Optimization of a bridging enzyme-linked immunosorbent assay for a more efficient anti-infliximab antibody detection in serum of patients with chronic inflammatory bowel disease

Kronične vnetne črevesne bolezni so večfaktorska, doživljenjska obolenja, ki jih zaznamuje kronični vnetni proces v črevesju. Med najpomembnejši spadata Crohnova bolezen in ulcerozni kolitis. Zdravljenje temelji na uporabi različnih kombinacij zdravil, med drugim so v primeru neuspeha standardne terapije v uporabi biološka zdravila, predvsem zaviralci dejavnika tumorske nekroze alfa, kamor spada infliksimab. Proti biološkim zdravilom, kot je infliksimab, lahko začne bolnikov organizem proizvajati specifična protitelesa, kar povzroči zmanjšan odziv na zdravljenje in reaktivacijo bolezni oz. prekinitev stanja remisije. Določanje teh protiteles v krvi bolnikov (še) ni univerzalno vpeljano v rutino, kljub temu da je velikega pomena pri odločanju o nadaljnem zdravljenju. Klasične metode (npr. premostitvena ELISA), ki se uporabljajo za njihovo določanje, pa imajo to pomanjkljivost, da protiteles proti zdravilu ob sočasni prisotnosti zdravila v krvi ne zaznajo. Naš namen je bil optimizirati deplecijsko premostitveno ELISA metodo s predhodno kislinsko ločbo, prvotno objavljeno v strokovnem članku raziskovalne ekipe iz Belgije, pri kateri infliksimab v serumu bolnika ne moti detekcije specifičnih protiteles proti infliksimabu, ter nadaljnje testiranje optimizirane metode na vzorcih bolnikov s kronično vnetno črevesno boleznijo, zdravljenimi z infliksimabom. Predpostavili smo, da bomo z optimizacijo omenjene metode lahko v vzorcih z višjimi koncentracijami infliksimaba zaznali tudi protitelesa proti infliksimabu, ki so bila pred ločbo vezana v imunske komplekse z infliksimabom. Z uporabo predpripravljenih vzorcev smo ovrednotili uspešnost deplecije infliksimaba ter uspešnost detekcije protiteles proti infliksimabu ob prisotnosti infliksimaba v serumu. Z optimizirano metodo pri testiranih 42 kliničnih vzorcih protiteles proti infliksimabu sicer nismo zaznali, smo pa pri predpripravljenih vzorcih pokazali, da lahko v vzorcu zaznamo protitelesa proti infliksimabu že pri višji koncentraciji infliksimaba v vzorcu, kot je to uspelo belgijskim raziskovalcem. Z nadaljnjimi modifikacijami, longitudinalnim spremljanjem in dobljenimi pozitivnimi rezultati po analizah bi metoda imela dober potencial za vpeljavo v klinično rutino glede na to, da si želimo pri zdravljenju kroničnih vnetnih črevesnih bolezni v prihodnosti predvsem pospešiti proces zgodnjega prepoznavanja neuspešnosti zdravljenja ter posledično izboljšati prognozo bolezni.Inflammatory bowel diseases are a multifactorial, lifetime ailments, characterized by a chronic intestinal inflammation. The two most notable diseases are Crohn\u27s disease and ulcerative colitis. Treatment is based on a combination of different types of medicine, and – in the case of standard therapy failure – it includes biological drugs such as tumor necrosis factor alpha inhibitors, for instance infliximab. The patient can start to produce anti-infliximab antibodies, which leads to a lower drug response and a possible reactivation of the disease that was already in remission. Determining these antibodies in the patients\u27 blood is not universally routinely practiced (yet), but it is nevertheless of great importance in deciding how to pursue with the treatment. The main issue with commonly used assays (e.g. bridging ELISA) for determining these antibodies is that they lack the ability to detect them in the presence of drug. Our main purpose was to optimize a bridging enzyme-linked immunosorbent assay with an acidic pre-treatment protocol as an efficient, drug tolerant anti-infliximab antibody detection method, previously studied by a Belgian research group, and to test this optimized method on a cohort of samples of patients with inflammatory bowel disease, treated with infliximab. We assumed that after optimising this method we would also be able to detect antibodies against infliximab, previously bound in immune complexes with infliximab, in samples with higher concentrations of infliximab. We optimized the already published method by evaluating the success of the infliximab depletion in the pre-treatment samples and the success of the drug tolerant anti-ifliximab antibody detection. Even though we did not detect anti-infliximab antibodies in any of the 42 patients\u27 samples with the optimized method, we did howewer prove that we could detect anti-infliximab antibodies at a higher primary infliximab sample concentration than the Belgian research group could. Following new modifications, longitudinal monitoring and receiving positive results after testing, this method could have potential for getting inducted into routine clinical practice, considering that the medical community aims to improve the process of early detection of unsuccesful treatment and improve the patient\u27s prognosis in the future