Surface plasmon resonance unveils important pitfalls of enzyme-linked immunoassay for the detection of anti-infliximab antibodies in patients\u2019 sera

Marten Beeg,Alessandro Nobili,Clorinda Ciafardini,Eleonora Allocati,Silvio Garattini,Marco Gobbi,Flavio Caprioli,Rita Banzi,Cesare Burti

doi:10.1038/s41598-021-94431-x

Abstract

Measurements of serum concentrations of therapeutic antibodies and anti-drug antibodies (ADA) can support clinical decisions for the management of non-responders, optimizing the therapy. In the present study we compared the results obtained by classical ELISA and a recently proposed surface plasmon resonance (SPR)-based immunoassay, in 76 patients receiving infliximab for inflammatory bowel diseases. The two methods indicated very similar serum concentrations of the drug, but there were striking differences as regards ADA. All the sera showing ADA by ELISA (14) also showed ADA by SPR, but the absolute amounts were different, being 7–490 times higher with SPR, with no correlation. Eight patients showed ADA only with SPR, and these ADA had significantly faster dissociation rate constants than those detectable by both SPR and ELISA. The underestimation, or the lack of detection, of ADA by ELISA is likely to reflect the long incubation steps which favor dissociation of the patient’s low-affinity ADA, while the commercial, high-affinity anti-infliximab antibodies used for the calibration curve do not dissociate. This problem is less important with SPR, which monitors binding in real time. The possibility offered by SPR to detect ADA in patients otherwise considered ADA-negative by ELISA could have important implications for clinicians.

Highlights

Measurements of serum concentrations of therapeutic antibodies and anti-drug antibodies (ADA) can support clinical decisions for the management of non-responders, optimizing the therapy
IFX trough levels and ADA serum concentrations were measured with a commercial enzyme-linked immunosorbent assays (ELISA) (Theradiag’s LISA-TRACKER Duo Infliximab) and by surface plasmon resonance (SPR)
The concentrations of IFX and ADA in each serum sample were determined by SPR in triplicate, with ex-novo preparation of samples and calibration curves, by two separate researchers with different experience, and the results confirmed that SPR is highly reproducible and robust (Suppl Fig. S1)

Summary

Introduction

Measurements of serum concentrations of therapeutic antibodies and anti-drug antibodies (ADA) can support clinical decisions for the management of non-responders, optimizing the therapy. The underestimation, or the lack of detection, of ADA by ELISA is likely to reflect the long incubation steps which favor dissociation of the patient’s low-affinity ADA, while the commercial, high-affinity anti-infliximab antibodies used for the calibration curve do not dissociate This problem is less important with SPR, which monitors binding in real time. Patients receiving therapies with monoclonal antibodies (mAb) often differ widely in their drug pharmacokinetics, and inadequate drug concentrations are a major cause of primary or secondary loss of r esponse[1,2] The latter may be a consequence of the development of anti-drug antibodies (ADA) which can affect clinical efficacy by either neutralizing the therapeutic antibodies or increasing their clearance[3,4,5]. Guidelines recommend TDIM as a reactive strategy when patients develop a loss of r esponse[24,25,26], it has not yet been commonly adopted in routine practice

Methods

Results

Conclusion