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. 2018 Feb 26;18(3):691.
doi: 10.3390/s18030691.

Label-Free Quantification of Anti-TNF-α in Patients Treated with Adalimumab Using an Optical Biosensor

Rosa Helena Bustos  1 , Carlos Zapata  2 , Efraín Esteban  3 , Julio-César García  4 , Edwin Jáuregui  5 , Diego Jaimes  6
Affiliations

Label-Free Quantification of Anti-TNF-α in Patients Treated with Adalimumab Using an Optical Biosensor

Rosa Helena Bustos et al. Sensors (Basel). .

Abstract

This study describes the development of an immunosensory label-free quantification methodology based on surface plasmon resonance (SPR) and its applicability in measuring/evaluating therapeutic drug monitoring (TDM) of anti-TNF-α monoclonal antibody (adalimumab) in rheumatoid arthritis (RA) patients. The experimental parameters evaluated in this study were immobilising ligands by pre-concentration assays, sensor surface regeneration, ascertaining the method's sensitivity and correlating the results from quantifying plasma samples by ELISA immunoassay. The results showed that TNF-α quantification values (in RU) were significantly different when comparing patients (~50-250 RU) to controls (~10-20 RU). Likewise, there was 0.97 correlation for patients and 0.91 for healthy volunteers using SPR and ELISA comparison methodologies. SPR immunosensory detection provided a precise, sensitive strategy, along with real-time determination, for quantifying adalimumab, having great potential for clinical routine regarding TDM.

Keywords: biological drug; optical biosensor; therapeutic drug monitoring (TDM).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Calibration curve obtained using a commercial ELISA kit.
Figure 1
Figure 1
Schematic overview of the direct bioassay. (a) Schematic representation of activation via amino coupling on the CMD 200 surface. Carboxyl groups have been activated for covalently immobilising the TNF-α protein. Ab anti-TNF-α is detected once the protein has been immobilised. (I) Plasma samples from patients taking adalimumab and their controls; (II) Procedure for obtaining adalimumab spiked calibration curve in vitro. (b) A typical sensorgram obtained by SPR, showing all the stages involved in adalimumab quantification.
Figure 2
Figure 2
The pH scouting of human recombinant TNF-α (17 KDa molecular mass and 5.8 pI). The protein was diluted in 10 mM sodium acetate buffer, pH ranging from 4.1 to 5.5. Ligand surface concentration on the chip at pH 4.1 was much higher than that obtained at pH 5.5. Optimum pre-immobilisation pH was 4.1 (10 µg/mL).
Figure 3
Figure 3
Immobilising TNF-α protein by covalent amine coupling onto the CMD 200 chip. The sensorgram shows TNF-α used as ligand, with its characteristic parts: surface activation (NHS/EDC), ligand attraction and covalent coupling, blocking unoccupied sites (ethanolamine HCl) and final immobilisation response (10,449 RU).
Figure 4
Figure 4
(a) Association and dissociation curves for the different adalimumab spiked concentrations in vitro. (b) Standard curve for resonance units (RU) compared to anti-human TNF-α. The concentration values used for the standard curve were 1.0, 2.5, 5.0, 10.0, 15.0 and 20.0 µg/mL. R-squared was 0.9311.
Figure 5
Figure 5
Association and dissociation sensorgrams for replicas of patient (a) and control (b) samples. Regeneration level with 0.0005% SDS. The RU obtained for patients having anti-TNF-α plasma levels was greater than that for controls (without anti-TNF-α).
Figure 6
Figure 6
Resonance units (RU) obtained for samples from patients and healthy volunteers; the results were significantly different (p < 0.005).
Figure 7
Figure 7
Correlating SPR and ELISA concentrations. (a) Patients (R2 = 0.97) and (b) healthy volunteers (R2 = 0.91) sample.
See this image and copyright information in PMC

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