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TECHNICAL FIELD

The present invention relates to a construct comprising a Ni and/or Au substrate provided of a hexa- histidine tagged protein A/G, as well as the use of said construct in order to build an antibody construct featuring an enhanced antigen binding abitlity.

More particularly, the present invention relates to the use of protein A/G tagged with a hexa-histidine tail at one protein terminus for the formation, on suitable substrates, of densely packed and uniformly oriented ( sub) -monolayers of proteins capable of binding functionally active antibodies in an optimized and highly effective fashion.

BACKGROUND ART

Protein A (or G) is a membrane bound protein found initially on the outer membrane of the bacterium Staphylococcus aureus. It has typically 5 IgG-binding domains which show a marked affinity for the Fc fragment of several antibodies, through its interaction with the heavy chain. Its affinity to antibodies is particularly marked for IgG and more moderate for IgA, IgM, and IgE (see Moks, T., Abrahmsen, L., Nilsson, B., Hellman, U., Sjoquist, J. and Uhlen, M ,1986, Eur. J. Biochem. 156, 637-643) . As a matter of fact, it is widely used for that reason in antibody purification assays. It can be now produced heterologously (e.g. in E. coli) and can be modified by protein engineering to bear a his-tag at its N-terminus for purification purposes by affinity chromatography .

In fact his-tag has a marked affinity for Ni complexes such as Ni-NTA, an organometallic compound which is often used in chromatography columns bound to resin beads. The chelated Ni can bind the 6-his tag and can release it by elution with imidazole-containing aqueous solutions at concentration in the range 100-1000 mM.

Recently, it has been discovered that the 6-his tail can bind effectively also metal Ni, enabling it as a suitable substrate to immobilize his-tagged proteins (see Alessandrini , A.; Bortolotti, C. A.; Bertoni, G.; Vezzoli, A.; Facci, P., J. Phis. Chem. C 2008, 112, 3747- 3750) .

Metal-coated quartz crystal microbalance (qcm) transducers (see Sauerbrey, G. Z. Phis., 1959, 155, 206- 222) have been used both in air for measuring protein surface density and in liquid (flow chamber) to measure adsorption kinetics. Furthermore, AFM measurements have been performed on Ni flat substrates evaporated on freshly cleaved mica sheets in order to assess protein surface density by direct visualization. In order to validate the proposed immobilization approach, control measurements exploiting different immobilization techniques have been set up.

Description of the invention

Primarily, the present invention aims to provide a construct comprising a Ni and/or Au substrate provided of Hexa-histidine-tagged protein A or G suitable for building a layer of antibodies featuring an enhanced antigen binding ability.

This is achieved by a construct having the features disclosed in claim 1.

The present invention aims to provide a a highly dense, sensitive layer of properly oriented antibody molecules (e.g. IgG, IgM, IgE, IGA or any kimeric protein obtained through fusion with a Fc fragment of an antibody) in order to maximize its performances as to capturing antigens from a liquid or gaseous sample.

This is achieved by adding the features disclosed in claim 2.

Furthermore, the present invention aims to provide a method for providing a his-tagged protein A/G layer that enables a uniform orientation of IgGs, thereby exposing effectively their antigen-binding sites, at variance with other antibody immobilization techniques.

This is use is disclosed in claim 3 and in the claims dependent thereon.

The invention appears to provide important advantages in respect of the prior art solutions, since it maximize the performances of any biosensor/biodevice using antibodies irrespectively of the detection techniques used in the specific device.

As a result, the invention can be used in any artificial molecular edifices forming the active part of a biosensor or a biodevice.

For instance, it can be used for capturing molecules or antigen-bearing cells from blood (e.g blood grouping), or any other biologic fluid, or for implementing sensitive sensors against e.g pollutants (in air, water, etc.), immunosensors against pollens, against drugs, against explosives, etc.

Illustration of drawings

Other features and advantages of the invention will become apparent by reading the following description of a form of embodiment of the invention, given as a non- limiting example, with the help of the figures illustrated in the attached drawings, in which:

· Fig. 1 illustrates the results of qcm experiments in air showing the difference binding efficiency and coverage for an oriented and a randomly oriented construct ;

• Fig. 2 shows the binding kinetics of protein A in case of interaction by the 6Xhis tag (light blue) and by surface amines (violet) ;

• Fig. 3 shows a AFM investigation of a Ni film incubated with 6Xhis-tagged protein A solution;

• Fig. 4 is a graph showing binding curves of antibodies on molecular constructs with different orientation outlining a 20% higher efficiency of the ordered construct;

• Fig. 5 is a graph showing binding curves of antigens on molecular constructs with different orientation outlining a 30% higher efficiency of the ordered construct; and

• Fig. 6 illustrated the various steps for the assembling of an oriented antibody construct amenable of enhanced antigen binding ability according to the present invention.

Description of a form of embodiment of the invention In figure 1, the results are shown of measurements performed with a qcm in air comparing the effect of a his-tagged protein A monolayer to that of a sample in which protein A is bound to the surface (gold, in this case) by exploiting its surface primary amines, hence involving potentially a higher number of covalent binding sites .

Particularly, the control construct was assembled by binding first a self-assembled monolayer (SAM) of 2- mecaptoethilamine on the surface of qcm Au electrodes.

Secondly, the SAM was incubated with glutharaldheyde, which on its turn was exposed to protein A.

The free aldheyde groups exposed off the surface- bound molecular edifice were able to react with the exposed primary amines of protein A forming immide bonds to give a randomly oriented protein A layer.

These results show that the transferred mass in case of the his-tagged monolayer on Ni is higher and corresponds to a coverage of 90% at variance with a figure of 22.5% for the construct not exploiting Ni-his- tag interaction.

Similarly, as shown in Fig. 2, adsorption kinetics performed by liquid chamber qcm indicate that his-tagged protein A reaches a higher saturation level if compared to a randomly oriented construct.

Surprisingly, the characteristic adsorption times are different in the tagged vs non-tagged sample, pointing to a different mechanism in the adsorption at the surface.

In fact, whereas his-tagged protein A can bind the surface only with the 6xhis tail forming coordination bonds between the imidazole rings and Ni at surface, the not tagged proteins have many more binding sites all around the protein globule and give rise to covalent bonds .

The result of the described difference in the binding mechanisms is that the non tagged proteins bind more readily the surface and in a "ball on a sticky surface" fashion, at variance with the his-tagged molecule that has to sit properly on the surface before achieving a stable binding.

As a consequence, the surface density of the two constructs is also different since the latter is more influenced by intermolecular interactions which can play an active role in compacting the molecular layer at the surface due to a sort of surface-diffusion/annealing.

Figure 3 shows the surface coverage as visualized by liquid AFM in alternating contact mode. Visible bumps are single protein A molecules protruding off the ultraflat Ni surface. These data confirm the higher density of the his-tagged layer and are consistent with the results obtained by both qcm measurements performed in air and in liquid .

The prepared protein A layer has then been used for binding IgG antibodies.

Figure 4 compares the results of the binding kinetics of human IgG antibodies on a protein A layer prepared with the two different aforementioned strategies .

Whereas the characteristic adsorption times are very similar, the saturation level is quite different resulting in a figure of 20% higher immobilization level in case of his-tagged protein A with respect to the randomly oriented construct. These results are readily understandable in terms of the fact that, whereas the kind of bond that IgGs make with protein A is the same irrespectively of protein A orientation at the surface, the surface density of his- tagged protein A is remarkably higher, giving rise to an higher IgG density at the surface.

Finally, the prepared constructs have been tested with the specific antibodies in order to assess their specific binding performances.

Figure 5 reports the results obtained again by measuring adsorption kinetics with the qcm in liquid.

Again the characteristic binding times are similar, as expected being the bonds involved in the specific recognition event the same in the two cases.

Nevertheless, the saturation level is markedly different in the two curves, showing a 30% higher level in case of the his-tagged construct.

This result is understandable considering not only the higher surface density of the tagged construct but also the fact that it bears a unique orientation.

In fact, being endowed with a unique binding tail, his-tagged protein A can assume barely a unique orientation at the surface.

This fact both helps improving its 2d packing (surface density) and promotes the exposition of an ordered array of IgG binding sites at the surface.

As a consequence the immobilized IgGs will be prevalently oriented in the same fashion. Since they bind protein A by their Fc fragment, they will arrange with their Fab domains exposed off the surface, in a fashion amenable for effective antigen binding.

On the contrary, the randomly oriented protein A construct will bind in a more disordered fashion IgGs and this fact will result in a worse exposure of the Fab fragments, hence of the specific binding sites for the antigens .

Steric hindrance will limit antigen binding to an extent even higher that that dictated by the lower antibody surface density.

In light of all these results Fig. 6 summarizes the scheme which describes the molecular edifice that has been built.

Said Fig. 6 reports the various steps by which the final molecular construct is realized:

step A) structure of his-tagged protein A;

step B) Self-assembling of protein A monolayer on Ni or Au substrate by the high affinity of the 6Xhis tag for the specified metal;

step C) oriented binding of antibodies by the oriented protein A;

step D) exposition and binding of antigens by the pre ^¬ formed oriented antibody layer.

The invention has been described with reference to a preferred form of embodiment thereof. However, it is clear that the invention encompasses several different equivalent embodiments, in particular those where the antibodies which are used are of different nature, e.g. IgG, IgM, IgE, IGA or any kimeric protein obtained through fusion with a Fc fragment of an antibody.