Antigen-antibody Pairs Research Articles

Evaluation of immune response and protective effect of four vaccines against the tick-borne encephalitis virus

Among three main subtypes of the tick-borne encephalitis virus (TBEV), the Siberian subtype is currently dominant in a majority of the endemic regions of Russia. However, inactivated vaccines are based on TBEV strains of the heterologous Far Eastern or the European subtypes isolated 40–77 years ago. To analyze the efficacy of the available vaccines against currently prevailing TBEV isolates of the Siberian subtype, mice were immunized subcutaneously three times (one group per each vaccine). The expression of seven cytokine genes was determined using RT-PCR. Sera were studied using homologous and heterologous ELISA, hemagglutination inhibition (HI) and neutralization tests with TBEV strains of the Far Eastern, Siberian and European subtypes. Cross-protective efficacy of the vaccines was evaluated with the TBEV strain 2689 of Siberian subtype isolated from an ixodid tick from the Novosibirsk, South-Western Siberia, Russia in 2010. The cytokine gene expression profile indicates a predominantly Th2 response due to exogenous antigen presentation. Titers for homologous combinations of vaccine strain and strain in ELISA, HI and neutralization tests exceeded those for heterologous antigen-antibody pairs. Despite antibody detection by means of ELISA, HI and neutralization tests, the mouse protection afforded by the vaccines differed significantly. Complete protection of mice challenged with 100 LD50 virus of the Siberian subtype was induced by the vaccine “Encevir” (“Microgen”, Tomsk, Russia). The minimal immunization doze (MID50) of “Encevir” protecting 50% of the mice was less than 0.0016ml. Partial protective effect of vaccines produced in Moscow, Russia and Austria revealed MID50 within recommended intervals (0.001–0.017ml). However, the MID50 for the vaccine “Encepur” (Novartis, Germany) 0.04ml exceeded acceptable limits with total loss of mice immunized with vaccine diluted 32, 100 and 320 fold. These results suggest regular evaluation of TBEV vaccines in regions where heterologous virus subtypes prevail.

Investigation of targeted biomolecules in a micro-fluxgate-based bio-sensing system

An investigation of targeted biomolecules was accomplished by combining a micro-fluxgate-based bio-sensing system and Dynabeads. The fluxgate sensor for biomolecule detection was fabricated by Micro Electro-Mechanical system technology, including thick photoresist lithography, electroplating and chemical wet etching. The magnetic core of the sensor was made of Fe-based amorphous ribbon core and three dimension solenoid coils were used as magnetic sensitive elements. The micro-fluxgate-based bio-sensing system was characterized firstly in different concentrations of Dynabeads, and a concentration as low as 100ng/ml was detected with an external dc magnetic field in the range of 525 μT to 875 μT. Sandwich assays are performed using antibody-antigen pair combination of biotin-streptavidin on a separated Au film substrate surface with a self-assembled layer. Detection of Alpha Fetoprotein antigens with different concentrations was performed and a minimum detectable concentration of 1pg/ml was achieved by the bio-sensing system. It is of considerable interest due to its potential application in the biomedical field based on known specific binding of target and labels.

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor-like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane-engineered cells. In the present study, purified anti-biotin antibodies from a rabbit antiserum along with in-house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody-antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane-engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay.

Detection of targeted carcinoembryonic antigens using a micro-fluxgate-based biosensor

In this work, a micro-fluxgate-based biosensor was designed for the detection of carcinoembryonic antigen (CEA) labeled by Dynabeads. The sensor with Fe-based amorphous core and three dimension solenoid coils was fabricated by Micro Electro-Mechanical system technology. Sandwich assays are performed using antibody-antigen pair combination of biotin-streptavidin assay on a separated Au film substrate surface with a self-assembled layer. With dc magnetic fields in the range of 560 μT to 875 μT, detection of CEAs with different concentrations was performed and a minimum detectable concentration of 1 pg/ml was achieved. Furthermore, CEA samples with different concentrations can be distinguished.

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.

An integrated solution for rapid biosensing with robust linker free covalent bindingsurfaces

A novel integrated biosensor methodology is proposed and demonstrated. The methodology utilizes a nitrogen-containing plasma polymer to achieve linker-free binding of a layer of biorecognition molecules. The sensor surface has been shown to maintain its performance after freeze-drying providing a long shelf life under ambient conditions. The sensor is configured for single wavelength ellipsometric detection providing a low cost, versatile, and rapid sensing and diagnosis platform suitable for a wide range of applications and end-users. The merits of the methodology are demonstrated using three antigen–antibodypairs.

Label-Free and Real-Time Detection of Antigen-Antibody Capture Processes Using the Oblique-Incidence Reflectivity Difference Technique

We successfully label-free and real-time detect the capture processes of human immunoglobulin G (IgG)/goat anti-human IgG and mouse IgG/goat anti-mouse IgG antigen-antibody pairs with different concentrations using the oblique-incidence reflectivity difference (OIRD) method, and obtain the interaction kinetics curves and the interaction times. The experimental results prove that the OIRD method is a promising technique for label-free and real-time detection of the biomolecular interaction processes and achieving the quantitative information of interaction kinetics.

An integrated solution for rapid biosensing combining linker free binding, freeze drying and high sensitivity ellipsometric detection

AbstractA novel integrated biosensor methodology is proposed and demonstrated. The methodology utilizes a nitrogen-containing plasma polymer to achieve linker-free binding of biorecognition molecules that allows the sensor surface to be freeze dried for long shelf life under ambient conditions. The sensor is configured for single wavelength ellipsometric detection providing a low cost, versatile, and rapid sensing and diagnosis platform suitable for a wide range of applications and end-users. The merits of the methodology were demonstrated using three antigen-antibody pairs.

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO2 or epoxy slides (purification-independent microarray – PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31days after spotting. We finally demonstrated that the method is suitable for other antigen–antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.

Tapered Optical Fiber Sensor for Label-Free Detection of Biomolecules

This paper presents a fast, highly sensitive and low-cost tapered optical fiber biosensor that enables the label-free detection of biomolecules. The sensor takes advantage of the interference effect between the fiber’s first two propagation modes along the taper waist region. The biomolecules bonded on the taper surface were determined by demodulating the transmission spectrum phase shift. Because of the sharp spectrum fringe signals, as well as a relatively long biomolecule testing region, the sensor displayed a fast response and was highly sensitive. To better understand the influence of various biomolecules on the sensor, a numerical simulation that varied biolayer parameters such as thickness and refractive index was performed. The results showed that the spectrum fringe shift was obvious to be measured even when the biolayer was only nanometers thick. A microchannel chip was designed and fabricated for the protection of the sensor and biotesting. Microelectromechanical systems (MEMS) fabrication techniques were used to precisely control the profile and depth of the microchannel on the silicon chip with an accuracy of 2 μm. A tapered optical fiber biosensor was fabricated and evaluated with an Immune globulin G (IgG) antibody-antigen pair.

Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 ± 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.

Antibody-mediated purification of co-expressed antigen–antibody complexes

Immunoaffinity is an established chromatographic method for isolating macromolecules independently on the presence of specific tags while the tight interaction between antigen and antibody has been exploited to stabilize proteins during crystallization trials. Therefore, it seems reasonable to try to combine the two protocols, namely to co-express the target proteins together with their specific antibodies to obtain stable complexes suitable for direct purification and further analyses. Using the variable region of single domain llama antibodies, we showed that the co-expression of antigen–antibody pairs is feasible in both the periplasm and the cytoplasm of bacteria. Moreover, the complexes that were formed in vivo could be purified using a tag fused to the recombinant antibody and remained stable during gel-filtration. The co-expression and co-purification strategy significantly increased the final protein yields promoting the accumulation of functional intrabodies. The described method may offer a suitable alternative for the purification of proteins intended for crystallization trials and it may also be used as a general purification protocol for both antigens and recombinant antibodies.

High-throughput surface plasmon resonance imaging-based biomolecular kinetic screening analysis

In this paper we show a high-throughput method to screen the kinetics and affinity constants of many biomolecular interactions simultaneously. During the preparation of the sensor chip, ligands were serially diluted and spotted in a 6 × 4 microarray on the sensor surface. A multi-analyte sample was injected and the real-time surface plasmon resonance (SPR) responses of all the 24 microarray spots were obtained using a commercial SPR imaging instrument. The multi responses of the association and dissociation processes obtained from a single analyte injection are sufficient to calculate the rates and affinity constants of the interactions between three interactant antigen-antibody pairs using a simple monophasic kinetic model. The method drastically reduces the measurement time and cost in the benefit of increased throughput.

Libraries against libraries for combinatorial selection of replicating antigen–antibody pairs

Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen-antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen-antibody clones in a single experiment.

DARS (Decoys As the Reference State) Potentials for Protein-Protein Docking

Decoys As the Reference State (DARS) is a simple and natural approach to the construction of structure-based intermolecular potentials. The idea is generating a large set of docked conformations with good shape complementarity but without accounting for atom types, and using the frequency of interactions extracted from these decoys as the reference state. In principle, the resulting potential is ideal for finding near-native conformations among structures obtained by docking, and can be combined with other energy terms to be used directly in docking calculations. We investigated the performance of various DARS versions for docking enzyme-inhibitor, antigen-antibody, and other type of complexes. For enzyme-inhibitor pairs, DARS provides both excellent discrimination and docking results, even with very small decoy sets. For antigen-antibody complexes, DARS is slightly better than a number of interaction potentials tested, but results are worse than for enzyme-inhibitor complexes. With a few exceptions, the DARS docking results are also good for the other complexes, despite poor discrimination, and we show that the latter is not a correct test for docking accuracy. The analysis of interactions in antigen-antibody pairs reveals that, in constructing pairwise potentials for such complexes, one should account for the asymmetry of hydrophobic patches on the two sides of the interface. Similar asymmetry does occur in the few other complexes with poor DARS docking results.

A novel label-free multi-throughput optical biosensor based on localized surface plasmon resonance

A novel and sensitive multi-throughput localized surface plasmon resonance (MLSPR) biosensor was developed for the first time. Various gold nanorods with different aspect ratios were used to fabricate the optical sensor. Five kinds of gold nanorods with different aspect ratios were chosen to construct five throughputs of MLSPR. Various LSPR peaks imply that different acceptor–ligand pairs can be detected simultaneously in the wavelength range from 530 to 940nm. The biosensor immobilized on glass slides was applied to label-free detection between acceptor and ligand. The MLSPR-based optical biosensor can be used to detect three antigen–antibody pairs simultaneously. The biosensor proposed herein is easy to fabricate, and its operation procedure is convenient as labeling procedure is unnecessary.

Review of Serologically Confirmed Cases of Transfusion-Related Acute Lung Injury Reported to a Blood Center.

We reviewed 13 cases of serologically confirmed Transfusion-Related Acute Lung Injury (TRALI) reported to our blood center over a five-year period. Cognate antigen-antibody pairs were noted, as well as product transfused, indication for transfusion, onset of symptoms related to transfusion and time to resolution. All patients demonstrated bilateral pulmonary edema on CXR. Onset of symptoms for all cases occurred during transfusion of implicated product, or within one hour post-transfusion. All donors of implicated products were multiparous females. The majority of implicated products (77%) were FFP. There were 3 TRALI deaths, all resulted from transfusion of FFP. Anti-neutrophil, HLA Class I and/or Class II antibodies were identified in all fatal cases. Platelets comprised 15% of implicated products, and RBC's only a single case (7 %). Among patients with TRALI receiving FFP, the most common indication was reversal of anticoagulation (50%). Chart review in two cases indicated Vitamin K in lieu of FFP may have been appropriate.Comparison of Serologically Confirmed TRALI CasesPatientProductReason for TxTime to ResolutionNeutrophil AntibodyHLA IHLA IIHLA PRA1FFPReverse anticoag.Death @ 7 hoursHNA-3aNeg.Neg.02PlateletThrombocytopenia<1 day+Neg.Neg.03FFPReverse anticoag.2 days+Neg.Neg.04PlateletPancytopenia<1 day+Neg.Neg.05FFPReverse anticoag.4 days+A2 B3DR 52I - 90% II - 56%6FFPNot availableunknown+Neg.Neg.07FFPPeriop. BleedingunknownNot testedA2Neg.40%8FFPReverse anticoag.2 weeksNot testedNeg.DR 1763%9Cryopoor plasmaTTP4 daysNot testedNeg.DR 5241%10FFPReverse anticoag.Death @ 4 hoursNot testedB 50DR 16I - 95% II - 89%11RBCBleeding2 daysNot testedNeg.DR 5260%12FFPGI BleedDeathNot testedNeg.DR 53 DQ 560%13FFPGI Bleed2 daysNot testedNegDR 5260%In our small series, the majority of implicated products were FFP, and all products were from multiparous females. Production of FFP exclusively from male donors is currently under consideration. Although male donor plasma can be expected to reduce the risk of TRALI associated with transfusion of FFP, it will not eliminate it entirely. Therefore, the risk of TRALI should be factored into the decision to transfuse all products. FFP should be administered cautiously for all indications, including reversal of anticoagulation.

Combining optical tweezers and scanning probe microscopy to study DNA–protein interactions

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.

Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations: Theory and Experiment

In this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the am and low fm ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios.

Antigen Binding Forces of Single Antilysozyme Fv Fragments Explored by Atomic Force Microscopy

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.