LAUSR

Background: Therapeutic drug monitoring is used to guide treatment in patients treated with TNF-α inhibitors. Current bridging ELISA (bELISA), mostly used in routine analysis, cannot detect anti-drug antibodies (ADA) in immune complexes and differentiate between neutralizing and non-neutralizing ADA. Reporter Gene Assay (RGA), which detects only neutralizing ADA, is both costly and labour-intensive. Therefore, alternative assays are warranted to obviate these limitations. Objectives: To develop an in-house competitive ELISA (cELISA) for detection of neutralizing ADA, to compare results between four different assays for ADA detection and to propose an algorithm to assist clinicians in personalized therapeutic drug monitoring of Infliximab (IFX) and Adalimumab (ADL). Methods: Samples from 105 patients on IFX or ADL therapy (nIFX=61, nADL=44) from the Departments of Rheumatology and Gastroenterology, University Medical Centre Ljubljana, with undetectable drug levels, were tested with in-house cELISA, in-house bELISA, in-house bELISA with acetic acid dissociation (acid bELISA (1)) and RGA. cELISA was developed following the principles of RGA, briefly, diluted samples were pre-incubated with a fixed amount of IFX or ADL, linked to horseradish peroxidase. After incubation on a TNF-α plate, the reaction was detected using TMB substrate. Within and between-run imprecisions for cELISA were determined. Correlation coefficient and agreement between results from different assays were calculated. Results: Within and between-run imprecisions in cELISA met the validation criteria (<20%). We found high correlation between cELISA and RGA (anti-IFX r=0.932 (p<0.0001) and anti-ADL r=0.948 (p<0.0001)) and 100% agreement between results. cELISA also correlated with bELISA (anti-IFX r=0.663 (p=0.0002) and anti-ADL r=0.896 (p<0.0001)). Agreement between bELISA and cELISA was 79% for anti-IFX and 82% for anti-ADL samples. The more sensitive cELISA and functional RGA detect 13% (8/61) more positive samples in anti-IFX group and 18% (8/44) more samples in anti-ADL group. Acid bELISA found 3% (2/61) more positive samples in anti-IFX group and 14% (6/44) of samples in the anti-ADL group. In total, 16% (10/61) more samples in anti-IFX and 30% (13/44) more samples in anti-ADL group were confirmed having ADA. Based on our results we propose a sequential algorithm, which enables reliable, affordable and increased detection of ADA (figure 1).Figure 1 Algorithm for therapeutic drug monitoring of IFX and ADL. IFX – Infliximab, ADL – Adalimumab, ADA – anti-drug antibodies, bELISA – bridging ELISA, acid bELISA – bELISA with acid dissociation, cELISA – competitive ELISA; Conclusions: cELISA and acid bELISA, together, can detect ADA in 16% more samples in anti-IFX and 30% in anti-ADL group than classical bELISA used in current practice. The proposed algorithm can assist in everyday practice and enables better evaluation of patients treated with TNF-α inhibitors. Reference [1]Van Stappen, et al. Drug Test Anal2017;9(2):243–7. Disclosure of Interest: None declared