addition, patients who had been re-treated with infliximab (defined as a minimum of 2 separate infliximab treatment series occurring ≥6 months apart) were significantly more likely to have developed detectable anti-infliximab antibody trough levels compared to patients who were receiving infliximab for the first time (odds ratio, 3.3; 95% CI, 1.2–9.2; P=.03). In contrast, neither concomitant immunosuppressive therapy nor infliximab re-treatment was significantly associated with trough levels of infliximab.
The threshold levels established for both infliximab and anti-infliximab antibodies were evaluated in combination in 69 CD patients (48 patients who had maintained response and 21 patients who had lost response). This combination was shown to be highly accurate (90%) for the association between trough levels and clinical response. The sensitivity and specificity of the combined analysis were 81% (95% CI, 57–94) and 94% (95% CI, 82–98), respectively. Among CD patients with both low infliximab trough levels (<0.5 µg/mL) and high anti-infliximab antibody trough levels (≥10 U/mL), a majority (85%) were classified as having lost response to infliximab maintenance therapy. Similarly, the vast majority (92%) of CD patients with high infliximab trough levels (≥0.5 µg/mL) and low anti-infliximab antibody trough levels (<10 U/mL) maintained their response to treatment.
Similar determinations of infliximab and anti-infliximab antibody trough levels were made in UC patients, although this group of patients was much smaller (n=21). The association between trough levels and infliximab response in UC patients showed a trend comparable to that observed in CD patients. High infliximab concentrations (median trough level, 3.8 µg/mL) and low anti-infliximab antibody concentrations (median trough level, 0 U/mL) were associated with maintenance of response, while low infliximab concentrations (median trough level, 0 µg/mL) and high anti-infliximab antibody concentrations (median trough level, 85 U/mL) were associated with a loss of response (P=.0083 and P=.0007 for association of infliximab and anti-infliximab antibody trough levels, respectively). The cutoff values for infliximab and anti-infliximab antibody trough levels were also determined for UC patients: 0.8 µg/mL for infliximab (75% sensitivity [95% CI, 35–97] and 100% specificity [95% CI, 48–100]) and 10 U/mL for anti-infliximab antibodies (80% sensitivity [95% CI, 44–97] and 100% specificity [95% CI, 69–100]).
Clinical Relevance
This study was significant because it defined clinically relevant threshold values for trough concentrations of both infliximab and anti-infliximab antibodies in IBD patients. Separately, these thresholds were shown to each have an 87% accuracy rate for identifying patients who showed maintenance of response versus loss of response to infliximab maintenance therapy. When the 2 threshold values were combined, the accuracy rate increased to 90%. Overall, the majority of patients with high infliximab trough levels and low anti-infliximab antibody trough levels maintained response to infliximab maintenance treatment; in contrast, most patients with low infliximab trough levels and high anti-infliximab antibody trough levels lost response to infliximab during maintenance treatment.
These findings are important because they provide insight into the clinical relevance of infliximab and anti-infliximab serum trough levels during maintenance therapy. While previous studies have also reported these trough levels, their association with response to infliximab therapy has been ambiguous. Some of this confusion may be due to the use of enzyme-linked immunosorbent assays (ELISAs) for the measurement of infliximab and anti-infliximab antibody levels, as use of these assays has met with some controversy in the field. For example, it is unclear whether ELISAs assess the amount of functional (bioactive) infliximab or if they detect only a subset of the total anti-infliximab antibodies formed; also, detection of anti-infliximab antibodies suffers from interference by infliximab present in serum. Additionally, ELISAs are subject to false-positive and false-negative results related to nonspecific binding or epitope masking, among other causes.
To overcome the shortcomings associated with the use of ELISA, Steenholdt and colleagues used a commercially available radioimmunoassay technique to measure infliximab and anti-infliximab antibody trough levels.17 For measurement of infliximab concentration, the radioimmunoassay selectively detects only functional (bioactive) infliximab by isolating the fraction of infliximab capable of binding TNF-α. When measuring anti-infliximab antibody concentrations, the radioimmunoassay is capable of detecting all immunoglobulin isotypes that bind infliximab. It should be noted that, unlike ELISAs, the methods for radioimmunoassay require more specialized equipment and include the use of a radioactive reagent; thus, the use of radioimmunoassays is limited to laboratories with the required equipment and facilities for handling radioactive materials. Also, because the cutoff values defined in this study were obtained using radioimmunoassays, it may not be possible to extrapolate them to values obtained using ELISA. The authors also acknowledged other important limitations of this study, including the lack of definitive criteria for response to infliximab maintenance therapy (eg, clinical activity index score or endoscopic evaluation, both of which have been previously validated for this purpose).
While cutoff values were established and found to be comparable (although not identical) in both CD and UC patients, limited conclusions can be drawn from this small and retrospective study. Prospective follow-up studies with larger patient sets will potentially help to confirm and validate these results.
References
1. Hanauer SB. Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic opportunities. Inflamm Bowel Dis. 2006;12(suppl 1):S3-S9.
2. Hanauer SB, Feagan BG, Lichtenstein GR, et al. Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet. 2002;359:1541-1549.
3. Colombel JF, Sandborn WJ, Reinisch W, et al. Infliximab, azathioprine, or combination therapy for Crohn’s disease. N Engl J Med. 2010;362:1383-1395.
4. Targan SR, Hanauer SB, van Deventer SJH, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease.
N Engl J Med. 1997;337:1029-1035.
5. Sands BE, Anderson FH, Bernstein CN, et al. Infliximab maintenance therapy for fistulizing Crohn’s disease. N Engl J Med. 2004;350:876-885.
6. Rutgeerts P, Sandborn WJ, Feagan BG, et al. Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med. 2005;353:2462-2476.
7. Baert F, Noman M, Vermeire S, et al. Influence of immunogenicity on the long-term efficacy of infliximab in Crohn’s disease. N Engl J Med. 2003;348:601-608.
8. Farrell RJ, Alsahli M, Jeen YT, et al. Intravenous hydrocortisone premedication reduces antibodies to infliximab in Crohn’s disease: a randomized controlled trial. Gastroenterology. 2003;124:917-924.
9. Ternant D, Aubourg A, Magdelaine-Beuzelin C, et al. Infliximab pharmacokinetics in inflammatory bowel disease patients. Ther Drug Monit. 2008;30:523-529.
10. Candon S, Mosca A, Ruemmele F, Goulet O, Chatenoud L, Cézard JP. Clinical and biological consequences of immunization to infliximab in pediatric Crohn’s disease. Clin Immunol. 2006;118:11-19.
11. Maser EA, Villela R, Silverberg MS, Greenberg GR. Association of trough serum infliximab to clinical outcome after scheduled maintenance treatment for Crohn’s disease. Clin Gastroenterol Hepatol. 2006;4:1248-1254.
12. Van Assche G, Magdelaine-Beuzelin C, D’Haens G, et al. Withdrawal of immunosuppression in Crohn’s disease treated with scheduled infliximab maintenance: a randomized trial. Gastroenterology. 2008;134:1861-1868.
13. Seow CH, Newman A, Irwin SP, Steinhart AH, Silverberg MS, Greenberg GR. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut. 2010;59:49-54.
14. Cornillie F, Shealy D, D’Haens G, et al. Infliximab induces potent anti-inflammatory and local immunomodulatory activity but no systemic immune suppression in patients with Crohn’s disease. Aliment Pharmacol Ther. 2001;15:463-473.
15. Bendtzen K, Ainsworth M, Steenholdt C, Thomsen OØ, Brynskov J. Individual medicine in inflammatory bowel disease: monitoring bioavailability, pharmacokinetics and immunogenicity of anti-tumour necrosis factor-alpha antibodies. Scand J Gastroenterol. 2009;44:774-781.
16. Afif W, Loftus EV Jr, Faubion WA, et al. Clinical utility of measuring infliximab and human anti-chimeric antibody concentrations in patients with inflammatory bowel disease. Am J Gastroenterol. 2010;105:1133-1139.
17. Steenholdt C, Bendtzen K, Brynskov J, Thomsen OØ, Ainsworth MA. Cut-off levels and diagnostic accuracy of infliximab trough levels and anti-infliximab antibodies in Crohn’s disease. Scand J Gastroenterol. 2011;46:310-318.
Commentary
Optimizing Infliximab Therapy for Inflammatory Bowel Disease—The Tools Are Getting Sharper
Marie-France Dubeau, MD, FRCPC
Subrata Ghosh, MD (Edin.), FRCP, FRCPE, FRCPC
Inflammatory Bowel Disease Clinic
Alberta IBD Consortium
Gastroenterology Division, Department of Medicine
University of Calgary
Calgary, Alberta
Address correspondence to: Dr. Subrata Ghosh, Alberta Health Services–Calgary Zone, Foothills Medical Centre, North Tower Room 929, 1403 29th St. NW, Calgary, AB T2N 2T9, Canada; Tel: 403-944-8222; Fax: 403-944-1095; E-mail: subrata.ghosh@albertahealthservices.ca
Infliximab (Remicade, Janssen Biotech) is a chimeric (75% human and 25% murine), monoclonal, immunoglobulin (Ig)G1 antibody that binds to soluble tumor necrosis factor (TNF)-α and prevents the cytokine from triggering the cellular TNF receptor complex and its effects. Infliximab also binds to transmembrane TNF-α and results in apoptosis of TNF-α–producing cells.1 Up to 40% of Crohn’s disease (CD) patients who initially respond to infliximab lose response within the first year.2 Secondary nonresponse may be due to disease-related factors or drug-related factors, including neutralizing antibodies, altered clearance of the drug, or immunologic escape from TNF-driven inflammation. Recent guidelines from the World Congress of Gastroenterology suggest that a diminished or suboptimal response to infliximab can be managed in 1 of 3 ways: shortening the interval between doses, increasing the dose to 10 mg/kg, or switching to a different anti-TNF agent (in patients who continue to have loss of response after receiving the increased dose).3
Failure of infliximab therapy may be due to pharmacokinetic or pharmacodynamic mechanisms or immunogenic mechanisms. Serum albumin may be predictive of infliximab pharmacokinetics.4 All exogenous proteins have the potential to induce immunogenicity.5 The formation of anti-infliximab antibodies (ATIs) is associated with a lower serum infliximab level, diminished clinical response, and infusion reactions.6 In the SONIC study, ATIs were detected at Week 30 in 0.9% of patients receiving combination therapy with azathioprine plus infliximab and 14.6% of patients receiving infliximab monotherapy.7 Median serum trough levels of infliximab were higher in the combination therapy group than the infliximab monotherapy group.
The most commonly used method for detection of ATIs is a double-antigen enzyme-linked immunosorbent assay (ELISA) that uses specific antibodies for capture and detection.8 Serum infliximab interferes with ATI measurement in this method. Infliximab is an IgG construct containing κ light chains. An alternative ELISA using an anti–human λ chain antibody for ATI detection is less amenable to interference and may be able to detect ATIs in patients with detectable serum infliximab. The presence of ATIs and detectable serum infliximab by this method may be a harbinger of evolving loss of response.9 The immunogenic part of infliximab is the Fab fragment, but measuring ATIs is more useful than measuring antibodies against Fab(2) or Fab fragments.10
Solid-phase ELISAs have a risk of false-positive results due to nonspecific binding to immunoglobulins other than infliximab.11 The use of fluid-phase radioimmunoassay (RIA) rather than solid-phase tests (RIA or ELISA) improves the specificity of the assay.12 RIA is not influenced by artifacts induced by solid-phase adsorption of proteins. Fluid-phase RIA measures the functional bioactive infliximab concentration that is not neutralized by ATIs and therefore remains capable of neutralizing TNF-α. Fluid-phase RIA reports the TNF-α binding capacity expressed as infliximab equivalents (µg/mL). ATIs (all isotypes) are detected when they bind to 125I-infliximab, after which they are separated by anti–human λ light chain antibodies.
A retrospective study published by Afif and colleagues in 2010 examined the utility of measuring ATIs and infliximab concentrations (by ELISA) in the management of inflammatory bowel disease patients.13 The authors found that increasing the infliximab dose in patients who have ATIs was ineffective, but increasing the dose in patients with subtherapeutic infliximab concentrations might be effective. Because the presence of infliximab in the sample interferes with the ATI assay, any patient with a detectable ATI concentration is considered by definition to have an undetectable infliximab concentration. Thus, 3 scenarios are possible: The patient can have a positive ATI test result; the patient can have a therapeutic infliximab concentration (defined as >12 mcg/mL at
4 weeks or a detectable trough level); or the patient can have a subtherapeutic infliximab concentration (defined as <12 mcg/mL at 4 weeks or an undetectable trough level). Afif and coauthors suggested a treatment algorithm for each situation, but interference in the ATI assay by infliximab limited the precision of interpretation.13 Reliable cutoff levels are necessary for both infliximab trough levels and ATI levels in order to anchor clinical decisions, but such cutoff levels were unavailable until recently.
In the current study by Steenholdt and colleagues, the authors attempted to determine clinically relevant cutoff values for infliximab trough levels and ATI levels associated with clinical response in patients with CD and ulcerative colitis (UC) by using fluid-phase RIA.14 Optimal cutoff levels to separate patients who maintained response from those who lost response were determined by using receiver operating characteristics analysis. The authors determined that a cutoff value of 0.5 µg/mL for infliximab trough level in CD patients provided a sensitivity of 86% and a specificity of 85%, with an accuracy of 87%. For UC patients, the cutoff level was 0.8 µg/mL, with a sensitivity of 75% and a specificity of 100%. The cutoff level for ATIs was 10 U/mL in both groups; this level corresponded to the detection limit of the assay. This level showed a sensitivity of 81% and a specificity of 90% in CD patients; in UC patients, this cutoff value yielded sensitivity and specificity values of 80% and 100%, respectively. The authors concluded that combining measurements of infliximab and ATIs had the highest overall accuracy (90%) in CD patients, with a sensitivity of 81% and a specificity of 94%. In this study, 20% of CD patients who lost response to infliximab had undetectable ATI levels; half of these patients had infliximab trough levels lower than the established level, while the other half had normal infliximab levels but still lost response. Unlike a previous Canadian study of UC patients, which showed a correlation between serum infliximab level and clinical response, the current study showed both low infliximab trough levels and high ATI levels in UC patients who lost response to maintenance infliximab therapy.14,15
In terms of the study’s limitations, the Steenholdt study was retrospective, and maintenance or loss of response was determined by chart reviews.14 The patient numbers were relatively small, especially for the UC group. The decision to continue or discontinue infliximab was based on clinical assessment by the gastroenterologist, not on infliximab trough level or ATI status. In addition, as in most studies, infliximab serum levels were measured as trough levels just prior to infliximab infusions but not at any other time point between infusions.
This study demonstrates that determination of clinically relevant, quantitative cutoff levels of infliximab and ATIs can be made with improved next-generation assays. In addition, the current study reinforces optimization of immunogenicity through the use of concomitant immunomodulators. Prospective studies are now required to base decision analysis on these cutoff levels and see whether they support intuitive treatment algorithms: increase in infliximab dosage (low infliximab trough levels, no ATIs), change to another anti-TNF monoclonal antibody (high ATI levels), or switch to another class of TNF inhibitors (adequate infliximab trough levels, no ATIs). A recent French study suggested that increasing the dose of infliximab may be effective irrespective of serum infliximab or ATI levels in patients who are losing response.16 This recommendation is current clinical practice in the majority of patients losing response to infliximab. Whether use of newer-generation assays with defined cutoff levels will provide better clinical decisions and outcomes will require prospective randomized trials, but at least the assays to conduct these trials optimally are becoming available.
References
1. Feldmann M, Elliott MJ, Woody JN, Maini RN. Anti-tumor necrosis factor-alpha therapy of rheumatoid arthritis. Adv Immunol. 1997;64:283-350.
2. Hanauer SB, Feagan BG, Lichtenstein GR, et al. Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet. 2002;359:1541-1549.
3. D’Haens GR, Panaccione R, Higgins PD, et al. The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD with the European Crohn’s and Colitis Organization: when to start, when to stop, which drug to choose, and how to predict response? Am J Gastroenterol. 2011;106:199-212.
4. Fasanmade AA, Adedokun OJ, Olson A, Strauss R, Davis HM. Serum albumin concentration: a predictive factor of infliximab pharmacokinetics and clinical response in patients with ulcerative colitis. Int J Clin Pharmacol Ther. 2010;48:297-308.
5. Cassinotti A, Travis S. Incidence and clinical significance of immunogenicity to infliximab in Crohn’s disease: a critical systematic review. Inflamm Bowel Dis. 2009;15:1264-1275.
6. Maharshak N, Plevy SE. A Crohn’s disease patient who does not respond to infliximab: what is next? Clin Gastroenterol Hepatol. 2011;9:1033-1038.
7. Colombel JF, Sandborn WJ, Reinisch W, et al; SONIC study group. Infliximab, azathioprine, or combination therapy for Crohn’s disease. N Engl J Med. 2010;362:1383-1395.
8. Svenson M, Geborek P, Saxne T, et al. Monitoring patients treated with anti-TNF-alpha biopharmaceuticals: assessing serum infliximab and anti-infliximab antibodies. Rheumatology. 2007;46:1828-1834.
9. Kopylov U, Mazor Y, Yavzori M, et al. Clinical utility of antihuman lambda chain-based enzyme-linked immunosorbent assay (ELISA) versus double antigen ELISA for the detection of anti-infliximab antibodies. Inflamm Bowel Dis. 2011 Oct 29. Epub ahead of print.
10. Ben-Horin S, Yavzori M, Katz L, et al. The immunogenic part of infliximab is the F(ab’)2, but measuring antibodies to the intact infliximab molecule is more clinically useful. Gut. 2011;60:41-48.
11. Hennig C, Rink L, Fagin U, Jabs WJ, Kirchner H. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods. 2000;235:71-80.
12. Bendtzen K, Geborek P, Svenson M, Larsson L, Kapetanovic MC, Saxne T. Individualized monitoring of drug bioavailability and immunogenicity in rheumatoid arthritis patients treated with the tumor necrosis factor alpha inhibitor infliximab. Arthritis Rheum. 2006;54:3782-3789.
13. Afif W, Loftus EV Jr, Faubion WA, et al. Clinical utility of measuring infliximab and human anti-chimeric antibody concentrations in patients with inflammatory bowel disease. Am J Gastroenterol. 2010;105:1133-1139.
14. Steenholdt C, Bendtzen K, Brynskov J, Thomsen OØ, Ainsworth MA. Cut-off levels and diagnostic accuracy of infliximab trough levels and anti-infliximab antibodies in Crohn’s disease. Scand J Gastroenterol. 2011;46:310-318.
15. Seow CH, Newman A, Irwin SP, Steinhart AH, Silverberg MS, Greenberg GR. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut. 2010;59:49-54.
16. Pariente B, de Chambrun GP, Krzysiek R, et al. Trough levels and antibodies to infliximab may not predict response to intensification of infliximab therapy in patients with inflammatory bowel disease. Inflamm Bowel Dis. 2011 Aug 29. Epub ahead of print.