A Surface Plasmon Resonance-based assay to measure serum concentrations of therapeutic antibodies and anti-drug antibodies

Abstract

Therapeutic drug and immunogenicity monitoring (TDIM) is increasingly proposed to guide therapy with biologics, characterised by high inter-individual variability of their blood levels, to permit objective decisions for the management of non-responders and reduce unnecessary interventions with these expensive treatments. However, TDIM has not yet entered clinical practice partly because of uncertainties regarding the accuracy and precision of enzyme-linked immunosorbent assays (ELISA). Here we report the characterisation of a novel surface plasmon resonance (SPR)-based TDIM, applied to the measurement of serum concentrations of infliximab, an antibody against tumour necrosis factor α (anti-TNFα), and anti-infliximab antibodies. SPR has the obvious advantages of directly detecting and measuring serum antibodies in minutes, avoiding the long incubation/separation/washing/detection steps of the methods proposed so far, reducing complexity and variability. Moreover, drug and anti-drug antibodies can be measured simultaneously. This new method was validated for sensitivity and reproducibility, and showed cost-effectiveness over commercial ELISA kits. This method may be applied to other biotherapeutics. These data pave the way for the development of SPR-based point-of-care devices for rapid on-site analysis.

Figure 1

General scheme of the SPR-based assay for simultaneous determination of IFX and ATI concentrations in serum. The SPR apparatus was the ProteOn XPR36 Protein Interaction Array system (Bio-Rad), which has six flow channels which can immobilise up to six ligands on parallel strips of the same sensor surface (e.g. TNFα, IFX, suitable controls including IgG or an “empty” channel). The flow channels can be rotated 90° so that up to six analyte solutions can be flowed in parallel on all the immobilised ligands, creating a multi-spot interaction array (each spot indicated by *): for example, injection of six concentrations of the analyte standards gives interaction affinities or the calibration curves; and the injection of six serum samples can simultaneously evaluate their SPR-binding signals on TNFα, IFX and appropriate controls. The terms “ligands” and “analytes” are used throughout the manuscript as indicating the immobilised and the flowing interactants, respectively.

Figure 2

Linearity between specific SPR signal and serum concentrations of IFX (A) or ATI (B). Serum from healthy volunteers was spiked with IFX (CT-P13) or ATI to the indicated concentrations then diluted 30-fold before injection into the SPR instrument. (a) Representative sensorgrams obtained injecting simultaneously the six dilutions of IFX-spiked sera over immobilised TNFα, after subtraction of the SPR signals from the parallel empty surfaces (see Fig. 1) for the experimental design and Fig. S1 for raw data). (b) Representative sensorgrams obtained injecting simultaneously the six dilutions of ATI-spiked sera over immobilised IFX, after subtraction of the SPR signals from the parallel surfaces coated with IgG. (c) Linearity between IFX concentration and specific SPR signal (at the end of the dissociation phase). Points show the mean ± SD for six consecutive injections, each as in panel a. The mean r² of the six straight lines is 0.997 ± 0.001; the equation obtained from the linear regression is: y = 69.01(±1.37) x + 8.36(±5.16). (d) Linearity between ATI concentration and specific SPR signal (at the end of the dissociation phase). Points show the mean ± SD for six consecutive injections, each as in panel b. The mean r² of the six straight lines is 0.998 ± 0.002; the equation obtained from the linear regression is: y = 4.65(±0.07) x + 7.30(±1.34).

Figure 3

Effect of IFX on determination of ATI concentration. Aliquots of human serum from healthy volunteers were spiked with commercial ATI, to the final concentrations indicated, and with IFX (CT-P13, final concentration 8 μg/mL) or its vehicle. Serum aliquots were diluted 30-fold, subjected to acidic pretreatment, and injected over immobilised IFX and IgG (the latter for evaluation of non-specific binding). The points indicate the specific SPR signal due to the binding of flowing ATI to immobilised IFX.