Biosensors and Bioelectronics 22 (2006) 268–274 Optimizing recombinant antibody function in SPR immunosensing The influence of antibody structural format and chip surface chemistry on assay sensitivity S. Townsend a , W.J.J. Finlay a,b , S. Hearty a,c , R. O’Kennedy a,b,∗ a School of Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland b Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin, Ireland c National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin, Ireland Received 9 November 2005; received in revised form 5 January 2006; accepted 9 January 2006 Available online 17 February 2006 Abstract Background: Recombinant antibody fragments are valuable tools for SPR-based detection of small molecules such as illicit drugs. However, the multiple structural formats of recombinant antibody fragments are largely uncharacterised with respect to their respective performance in SPR sensing. We have expressed a model anti-M3G antibody in both scFv and chimeric Fab formats to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore TM . We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. Results: Under optimised competition ELISA conditions, the anti-M3G scFv was found to have an IC 50 value of 30 ng/ml, while the most stable Fab construct exhibited an IC 50 value of 2.4ng/ml. In SPR competition assay on an M3G-OVA-coated SPR chip surface, the two constructs again differed in sensitivity, with IC 50 values of 117 and 19ng/ml for the scFv and Fab, respectively (the scFv also exhibiting poor linearity of response). However, when the SPR chip surface was directly coated with M3G, both antibody constructs exhibited good linearity of response, similar high sensitivity IC 50 values (scFv 30ng/ml, Fab 14ng/ml) and high reproducibility (50 effective regenerations for M3G-OVA, 200 for M3G direct). During SPR assay development it was noticed that scFv and Fab constructs gave differing off-rate profiles. Subsequent HPLC, ELISA and electrophoretic analyses then confirmed that a portion of the scFv population multimerises. Bivalent scFv was found to profoundly affect the dissociation curve for scFv in stringent SPR kinetic analyses, leading to a 40-fold difference in calculated off-rate values (Fab off rate 4.7 × 10 -3 S -1 , scFv off rate 1.03 × 10 -2 S -1 ). Conclusion: The structural format of recombinant antibody fragments and chip functionalisation methodology can both profoundly affect the function of anti-M3G SPR assay, with direct coating and Fab format proving to be optimal. The confirmation of scFv multimerisation and resulting changes in SPR kinetics profile, in comparison with a Fab, further suggest that caution must be taken in the interpretation of SPR sensorgrams, which are commonly used in the ‘affinity ranking’ of scFv panels in which the extent of dimerisation in each sample is unknown. © 2006 Elsevier B.V. All rights reserved. Keywords: Fab; scFv; SPR; Surface chemistry; Kinetics 1. Introduction With the increased importance of regular testing for illicit drug content in clinical or forensic samples, a clear goal is the development of simple, rapid and highly specific multi-analyte detection methods. Traditional methods for detecting small drug molecules, such as thin layer chromatography (TLC) and high- ∗ Corresponding author. Tel.: +353 1700 5319; fax: +353 1700 5412. E-mail address: Richard.Okennedy@dcu.ie (R. O’Kennedy). performance liquid chromatography (HPLC) have become out- dated due to their relative expense, complexity and low through- put (Hall et al., 1993). Automated ‘real-time’ immunoassays are an emerging candidate to take their place (Yau et al., 2003). To achieve real-time analysis of small molecules, surface plas- mon resonance (SPR) biosensors, such as Biacore TM , are being applied to develop highly sensitive competition immunoassays (Rich and Myszka, 2005). The capacity for SPR to monitor label- free binding events in real time has made it a popular method to examine molecular activity. However, the efficacy of the biosen- sor is heavily dependant on ligand immobilisation methodology 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.01.010