AIM:To evaluate the prevalence of double negative(DN)sera and the mechanisms responsible for DN status.METHODS:Sera of inflammatory bowel disease patients treated with infliximab(IFX)were tested for drug/antibodies to infliximab(ATI)trough levels and the proportion of DN results was compared between a commercially available double antigen ELISA(with labeled IFX as the detection antibody)and an antilambda ELISA(with anti-human lambda chain detection antibody).Repeat testing with lower than customary serum dilution(1:10)was performed.Patients with DN status were matched with IFX+/ATI-controls and were followed-up for subsequent development of nontransient ATI to investigate if DN status precedes ATI.RESULTS:Of 67 sera obtained at time of loss of response,only 6/67(9%)were DN by anti-lambda ELISA compared to 27/67(40%)with double antigen ELISA(P<0.001,Fisher’s Exact test).Of the latter27 sera,22%were also DN by anti-lambda ELISA,whereas 44%were actually IFX positive(IFX+ATI-),30%were ATI positive(IFX-ATI+)and 4%were double positive(IFX+ATI+).Re-testing using a 1:10 dilution converted most DN results into IFX+and/or ATI+status.Patients with DN status had shorter survival free of non-transient ATI compared with matched controls(log rank test,P<0.001).In 9/30(30%)of these patients,non transient ATI occurred before and after the event at which the DN serum was obtained,supporting the view that a DN result may represent aparticular time-point along the two curves of ATI titer rise and infliximab drug level decline.CONCLUSION:DN status may result from false negative detection of IFX or ATI by double antigen ELISA,suggesting a transitional state of low-level immunogenicity,rather than non-immunological clearance. AIM: To evaluate the prevalence of double negative (DN) sera and the mechanisms responsible for DN status. METHODS: Sera of inflammatory bowel disease patients treated with infliximab (IFX) were tested for drug / antibodies to infliximab (ATI) trough levels and the proportion of DN results was compared between a commercially available double antigen ELISA (with labeled IFX as the detection antibody) and an antilambda ELISA (with anti-human lambda chain detection antibody). Repeat testing with lower than customary serum dilution (1:10) was performed. Patients with DN status were matched with IFX + / ATI-controls and were followed-up for subsequent development of nontransient ATI to investigate if DN status precedes ATI .RESULTS: Of 67 sera obtained at time of loss of response, only 6 / 22% of also DN by anti-lambda ELISA (P <0.001, Fisher’s Exact test) .Of the latter 27 sera, 22% also also DN by anti-lambda ELISA , while 44% were actually IFX positive (IFX + ATI -), 30% were AT Re-testing using a 1: 10 dilution transformed most DN results into IFX + and / or ATI + status.Patients with DN status had shorter survival free of non-transient ATI compared with matched controls (log rank test, P <0.001) .In 9/30 (30%) of these patients, non transient ATI occurred before and after the event at which the DN serum was obtained, supporting the view that a DN result may represent aparticular time-point along the two curves of ATI titer rise and infliximab drug level decline. CONCLUSION: DN status may result from false negative detection of IFX or ATI by double antigen ELISA, suggesting a transitional state of low -level immunogenicity, rather than non-immunological clearance.