Significance of low level infliximab in the absence of anti-infliximab antibodies

Figure 1 Flow chart of the patients included in the two parts of this study. The analytical part (dashed lines) comprised a comparison of two different assays and of two different serum dilutions; the clinical part (solid lines) followed up, in a case-control study, double negative patients versus patients with adequate infliximab levels for subsequent antibodies to infliximab formation and clinical outcome. DN: Double negative; LOR: Loss of response; AL: Anti-lambda; DA: Double-antigen; IFX: Infliximab; ATI: Antibodies to infliximab.

Figure 2 ELISA tests of patients with loss of response. Out of 67 patients with loss of response, 27 (40%) were double negative with double antigen ELISA. Of those, only 6 (9%) were DN using anti-lambda ELISA. LOR: Loss of response; DN: Double negative; IFX: Infliximab; ATI: Antibodies to infliximab.

Figure 3 Re-testing of double negative sera to increase analytical sensitivity. To examine whether some of the double negative sera represented low-titer antibodies to infliximab or low-level infliximab, we randomly selected 45 double negative sera and re-tested them at a 1:10 dilution to increase analytical sensitivity (compared with standard anti-lambda testing using 1:100 serum dilution). A: IFX and ATI values of double negative vs healthy controls’ sera, analyzed at 1:100 dilution anti-lambda ELISA. Cut off values for double negativity: IFX < 1 μg/mL, ATI < 2.1 μg/mL-eq. DN sera - black squares, healthy controls - circles; B: IFX and ATI values of sera DN on 1:100 dilution vs healthy controls analyzed at 1:10 dilution anti-lambda ELISA. Cut off values for double negativity: IFX < 0.04 μg/mL, ATI < 0.6 μg/mL-eq. Previously DN sera (on 1:100 dilution) - black squares, healthy controls - circles. DN: Double negative; IFX: Infliximab; ATI: Antibodies to infliximab.

Figure 4 Temporal formation of antibodies to infliximab in nine patients in whom antibodies to infliximab positive sera preceded double negative sera. ATI formation before DN event (triangles), ATI formation after DN event (circles). ATI: Antibodies to infliximab; DN: Double negative.

Figure 5 Survival free of antibodies to infliximab and loss of response. A: ATI development in cases vs controls; B: Secondary LOR in cases vs controls. The asterisk shows that five of the controls experienced secondary LOR at a time-point comparable to the double negative event. ATI: Antibodies to infliximab; LOR: Loss of response.