Seroconversion panels are an important tool for investigating antibody responses and developing serological assays. A seroconversion panel was generated from a single SARS-CoV-2 positive plasma donor over 87 days. This seroconversion panel was tested against 6 SARS-CoV-2 antibody tests (IgG, IgM, and total Ig). All test kits utilized recombinant antigens that are specific to SARS-CoV-2. The seroconversion panel showed IgG responses for SARS-CoV-2 after day 50. IgM levels peaked on day 50 (prior to IgG) and declined in subsequent samples. This seroconversion panel is a useful tool for validation of SARS-CoV-2 antibody assays.

Clinical utility of infliximab and adalimumab therapeutic drug monitoring (TDM) is unquestionable. However, little is known on the clinical utility of TDM-guided patient management for vedolizumab (VDZ) in inflammatory bowel disease (IBD), and more studies are needed to understand the correlation between VDZ trough levels (VTL) and clinical response, and to establish solid cut-off therapeutic levels of VDZ. A prospective cross-sectional observational study is ongoing at La Paz University Hospital (Madrid, Spain) to explore the correlation between VTL and loss of clinical response in IBD patients treated with VDZ. Trough serum samples were collected from 21 IBD patients at baseline and during the course of treatment from 4 months to 2 years. VTL and anti-VDZ antibodies were measured with Promonitor®-VDZ and Promonitor® Anti-VDZ tests (Progenika, a Grifols company, Spain), based on ELISA technology, respectively. All statistical analysis performed used a non-parametric approach (JMP software 14.0). Here we report preliminary results for 21 patients (66.7% CD and 33.3% UC) recruited so far. VDZ was administered every 4 weeks (n = 3) and 8 weeks (n = 18). VTL measurement was performed at baseline (3.8%), induction therapy (28.2%) and maintenance therapy (67.9%) in 101 samples. None of the patients developed antibodies against VDZ. Median VTL was 9.1 µg/ml (range, 0–72.3). The median VTL during induction was significantly higher 32.4 µg/ml (IQR: 13.8, 43.1) vs. 8.2 µg/ml (IQR: 6.3, 16.2) during maintenance (p < 0.0001), therefore, all analyses accounted for this effect. Median VTL was higher in patients who received VDZ every 4 vs. 8 weeks (11.9 µg/ml vs. 8.0 µg/ml, respectively; p = 0.182). Significant inverse correlations between CRP and VTLs, and between VTL and platelets were observed during maintenance (Spearman Rho −0.27, p = 0.03; Spearman Rho −0.40, p < 0.001, respectively). A high statistically significant inverse correlation between VTLs and GOT/GPT was noted in the UC group (Spearman Rho −0.79; p < 0.0001). There were no significant differences between median VTLs and the rest of analytical variables tested (haemoglobin, leucocytes, albumin, ESR and creatinine). VTL are in line with those reported in other studies. Low CRP and platelets which are related with less disease are significantly associated with higher VTL.

Background: Therapeutic drug monitoring (TDM) of infliximab (IFX) and adalimumab is increasingly being used for the management of patients with inflammatory bowel disease (IBD) [1]. Enzyme-linked immunoassays (ELISA) is one of the most used techniques; however, often requires batching samples which can delay results, and be inconvenient in urgent situations where a quick action is needed. In this study we compare the performance of a new lateral flow (LF) rapid test to detect anti-IFX antibodies with ELISA in IBD patients treated with either Remicade® (RMC) only or CT-P13 only or switchers from RMC to CT-P13. Methods: Serum samples from IBD patients who participated in BIOSIM01, an observational retrospective study, in which patients were treated with either RMC only, or CT-P13 only, or CT-P13 following a switch from RMC, were used. In this study a total of 564 consecutive trough sera (corresponding to 103 patients) were collected just before the infusion to all patients. All samples were frozen for subsequent testing with ELISA (Promonitor® Anti-IFX, Progenika, Spain) and the LF rapid test (Promonitor® Quick Anti-IFX, Progenika, Spain). The rapid test is an immunochromatographic qualitative kit based on LF technology to detect anti-IFX antibodies in patients treated with any IFX molecule in human whole blood (fingerprick or venous) or serum. Antibody test results were read visually at 30 minutes. ELISA quantitative results were categorized as positive or negative to allow comparisons with the rapid test. Results: The rapid test allowed the detection of anti-IFX antibodies in a few minutes with just 15 μL of serum and showed an almost perfect agreement with the comparative ELISA method. Overall, positive and negative percent agreements between ELISA and the LF test were 97.9% (CI95: 96.3–98.8), 96.0% (CI95: 88.9–98.6) and 98.2% (CI95: 96.5–99.0), respectively. No significant differences in the detection of Anti-IFX antibodies against either drug in any of the three cohorts were observed (Table 1). All antibody positive patients showed no detectable IFX levels as measured with ELISA. Table 1. Detection of anti-IFX antibodies in the different cohorts with the LF rapid test and ELISA PPA, Positive Percent Agreement; NPA, Negative Percent Agreement. Conclusions: The almost perfect agreement reported here between the LF and ELISA tests provides support to clinicians and laboratory personnel involved in biological drug monitoring in the use of the rapid test. The rapid test shows detection of antibodies aganist either the innovator or the biosimilar with comparable analytical sensitivity and offers a good solution in case a qualitative quick analysis of anti-IFX antibodies is required. References: [1] Vande Casteele, (2015), Trough concentrations of infliximab guide dosing for patients with inflammatory bowel disease, Gastroenterology, 1320–9

Background Loss of clinical response and infusion reactions to infliximab (IFX) are associated to the development of antibodies to IFX (ATI). ATI detection is a key step of patient management. However, current techniques may require additional patient appointments for sample collection, processing and batching in centralised facilities. Test reporting usually takes several days or weeks impairing effective decision making. Objectives To clinically validate the use of a new rapid test to detect ATI in capillary blood in a real-life point-of-care (POC) setting where patients attend the infusion center for the reference IFX (Remicade®, RMC) or CT-P13 (Inflectra®, IFT, or Remsima®) infusions. Methods PQ-EF1 and PQ-EF2 are prospective, observational studies designed to evaluate and compare the performance of a rapid POC test (CE-marked Promonitor® Quick Anti-IFX, Progenika, Spain) to detect ATI in routine clinical practice in rheumatic and gastroenterology patients treated with the reference IFX or the biosimilar CT-P13 attending the infusion center with the ELISA technique as a reference. The POC test is a qualitative immunochromatographic assay based on lateral flow technology to detect ATI (including biosimilar CT-P13) in either fingerprick or serum or venous whole blood. Consecutive patients (initiating or under maintenance therapy) were recruited and tested in La Paz and Basurto University hospitals with the rapid test in capillary and venous whole blood specimens immediately before the infusion. ATI test results were read visually with the POC test in 30 min, just before the patient started the infusion. Trough sera were also collected for subsequent analysis with the rapid test and benchmarked with Promonitor®-Anti-IFX ELISA (Progenika, Spain). Follow-up time was 6 months. ELISA quantitative results were categorized as positive and negative to allow comparisons with the qualitative rapid test. Results Ninety consecutive patients were recruited (a total of 137 visits in the 6 months follow-up) accounting for a total of 137 sera, 137 fingerpricks and 71 venous whole blood samples. Overall, 8 (8.9%) patients developed ATI (5 ankylosing spondylitis, 1 Crohn9s disease, 1 ulcerative colitis and 1 juvenile idiopathic arthritis). ATI were detected in 5 patients treated with Remicade® and 3 treated with Inflectra®. Overall agreements between fingerprick vs venous whole blood and fingerprick vs serum measured with the rapid POC test were 100% and 99%, respectively. Positive (PPA) and negative (NPA) agreements between the POC test and ELISA were 91% and 99%, respectively. PPA and NPA between the ELISA and the POC test in serum was 100% and 99%, respectively. Conclusions ATI can be reliably detected in either venous or capillary circulation. Results show an almost perfect agreement between specimens and with the reference ELISA technique. ATI measurement with the POC test allows the clinician to detect ATI in a quick and fully decentralised mode facilitating immediate POC decision making. Disclosure of Interest A. Ametzazurra Employee of: Employee of Progenika Grifols, N. Rivera: None declared, A. Balsa: None declared, M. Arreba: None declared, E. Ruiz: None declared, C. Plasencia: None declared, J. Ortiz: None declared, D. Pascual-Salcedo: None declared, M. Muñoz: None declared, C. De Aysa: None declared, M. Allande: None declared, N. Torres Employee of: Employee of Progenika Grifols, A. Hernández Employee of: Employee of Progenika Grifols, X. Recalde Employee of: Employee of Progenika Grifols, A. Martínez Employee of: Employee of Progenika Grifols, D. Nagore Employee of: Employee of Progenika Grifols

Background: Loss of clinical response and infusion reactions to infliximab are associated to Anti-IFX antibodies (ATI). ATI detection is a key step of patient management algorithm [1]. However, current techniques require additional patient appointments for sample collection, processing and batching in centralised facilities. Test reporting usually takes several days or weeks impairing effective decision making. Here we validate the use of capillary blood in a real-life point-of-care (POC) setting where patients attend the infusion centre for Remicade® (RMC) or CT-P13 infusions.

Objectives The aim of this study was to determine whether antibodies to infliximab (IFX) in Remicade-treated patients cross-react with the biosimilar CT-P13. Methods 250 consecutive patients with rheumatic diseases under Remicade and 77 controls were retrospectively selected for the study. Anti-IFX antibodies at drug through levels were measured in parallel with three different bridging ELISA assays: Promonitor-ANTI-IFX kit, which uses Remicade to detect antibodies, and two more assays that use either Inflectra or Remsima with the same format. Correlation and association between each assay was studied. Results 50.4% of patients were tested positive with Promonitor-ANTI-IFX. All were antibodies to IFX (ATI)-positive when either Inflectra or Remsima assays were used. In all comparisons positive and negative percentage agreements were 100%, and correlation coefficients were ≥0.995. No differences between rheumatoid arthritis and spondyloarthritis, or between concomitant immunosuppressives, were observed. Conclusions Anti-IFX antibodies of Remicade-treated patients cross-react with either Inflectra or Remsima. Although additional epitopes may be present in the biosimilar, results suggest that epitopes influencing the immune response to IFX are also present in the biosimilar. Antibody-positive patients treated with Remicade should not be switched to the biosimilar, since antibodies will interact with the new drug and potentially lead to loss of response. This finding supports the utility for therapeutic drug monitoring before a switching strategy is considered.

Background Antibodies to infliximab (ATI) are associated to loss of response, adverse effects and discontinuation of treatment in rheumatic patients1. However, the clinical relevance of measuring free versus total anti-drug antibodies (free plus drug-complexed antibodies) remains to be established. Objectives To analyse the association between the clinical response and ATI measured as free and total content of immunoglobulins in a cohort of patients with spondyloarthritis. Methods A retrospective pilot study was conducted including 12 patients with spondyloarthropathies (9 ankylosing spondylitis (AS) and 3 spondyloarthritis (SpA)) under maintenance infliximab (IFX) therapy (3–5 mg/kg every 8 weeks). Disease activity was assessed by BASDAI score every 6 months. Trough serum samples were consecutively collected every two months during a follow up period of 1±0.1 years. In total 75 samples were collected (average of 6.3 per patient). Ten patients (83%) received concomitant therapy with methotrexate. IFX levels were measured with Promonitor®-IFX (Progenika-Grifols, Spain). Free and total-ATI were measured with Promonitor® Anti-IFX (Progenika-Grifols, Spain) (PR) and IDKmonitor® Infliximab total ADA (IDK) (Immundiagnostik, Germany) ELISA kits, respectively. Cut-point of PR and IDK ATI assays is 5 and 10 AU/mL, respectively. FDA guidance for comparing two diagnostic tests in the absence of a gold standard was followed2. Results Five (41.7%) and (83.3%) patients were positive for free and total-ATI using PR and IDK assays, respectively. Overall, positive (PPA) and negative (NPA) percent agreements between the two assays were 52%, 16% and 100%, respectively. All samples with positive free-ATI (7) had undetectable IFX levels (0.0±0.0 μg/mL); however, 72% of total-ATI positive samples (43) were also positive for IFX levels (1.7±1.7 μg/mL, min 0.035 μg/mL and max 6.4 μg/mL). Both outcomes are coherent with the intended use of each kit, which also explains the low PPA. However, total-ATI are positive in 40% of the samples even when the patient responds (BASDAI<4), which questions the utility of total-antibodies in a clinical setting. This evidences a low clinical specificity of total-ATI measurement using the cut-off of 10 AU/mL recommended by the manufacturer and therefore that the presence of total-ATI in the presence of detectable drug has little utility. Patients without free-ATI had significantly lower BASDAI than patients with free-ATI (2.7±2.0 without antibodies vs 5.0±0.9 with antibodies, p=0.049), whereas no statistically significant difference was observed between patients when total-ATI were used (3.1±2.2 without antibodies vs 2.9±2.0 with antibodies, p=0.969) (Fig. 1). Conclusions Total-ATI do not correlate with disease activity in spondyloarthritis. The fact that many samples are positive to total-ATI even in patients with low disease activity makes very difficult interpretation of results. The explanation is that the fraction of ATI bound to IFX is not clinically relevant as long as there is free therapeutic drug in circulation. References Ducourau et al. Arthritis Res Ther 2011;13:R105 Statistical guidance on reporting results from studies evaluating diagnostic tests. 2007.FDA Disclosure of Interest A. Ruiz del Agua Employee of: Employee of Progenika Grifols, J. Pascual Employee of: Employee of Progenika Grifols, N. Torres Employee of: Employee of Progenika Grifols, D. Pascual-Salcedo: None declared, A. Martínez: None declared, T. Jurado: None declared, C. Plasencia: None declared, A. Balsa: None declared, B. Ruiz-Argüello Employee of: Employee of Progenika Grifols, A. Maguregui Employee of: Employee of Progenika Grifols, A. Ametzazurra Employee of: Employee of Progenika Grifols, A. Martínez Employee of: Employee of Progenika Grifols, D. Nagore Employee of: Employee of Progenika Grifols

Background The options for treatment of rheumatic diseases is constantly growing. Tocilizumab (TCZ) is an anti-interleukin 6 receptor monoclonal antibody indicated for the treatment of severe active and progressive rheumatoid arthritis (RA) and active systemic juvenile idiopathic arthritis (sJIA). Monitoring of drug levels (DL) and anti-drug antibodies (ADA) in clinical practice is highly advisable in order to establish normal therapeutic ranges, complement clinical assessment and optimise patient treatment. As a consequence several tests and technologies have been developed. However, a common objection that limits implementation of biological drug monitoring in medical practice is the claim that the magnitude of measurements varies among assays and that assay standardisation is lacking. Objectives The purpose of this study was to evaluate the correlation and agreement between different commercially available tests to measure TCZ and antibodies to TCZ (ATT) in patients with rheumatic diseases. Methods Trough levels of TCZ and ATT were analyzed in a retrospective cohort of 26 rheumatic patients (42 serum samples, 88% RA and 12% sJIA) under maintenance therapy with TCZ for up to two years. Samples were assayed using Promonitor-TCZ and Promonitor-ANTI-TCZ ELISA kits (PR, Progenika, Spain), LISA-TRACKER Duo Tocilizumab ELISA kit (LT, Theradiag, France), and by Sanquin Blood Supply testing service (SQ, Amsterdam, The Netherlands) that uses ELISA and RIA technologies to measure TCZ and ATT levels, respectively. One-way analysis of variance (ANOVA) and Spearman9s test was used to study correlation between the DL tests; the difference (bias) in values obtained with DL assays was also assessed with Bland-Altman analysis. Percentage of positive and negative agreement was used to study the association between the ADA assays. Results TCZ trough level in patients are much higher than other biological treatments like anti-TNF infliximab and adalimumab. ANOVA analysis showed that the differences in the median values among the three DL assays are not statistically different (median 4.4 vs 4.7 vs 5.4 μg/mL, IQR 0 – 8.7 μg/mL vs 0 – 7.8 μg/mL vs 0 – 9.4 μg/mL for Promonitor-TCZ, LISA-TRACKER and Sanquin ELISA tests, respectively, p=0.693). Spearman9s coefficient showed a very high correlation between the each pair of assays (r=0.951, 0.935 and 0.944 for PR vs LT, PR vs SQ and LT vs SQ, respectively, p<0.0001). Bland-Altman analysis shows an excellent association between each pair of DL assays with no significant difference in bias (-0.3, -0.5 and -0.2 μg/mL for PR vs LT, PR vs SQ and LT vs SQ, p>0.05). Antibodies to TCZ were not detected with any of the assays in the patient cohort confirming the low immunogenicity of TCZ. Conclusions Despite different reagents, assay formats and technologies, all tests studied here provide exactly the same results and measure the same magnitude of TCZ in rheumatic patients. Assays can be safely exchanged in a clinical setting even in the absence of a gold standard test, and should lead to similar drug therapeutic ranges and actions. Acknowledgement This work was supported by the Basque Country Government (Dep. Education, Universities and Research, Ref. IT687–13) and ZabaldUZ 2012, UPV/EHU. Disclosure of Interest S. Martín Grant/research support from: PhD student research support from Progenika, A. Ruiz del Agua Employee of: Employee of Progenika Grifols, N. Torres Employee of: Employee of Progenika Grifols, D. Pascual-Salcedo: None declared, C. Plasencia: None declared, T. Jurado: None declared, A. Martínez: None declared, A. Balsa: None declared, B. Ruiz-Argüello Employee of: Employee of Progenika Grifols, A. Martínez Employee of: Employee of Progenika Grifols, R. Navarro: None declared, D. Nagore Employee of: Employee of Progenika Grifols

Background Rituximab effects in rheumatoid arthritis patients have been extensively described in the literature. However, kinetics of rituximab levels in serum and their association with the clinical and immunological changes in these patients remain unknown. Methods Thirty five patients were treated with three courses of rituximab (one course: 1000 mg administered intravenously on days 1 and 15). Levels of rituximab in the serum were determined by ELISA (Promonitor ®-RTX) before initiating each treatment course (t=0d) and at 30, 90 and 180 days. We then analyzed the association of serum rituximab levels with changes in DAS28, PCR, ESR, IgG, IgM and IgA levels, B cell frequency, RF and C3 and C4 complement. Results The peak of rituximab levels was observed at 30 days after initiating each course. Rituximab levels ranged from 13000 to115000 ng/ml at 30d. At 90d levels decreased significantly, but they were still detectable in 80% of patients. Those patients with higher levels in the first course of the treatment presented also higher levels in the second and third courses. No anti-rituximab antibodies were detected during follow-up. To analyze the association of rituximab levels with clinical and immunological parameters, patients were segregated in two groups according to 30d-rituximab levels: group H ( >56000 pg/ml) (52% of patients) and group L (<55000pg/ml) (48% of patients) (Table 1). Both groups of patients were comparable before treatment. EULAR response was achieved in 63.4% of Group H patients and 88.9% of group L patients. The decrease of DAS28 was 42% higher in Group L than in group H patients at 90d. Group L patients were also associated with a higher decrease of ESR (p<0.038) at 180d. Depletion of B cells was comparable in the two groups but CD19+ cell recovery was earlier in the peripheral blood of group H patients. Conclusions Lower levels of rituximab in serum were associated with a better clinical response and later B cell recovery. Circulating levels of rituximab may be conditioned by the relocation of rituximab to the extravascular compartment. In this extravascular compartment, rituximab could thus expand its mechanism of action beyond the peripheral blood. Disclosure of Interest None Declared