Study on interactions of human IgG with immobilized anti-IgG or recombinant Staphylococcal protein A using surface plasmon resonance spectrometry

A. O. Bakhmachuk, O. B. Gorbatiuk, O. G. Palyvoda © 2016 A. O. Bakhmachuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 543.424+577.27 Study on interactions of human IgG with immobilized anti-IgG or recombinant Staphylococcal protein A using surface plasmon resonance spectrometry


Introduction
The successful development of approaches and analytical tools of biotechnology will enable to upgrade substantially the quality of life by improving the methods of biomedical diagnostics, molecular engineering, environmental monitoring, food analysis, drug discovery, etc.For this purpose, in many cases, the biosensors could be helpful.Their design feature is the combination of a bioselective element that specifically reacts with an analyte and a physical transducer that transforms the result of biological process into the electrical signal suitable for further processing and characterization [1,2].The use of variety of immune components is very attractive in this area of research.The immunosensors demonstrate high selectivity through the use of immune molecules, simple operation, simple sample preparation and high

Molecular and
Cell Biotechnologies ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 1. P 54-60 doi: http://dx.doi.org/10.7124/bc.00090Dsensitivity [3].Furthermore, the biosensors based on the phenomenon of surface plasmon resonance (SPR) are applied to the label-free and real time detection of various intermolecular interactions.SPR spectrometer registers the changes of the refractive index of a thin layer (~ 200 nm) of medium, adjacent to the sensor surface.Immobilization of biomacromolecules and interaction of immobilized components with their partner molecules alter the refractive index of this layer and cause the SPR response [4].An important step to achieve high efficiency of the immunosensor is the preservation of the functional activity of immune components during their immobilization on the sensor surface.However, during the immobilization of antibodies on the surface of physical transducers their antigen-binding activity is usually much lower in comparison with the activity of the same antibodies in solution.The main reasons for this are believed to be random orientation of antibodies on the sensor surface and steric hindrance caused by the influence of the surface of a solid substrate [5].To prevent this, it is necessary to create an intermediate layer, which would include the immunoglobulin-binding proteins, such as Staphylococcal protein A (SPA).It is known that SPA selectively binds Fc-fragment of antibody, leaving available antigen-binding sites [6].The immunoglobulinbinding region of SPA does not contain cysteine residues [7,8], so the immobilization of SPA is possible only by physical sorption on the sensor surface, which is not always reliable.Genetically engineered introduction into the recombinant SPA of the cysteine residue, which interacts with the gold sensor surface through exposed SH-group, improves the reliability of SPA immobilization preserving its functional properties.In the Institute of Molecular Biology and Genetics NAS of Ukraine, the original genetically engineered construct was created and the recombinant Staphylococcal protein A with the introduced C-terminal cysteine residue (SPA-Cys) was obtained [9].Species-specific antibodies, such as goat anti-human IgG antibodies (anti-IgG), are more traditional immunoglobulin-binding proteins, which could also be immobilized on a sensor surface as an intermediate layer for further immobilization of IgG.
The aim of the present work is the comparison of IgG-binding activity of SPA-Cys or goat anti-human IgG antibodies (anti-IgG) after their immobilization on a gold sensor surface of the SPR spectrometer.
Human IgG was obtained also by affinity chromatography using SPA-Cys-modified silica as described in [9].The synthesis and purification of recombinant SPA-Cys were described in [9].The homogeneity of the proteins used was checked by 13% SDS-PAGE.
The SPR spectrometer ("Plasmon SPR-4m") was used to study the protein-protein interactions (the device and corresponding software have been developed at the Lashkaryov Institute of Semiconductor Physics NAS of Ukraine).This computer controlled optoelectronic spectrometer uses the optical phenomenon of SPR in Kretschmann configuration.A thin layer (50 nm) of gold, which was deposited on a glass plate, is used as a sensitive element of the SPR spectrometer.Just before the experiment, the gold surface of the glass plate was purified by incubation in a mixture of "piranha" (a mixture of 30% H 2 O 2 and concentrated H 2 SO 4 in 1:3 ratio; WARNING: "piranha" reacts violently with organic compounds and must be handled with extreme care) for 2 minutes.Then the plate repeatedly washed with water and dried in air.The treated glass plate is mounted on the prism of the device using the immersion liquid that has the same refraction index as the prism and the glass plate.The gold surface serves as a bottom of a flow measuring cell (~20 μl).A silicone rubber ring serves as the side walls.A plexiglass cover contains input and output pipes, which the buffer solution and investigated samples pass through [10].The flow rate of liquid (usually 40 µl/ min) is controlled by peristaltic pump «Ismatec» (Switzerland).
At the beginning of the experiment the measuring cell was washed with the working buffer solution (PBS, in some cases -CB or SSC), to obtain a stable signal of the device -the baseline.For immobilization of the proteins 120 µl of 1 µM protein solution in PBS, CB or SSC was injected into the measuring cell.After 25 min-incubation of the sample (the pump was switched off) a flow of the working buffer solution was used to remove the excess of the proteins that were not immobilized.
To prevent non-specific adsorption of studied components on the surface sites, which are left uncovered with the immobilized protein, a sensor surface was passivated with milk proteins.For this purpose, 120 µl of 200 µg/ml milk proteins in the working buffer solution (PBS) were injected into the measuring cell, then incubated at the pump switched off for 20 min, after that the surface was washed with PBS until stabilization of the sensor signal [11].
On the main stage of the experiment 120 µl of solutions of different concentrations of IgG in PBS were injected into measuring cell, incubated at the pump switched off for 10 min, then the cell was washed with PBS until stabilization of the sensor signal.
For regeneration of a bioselective element (destruction of bonds between the immobilized protein and IgG as well as a removal of the latter), 120 µl 40 mM citrate buffer (pH 2.5) was injected into the measuring cell, then the cell was washed with PBS until stabilization of the sensor signal [12].

Results and Discussion
In this paper the following proteins were investigated: recombinant Staphylococcal protein A with the introduced C-terminal cysteine residue (SPA-Cys), goat antibodies against human immunoglobulin (anti-IgG), human IgG obtained from "Serotec", and human IgG obtained by affinity sorbent.All of them are represented on the electrophoregram after electrophoresis in 13 % SDS-PAG (Fig. 1).The only one band, which moved a little faster than a marker protein of 35 kDa, is clearly seen on the lane 1.This is consistent with the mobility of SPA-Cys (34.5 kDa).All preparations of antibodies have two major bands corresponding to heavy and light chains of IgG.The presence of additional minor band (~ 33 kDa) in the "Serotec" preparation of human IgG can be explained by the presence of some degradation products or residual serum proteins.
Fig. 2 shows the SPR sensogram representing the immobilization of SPA-Cys on the gold sensor surface.After prolonged washing of the measuring cell by PBS and stabilization of sensor signal the difference between the signal before injection of 120µl of 1 µM SPA-Cys and after washing with PBS was almost 0,14 angular degrees (Fig. 2).This value represents the number of reliably immobilized SPA-Cys molecules.According to the conversion factor of SPR response into the value of the surface density of immobilized protein [13], it is 1.35 ng/mm 2 (Table ).Given the molecular weight of SPA-Cys (34.5 kDa), one can calculate that on average about 2.4 molecules of SPA-Cys were immobilized for every 100 nm 2 .It does not indicate the formation of a dense monolayer of proteins on the sensor surface.So, after the immobilization of SPA-Cys the free sites for the non-specific sorption on the gold surface were blocked with the injection of a solution of 200 µg/ml milk proteins in PBS.
After immobilization of SPA-Cys and passivation of the gold sensor surface the interactions of immobilized SPA-Cys with IgG were investigated.The level of the SPR sensor response was shown to be directly proportional to the concentration of IgG, at least in the range of 10-40 µg/ml (Fig. 3).This figure also illustrates a successful regeneration of the bioselective element.After treatment of the sensor surface with a solution of 40 mM citrate buffer (pH 2.5) [12] the SPR signal returned back almost to the values that preceded the injection of the sample.It demonstrates the effective destruction of the bonds between immobilized SPA-Cys and IgG and the removal of the latter.The subsequent injection of new samples of IgG showed that the regeneration did not affect the level of immunoglobulin-binding activity of the immobilized SPA-Cys.Thus, it becomes pos-sible to reuse the bioselective element of the immunosensor based on SPA-Cys.
At first, the immobilization of another immunoglobulin-binding protein, namely, goat antibodies against human immunoglobulin G (anti-IgG) was performed under the same conditions as those during the immobilization of SPA-Cys (1 µM of protein in PBS).The sensor response after immobilization of anti-IgG was more than two times higher than the response we received after immobilization of SPA-Cys (Fig. 2, Fig. 5, Table ).After blocking the nonspecific sorption sites with milk proteins the injection of IgG solutions of concentration from 10 to 40 µg/ml showed that the sensor registered the interactions of immobilized anti-IgG and IgG, but the level of SPR sensor response practically does not depend on the IgG concentration (Fig. 4).This may indicate a rapid saturation of all available antigenbinding sites of anti-IgG.In addition, the values of the SPR sensor response during the interaction of IgG with immobilized anti-IgG is significantly lower than during the interaction with the immobilized SPA-Cys (Fig. 4).
According to the conversion of the SPR response into the value of the surface density of immobilized protein [13], the values of the surface density were calculated for the immobilized SPA-Cys and anti-IgG, as well as for human IgG, which interacted with the above mentioned immobilized proteins (Table ).It should be noted that the increased sensor response on the IgG addition to the immobilized SPA-Cys can be also explained by vertical orientation of molecule-dipole, which increases the interaction of the molecules with the plasmon polariton electromagnetic field, which in its turn evokes some additional shift of the SPR curve toward larger angles [14].However, IgG molecule should not be regarded as a rigid rod, and it is not known how much the orientation of IgG molecule at the interaction with immobilized SPA-Cys differs from that at the interaction with immobilized anti-IgG.Therefore, it is quite difficult to determine to what extent the above explanation is applicable to this case.
It is well-known that at the immobilization of antibodies on the surface of immunological plate during ELISA a carbonate buffer is used.So, to look for more favorable conditions for the immobilization of anti-IgG on the gold sensor surface 100 mM carbonate buffer (pH 9.5) and SSC buffer solution (pH 7.2) were applied.Unfortunately, as shown in Fig. 5, the application of these buffer solutions does not lead to an increased level of the immobilized anti-IgG.
The application of CB and SSC buffer solutions at the anti-IgG immobilization did not also improve the level of interactions (in PBS) of the immobilized anti-IgG with human IgG: the binding level is in accordance with the level of anti-IgG immobilization (Fig. 6).It should again be noted, that in most cases there is no clear dependence of the sensor response of the bioselective anti-IgG element on the concentration of IgG.It may be associated with a relatively  The ratio of bound IgG molecules to the number of molecules of IgG-binding protein 0.45 0.17 small number of available antigen-binding sites of the immobilized anti-IgG, almost all of which become occupied by IgG at the injection of the smallest concentration of the latter.

Conclusions
Thus, the study of the immobilization of the recombinant SPA-Cys or the goat anti-human IgG antibodies on the gold sensor surface, and their interactions with IgG demonstrated the significant advantages of SPA-Cys.Although the level of the sensor response at the immobilization of anti-IgG was higher compared to that of SPA-Cys, but given the difference in the molecular weights of two proteins, there is almost twofold advantage of SPA-Cys in the number of immobi-lized molecules.As a result, after further injection of IgG solutions into the measuring cell the bioselective element of immunosensor based on SPA-Cys demonstrates a significantly higher ability to bind IgG, than bioselective element based on anti-IgG.
Obviously, the bacterial synthesis of recombinant SPA-Cys is much more convenient and profitable than obtaining antibodies through the immunization of animals.A consideration of bioethical issues also inclines in favor of SPA-Cys application.It also needs to take into account the fact that SPA binds different classes of immunoglobulins of many organisms (human, rabbit, mouse, etc.), therefore its application can create so-called "universal" immunosensor platform, while the specificity of anti-IgG is much narrower.All these considerations together with our results clearly indicate the good prospects of the recombinant protein SPA-Cys in the scientific research and for the practical applications in the immunosensors.

Fig. 2 .
Fig. 2. The SPR sensogram representing the immobilization of SPA-Cys (120 µl of 1 µM SPA-Cys was injected) on the gold sensor surface.Δ -the sensor response that represents the number of reliably immobilized SPA-Cys molecules.

Fig. 3 .R
Fig. 3. SPR sensogram representing the interactions of the SPA-Cys bioselective element with 120 µl of 10, 20 and 40 µg/ml IgG.R -the base line after regeneration of the bioselective element.

Fig. 4 .
Fig. 4. Comparison histogram of the SPR sensor responses at the interaction of the bioselective element based on SPA-Cys or anti-IgG with 120µl of 10, 20 and 40 µg/ml IgG

Fig. 5 .Fig. 6 .
Fig. 5. Histogram showing the dependence of the level of immobilized anti-IgG on a gold sensor surface on the composition of the buffer solutions at the immobilization