Asta Makaraviciute

Site-directed antibody immobilization techniques for immunosensors

Immunosensor sensitivity, regenerability, and stability directly depend on the type of antibodies used for the immunosensor design, quantity of immobilized molecules, remaining activity upon immobilization, and proper orientation on the sensing interface. Although sensor surfaces prepared with antibodies immobilized in a random manner yield satisfactory results, site-directed immobilization of the sensing molecules significantly improves the immunosensor sensitivity, especially when planar supports are employed. This review focuses on the three most conventional site-directed antibody immobilization techniques used in immunosensor design. One strategy of immobilizing antibodies on the sensor surface is via affinity interactions with a pre-formed layer of the Fc binding proteins, e.g., protein A, protein G, Fc region specific antibodies or various recombinant proteins. Another immobilization strategy is based on the use of chemically or genetically engineered antibody fragments that can be attached to the sensor surface covered in gold or self-assembled monolayer via the sulfhydryl groups present in the hinge region. The third most common strategy is antibody immobilization via an oxidized oligosaccharide moiety present in the Fc region of the antibody. The principles, advantages, applications, and arising problems of these most often applied immobilization techniques are reviewed.

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.

Development of a reusable protein G based SPR immunosensor for direct human growth hormone detection in real samples

Although it has been shown that protein G based site-directed antibody capture is an advantageous immobilization technique in immunosensor development, the application of such immunosensors for direct repeated real sample measurements has not been addressed. In this surface plasmon resonance analysis based study random covalent antibody immobilization was employed as a reference technique in contrast to site-directed protein G mediated affinity capture and protein G mediated antibody capture stabilized by chemical cross-linking aimed at direct repeated human growth hormone detection in real samples. Site-directed affinity capture increased the analytical signal 2.6 times compared to random antibody immobilization. However, this approach resulted in significant antibody dissociation from the sensor surface during the measurements, which was detrimental for signal evaluation, immunosensor reusability and formed a potential platform for non-specific binding of molecules present in real samples. In contrast, chemical cross-linking of the protein G and antibody complexes under optimal conditions enabled repeated measurements and improved signal evaluation. Moreover, the analytical signal was increased 3.5 times in comparison to random antibody immobilization. The obtained results put emphasis on sensing molecule orientation, stability surface mass density and remaining activity after the immobilization. Based on these findings an immunosensor model for human growth hormone detection was developed. The limit of direct detection of the developed immunosensor was 0.99 nM, the limit of quantification was 3.31 nM and the linear detection range was from 3 to 9 nM. It has been demonstrated that the immunosensor model was prone to non-specific binding of serum proteins that could be minimized following a one-step sample pre-treatment procedure.

Considerations in producing preferentially reduced half-antibody fragments

Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

A QCM-D Study of Reduced Antibody Fragments Immobilized on Planar Gold and Gold Nanoparticle Modified Sensor Surfaces

An immunosensor is an analytical system consisting of specific immune system molecules coupled to a signal transducer. Immunosensor sensitivity depends on the type of immunorecognition ligands used, immobilization influence on their activity and orientation on the surface. Quartz crystal microbalance with dissipation (QCM-D) was employed to investigate the immobilization of antibodies against bovine leukemia virus antigen gp51 (gp51). Disulphide bridges of antibody hinge region were reduced chemically to yield two half antibody fragments (Frag-Ab), each having a single antigen binding site and free sulfhydryl groups that were used for immobilization. Frag-Ab were immobilized on planar gold and gold nanoparticle (AuNP) modified QCM-D sensor surfaces from initial solutions of different concentrations. Higher Frag-Ab surface density values were obtained on AuNP modified surfaces at all tested antibody concentrations. Frag-Ab/gp51 specific interaction was registered and it was determined that the highest sensitivity was exhibited by Frag-Ab immobilized at the lowest surface desities on both types of investigated surfaces. Specific gp51 interaction with Frag-Ab and non-specific binding to bovine serum albumin modified surfaces were compared by employing Δf/ΔD plots, which could serve as fingerprints of different processes.

Capacitive micromachined ultrasound transducers (CMUT) for resonant gravimetric immunosensing

The array of the CMUT sensors was fabricated by the direct bonding of the silicon membranes provided as a device layer of the SOI wafer with the oxidized and pre-patterned highly doped silicon wafer. The active surface of the sensors was coated with thin gold film. We demonstrate the detection of the formed immune complex over the CMUT surface modified by the bovine leukemia virus antigen BLV gp51. The modified CMUT surface was allowed to interact with the specific antibody anti-gp51 labeled by the horseradish peroxidase. Antibody labels were actvated after the interaction by wetting the sensor surface with tetramethylbenzidine to provide the reference quantification of the formation of the immune complex. CMUT sensor readings (resonance frequency and the resonance value of the impedance real part, “resistance”) were obtained before and after modification by the BLV gp51 and after the interaction with antig-p51. The readings were interpreted and fitted by the finite element analysis. It was determined that increase of the elasticity modulus and stress of the sensor structure caused increase of the resonance frequency and some decrease of the resonance quality (resistance value). After the immune complex is established, we observe decrease of the resonance frequency by 4% on average from the initial value. Finite element analysis model fitting to the experimental results revealed the mass loading function of the CMUT structure in the presence of increased elasticity of the proteins.

Application of CMUT as immunosensor

Surface biofunctionalization of capacitive micromachined ultrasonic transducer (CMUT) based sensor can develop non-informative stress and elasticity, which are distorting sensor readings related with the mass of established immune complex. The prototype sensor calibration function, using polimethylmetacrylate (PMMA) films from 7 to 100 nm, was developed to relate the sensor readings with the changing mass, elasticity and stress of the film on the CMUT surface. The range of calibration in terms of mass is from 1 to 80 pg per single CMUT cell, 0 - 20 MPa in terms of stress with corresponding sensor resonance frequency and resistance readings from 10 to - 35 kHz and from 4.5 to -0.5 Ω. Antigen and antibody specific interaction sensing experiment was performed and obtained results showed that informativity of CMUT based immunosensor is much greater than expected, since most of the sensor readings were out of the calibration function, developed for PMMA, range. At the same time, immunosensing experiment demonstrated clear distinction between antigen and antibody mass, which is more than threefold.