Cardiac troponin I: a case study in rational antibody design for human diagnostics.

In vitro diagnostic (IVD) platforms provide rapid and accurate determination of disease status. The clinical performance of antibody-based diagnostic platforms is paramount as the information provided often informs the medical intervention taken and, ultimately, the patient's outcome. Breaking down such an immuno-IVD device into its component elements, the biorecognition entity is key to the analytical specificity of the test. Furthermore, tailored optimisation of the antibody is often necessary to impart the desired biophysical properties for the specific application. This tailoring is now widely facilitated by advances in combinatorial approaches to antibody generation, molecular evolution strategies and the availability of truly high-throughput (HT), refined surface plasmon resonance-based screening tools. In this paper, we demonstrate a rational, knowledge-driven approach to the generation of epitope-specific antibodies for the early detection of cardiovascular disease, discuss the merits of the approaches taken and offer a perspective on HT strategies to mining large antibody libraries. These results highlight the expedience of such methodologies for the development of truly superior cardiovascular disease biorecognition elements.

Optimizing recombinant antibody function in SPR immunosensing. The influence of antibody structural format and chip surface chemistry on assay sensitivity.

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. 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 2.4 ng/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 19 ng/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 30 ng/ml, Fab 14 ng/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 x 10(-3)S(-1), scFv off rate 1.03 x 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.