The specificity and affinity of Affimer binders has been showcased in a paper published in the journal Analyst. Co-authored by Dr Paul Ko Ferrigno and Dr Cassey McRae working with Dr Ashwin Seshia and Ms Nicole Weckman from the University of Cambridge, the study used quartz crystal microbalance measurements to analyse the binding of Affimer proteins to a gold surface and their subsequent specific binding to different mouse IgG antibody sub-types.
The results showed that Affimer proteins identified as specific binders for either IgG2a or IgG2b were able to specifically bind their target analytes. The murine antibody sub-types share approximately 82% of their sequence identity in the constant region of the heavy chain, but the Affimer binders were shown to be sufficiently selective to avoid any cross-reactivity in target binding. What’s more the affinities of these Affimer binders for their specific IgG2 target proteins were found to be in the nM range, similar to most antibodies, with affinities of 13nM and 24nM observed.
Beyond merely exemplifying the high level of specificity available with Affimer proteins, being able to specifically distinguish between different antibody isotypes and sub-types offers great potential for the development of diagnostic assays. The ability to monitor different antibody sub-classes could help identify antibody deficiencies in different disease states, allow us to monitor antibody responses to treatment in autoimmune diseases and adjust treatment accordingly, and to monitor antibody responses to vaccinations. Of course, a first step in creating a system to monitor antibody subclass variations is the development of tools that can specifically bind each of the highly homologous antibody molecules. This has proven tricky for affinity proteins immobilised on solid surfaces such as gold, as the metallic surface can interfere with the structure of the affinity protein resulting in a reduction in or loss of affinity for the target.
The high specificity binding of the anti-IgG2a and anti-IgG2b Affimer binders was first demonstrated by affinity purification of each target analyte. Biotinylated Affimer binders were conjugated to streptavidin beads and able to pull down their specific target analyte without showing any cross-reactivity to IgG1, IgG3 or the other IgG2 subclass.
But, the performance of Affimer binders immobilised on a gold surface may prove different to within an affinity purification situation. To analyse the performance of Affimer binders on gold they were modified with a cysteine residue at the C-terminus, ensuring the Affimer binders were bound to the gold chip in a single uniform orientation, which could increase target binding.
Affimer binding to the gold chip was determined by a negative frequency shift in the measurements of the quartz crystal microbalance (link is external) with dissipation monitoring (QCM-D), with very little change during rinsing, showing that the Affimer proteins were tightly bound to the gold surface.
QCM-D is a label-free technique that measures changes in mass by area via variations in frequency of the quartz crystal resonator, which is disturbed by the addition or removal of small masses with nanogram sensitivity at the surface of the resonator. The QCM-D is therefore a useful tool to directly investigate and quantify binding of Affimer proteins to surfaces and subsequently the binding of the analyte of interest to the immobilized Affimer proteins.
After functionalising the gold surface with Affimer proteins and blocking any bare surface with casein, the IgG2a or IgG2b proteins were flowed over the surface. The specificity of each Affimer for its specific subclass of IgG2 was clearly preserved even when it was bound to a solid surface- where IgG2a specific Affimer binders bound IgG2a but not IgG2b, and IgG2b specific Affimer binders bound to IgG2b but not IgG2a.
High affinities for the IgG2 subtype targets were noted for the Affimer binders of 13nM and 24nM. This compares easily with standard antibody-antigen complexes and highlights the ability of Affimer proteins to specifically bind their targets with high affinity while immobilised on gold surfaces. Applications for Affimer proteins in a range of gold-based assays, such as Raman scattering (SERS) and MEMS/NEMS, as well as the commonly used QCM-D and SPRi assays stem from this work. With the possibility to multiplex many of these assays, this study is an important first step in developing an assay that can monitor multiple sub-classes of IgG simultaneously.